Purification of enveloped or membranous biological particles

Abstract

The present disclosure is directed to a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety and use thereof for the separation of enveloped or membranous biological particles from impurities. Also disclosed are chromatography devices and a method for separating enveloped or membranous biological particles from impurities, comprising adding a liquid sample comprising enveloped or membranous biological particles and one or more impurities, to a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety, and eluting the enveloped or membranous biological particles from the chromatography material in at least one eluate fraction using an elution buffer, the elution buffer optionally comprising a poly-hydroxyl compound, such as sucrose or sorbitol.

Description

[0001] PURIFICATION OF ENVELOPED OR MEMBRANOUS BIOLOGICAL PARTICLES

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of purification of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, or virus-like particles. The disclosure is directed to use of a chromatography material for the separation of enveloped or membranous biological particles from impurities, as well as a method for separating enveloped or membranous biological particles from impurities, and a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety.

[0004] BACKGROUND

[0005] Viral vectors commonly used in medical products include enveloped virus particles, such as Lentivirus (LV). Extracellular vesicles (EV), which are produced and released by cells, is another example of a vector with potential in cell and gene therapy.

[0006] The Lentivirus is classified as a retrovirus, and it has a single stranded RNA genome with a reverse transcriptase enzyme. Lentiviruses have a viral envelope with glycosylated proteins acting as ligands that have affinity for receptors in the outer cell membrane surface of host cells. The virus effects transcription of the viral genetic material upon entering the cell. The benefit of using LV as a viral vector is that it can penetrate the nuclear envelope in dividing as well as non-dividing cells, unlike other retroviruses that only penetrate cells undergoing mitosis. Many cell types in adult individuals do not divide, and LV might be the only option to transfer genetic material into such cells.

[0007] To produce a lentivirus, several plasmids are transfected into a so-called packaging cell line. One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome and is marked by the presence of the IJJ (psi) sequence. This sequence is used to package the genome into the virion. To use lentivirus in gene therapy it is necessary to purify the virion from cell impurities like host cell proteins and DNA, and excess plasmids after transfection. Normally, the harvested host cells producing lentivirus are nuclease treated and the lentivirus is purified with several filtration techniques, such as normal flow microfiltration, ultrafiltration and diafiltration, to reduce the level of impurities to approved levels.

[0008] As mentioned in WO2023 / 274989 Al (Cytiva Bioprocess R&D AB), current downstream purification processes of lentivirus are often synonymous with low recovery of infectious viruses, since lentiviruses are unstable and sensitive to shear forces, buffer components such as salt, and degrade quickly in room temperature. WO2023 / 274989 is directed to a chromatography medium comprising weak anion exchange ligands, which may be used for purification of enveloped virus particles and exosomes.

[0009] However, there is a continuous need in the art for novel chromatography materials and purification methods, providing improved or at least alternative ways of separating enveloped or membranous biological particles from impurities.

[0010] SUMMARY OF THE DISCLOSURE

[0011] One object of the present disclosure is to provide a chromatography material for use in affinity chromatography for purifying enveloped or membranous biological particles, which chromatography material may be used to recover the enveloped or membranous biological particles at industrial scale and at sufficiently mild elution conditions to obtain a commercially relevant level of yield.

[0012] The present disclosure provides use of a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety, for the separation of enveloped or membranous biological particles from impurities.

[0013] Further provided is a method for separating enveloped or membranous biological particles from impurities, comprising: Adding a liquid sample comprising enveloped or membranous biological particles and one or more impurities, to a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety; Eluting the enveloped or membranous biological particles from the chromatography material in at least one eluate fraction using an elution buffer.

[0014] Additionally, the present disclosure provides a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety, wherein the ligand is defined by Formula I: wherein

[0015] X is selected from O, CH2-CH(OH)-CH2-O, CH2-CH(OH)-CH2-O-CH2-CH2-, CH2, and -O-CH2-CH2-SO2-CH2; n is an integer from 0 to 40, preferably 0 < n < 10; each Ro is independently selected from H and alkyl; each Zi is independently selected from 2-pyrrolidone, / V-isopropylformylamide, l-methoxy-2,3- propanediol, / V-[tris(hydroxymethyl)methyl] formylamide, methylamide, / V-alkylformamide, N,N'- dialkylformamide, and alkyl formate; m is an integer from 1 to 40, preferably 1 < m < 10; each Ri is independently selected from H and OH; each R2 is independently selected from H and CsH5; each Z2 is: each Y is independently selected from CH2, S, NH, O, -C(O)NH-, -NHC(O)-, NH-(CH2)u-C(O)-NH, and (CH2)U-NH-C(O), wherein u is an integer from 1 to 6; p is 0 or 1; q is an integer from 0 to 6; each R3is independently selected from H and and each R4, RS, RS, and R7is independently selected from NH2 or a salt thereof, H, Br, F, Cl, alkyl, OH, O- alkyl, and CF3.

[0016] The present disclosure is defined by the appended independent claims. Non-limiting embodiments will become apparent from the dependent claims, the appended drawings, and the following description. It is noted that the present disclosure relates to all possible combinations of features recited in the claims.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] These and other aspects of the present disclosure will now be described in more detail, with reference to the appended drawings showing embodiment(s) of the disclosure, in which:

[0019] Fig. 1 illustrates a method for separating enveloped or membranous biological particles from impurities according to the present disclosure.

[0020] Fig. 2 shows synthetic pathways for the coupling of boronate ligands to resin (Fig. 2A) and to Fibro (Fig. 2B).

[0021] Fig. 3 shows chromatograms from duplicate runs (Fig. 3A and Fig. 3B, respectively) for Lentivirus capture by a boronate resin prototype.

[0022] Fig. 4 shows an analysis of fractions from the duplicate runs (Fig. 4A and Fig. 4B, respectively) for the Lentivirus capture by boronate resin prototype. Fig. 5 shows the physical titer of virus particles recovered (Fig. 5A), total DNA (Fig. 5B), and total protein (Fig. 5C) removed, for Lentivirus capture using a boronate Fibro prototype.

[0023] Fig. 6 shows a comparison of binding capacity between the boronate Fibro prototype and the boronate resin prototype.

[0024] DETAILED DESCRIPTION

[0025] The present disclosure provides improved or at least alternative chromatography materials and their use for the separation of enveloped or membranous biological particles from impurities. More particularly, provided is a use of a chromatography material, comprising a support material functionalised with a ligand comprising a boronate moiety, for the separation of enveloped or membranous biological particles from impurities.

[0026] It is to be understood that the chromatography material referred to in the above-disclosed use of a chromatography material is as defined and exemplified in detail below in connection with the description of the chromatography material as such, including but not limited to the Formulas l-V defining the ligand.

[0027] A key advantage of lentivirus capture by use of a boronate ligand is that an efficient impurity removal is achieved. Mild elution conditions may be applied for boronate ligands, by use of a polyhydroxyl compound that functions as stabilizer for the virus. In contrast, the previously described anion exchange capture with salt elution required direct dilution into low salt condition with stabilizer (see WO2023 / 274989).

[0028] The functionalised support material is suitable for use as a chromatography material in affinity capture chromatography. In operation, the chromatography material comprising the functionalised support material is contacted with a mobile phase, such as a liquid sample or a feed, containing the target compounds, i.e., enveloped or membranous biological particles. The target compounds are retained in the chromatography material by the ligand in preference to other components also present in the mobile phase. Such other components in the mobile phase may comprise impurities such as cell host debris, protein, genomic DNA, serum protein, some elements of medium, helper DNA, or helper virus, etc.

[0029] The term "chromatography material" is used herein to denote a type of separation matrix. The term "separation matrix" is used herein to denote a material comprising a support material to which one or more ligands comprising functional groups have been coupled. The functional groups of the ligand(s) bind compounds herein also called analytes, which are to be separated from a liquid sample and / or which are to be separated from other compounds present in the liquid sample. A separation matrix may further comprise a compound which couples the ligand(s) to the support material. The terms "linker", "extender", and "surface extender" may be used to describe such a compound, as described in more detail further below. Herein, the term "support material" may be used interchangeably with the term "support".

[0030] In this context, "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group, or capturing agent, immobilized on its surface, whereas "analyte" includes any specific binding partner to the ligand. The term "ligand" may herein be used interchangeably with the terms "specific binding molecule", "specific binding partner", "capturing molecule" and "capturing agent". It is to be understood that "a ligand" is intended to mean a ligand species and that the singular form of the term may encompass a large number of individual ligands.

[0031] Herein, the analytes in a liquid sample which interact with a ligand may be referred to as "biological target compounds", "target compounds", or "targets" in short. Target compounds of interest according to the present disclosure are enveloped or membranous biological particles.

[0032] The term "enveloped or membranous biological particle" encompasses enveloped virus particles, extracellular vesicles (e.g., exosomes), and virus-like particles. Particularly interesting examples of enveloped virus particles are lentivirus particles.

[0033] Enveloped viruses generally have sizes ranging from 20 nm and up to 300 nm, depending on the type of virus. Lentiviruses may have a size in the range of 80-120 nm, often 100-120 nm. Enveloped viruses may be larger than non-enveloped viruses such as an adenovirus, which is packaged only in a capsid. Enveloped viruses are often larger than adeno-associated viruses, which are typically about 25 nm in size. Extracellular vesicles such as exosomes may have a size in the range of from 30 to 180 nm.

[0034] The term "virus particle" is herein used to denote a complete infectious virus particle. It includes a core, comprising the genome of the virus (i.e., the viral genome), either in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the core is surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the enclosed viral genome together constitute the so-called nucleocapsid. The nucleocapsid of enveloped viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapy, the genome of a virus particle is modified to include a genetic insert, comprising genetic material of interest.

[0035] The term "vector" is herein used to denote a virus particle, normally a recombinant virus particle, which is intended for use to achieve gene transfer to modify specific cell type or tissue. A virus particle can for example be engineered to provide a vector expressing therapeutic genes. Several virus types are currently being investigated for use to deliver genetic material (e.g., genes) to cells to provide either transient or permanent transgene expression. These include enveloped viruses such as retroviruses (y-retroviruses and lentiviruses), poxviruses, baculoviruses, and herpes simplex viruses, as well as non-enveloped viruses such as adenoviruses, and adeno-associated viruses (AAV). Herein, the term "vector" may be used interchangeably with the term "virus particle".

[0036] The term "virus-like particle" is intended to mean a virus-derived structure made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so it is not capable of infecting the host cell.

[0037] The term "impurities" is herein intended to mean any molecule or substance which is present in the liquid sample, and which is not the desired biological target compound. In the context of the present invention, "impurities" includes primarily host cell proteins (HCP), and host cell DNA. However, the term "impurities" generally also includes aggregates, such as aggregates of the biological target compound, and fragments of the biological target compound.

[0038] The term "surface" herein means all external surfaces and includes in the case of a porous support outer surfaces as well as pore surfaces.

[0039] The ligand of the herein disclosed chromatography material comprises a boronate moiety. As is well known in the art, boronate is a salt or ester of boronic acid. The ligand comprising a boronate moiety has a binding affinity for enveloped biological particles and for membranous biological particles. Thus, the ligand is capable of specific binding to enveloped biological particles and to membranous biological particles. In particular, the ligand is capable of binding to enveloped or membranous biological particles which have an envelope / membrane comprising glycosylated proteins which are accessible on the external parts of the envelope or membrane, respectively.

[0040] The ligand may alternatively be described as comprising an affinity group having a binding affinity for said enveloped or membranous biological particles. Said affinity group comprises a boronate moiety.

[0041] Herein, the term "moiety" is used to describe that the boronate is a part or portion of the ligand. In addition to boronate, the ligand may comprise an aromatic structure, an aliphatic structure, and / or a polymeric structure, as defined in detail further below.

[0042] More particularly, the present disclosure provides a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety, wherein the ligand is defined by Formula I: wherein

[0043] X is selected from O, CH2-CH(OH)-CH2-O, CH2-CH(OH)-CH2-O-CH2-CH2-, CH2, and -O-CH2-CH2-SO2-CH2; n is an integer from 0 to 40, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, preferably 0 < n < 10; each Ro is independently selected from H and alkyl; each Zi is independently selected from 2-pyrrolidone, / V-isopropylformylamide, l-methoxy-2,3- propanediol, / V-[tris(hydroxymethyl)methyl] formylamide, methylamide, / V-alkylformamide, N,N'- dialkylformamide, and alkyl formate; m is an integer from 1 to 40, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, preferably 1 < m < 10; each Ri is independently selected from H and OH; each R2 is independently selected from H and CsH5; each Z2 is: each Y is independently selected from CH2, S, NH, O, -C(O)NH-, -NHC(O)-, NH-(CH2)u-C(O)-NH, and (CH2)U-NH-C(O), wherein u is an integer from 1 to 6, i.e., 1, 2, 3, 4, 5, or 6; p is 0 or 1; q is an integer from 0 to 6, i.e., 0, 1, 2, 3, 4, 5, or 6; each R3 is independently selected from H and and each R4, Rs, Re, and R7is independently selected from NH2 or a salt thereof, H, Br, F, Cl, alkyl, OH, O-alkyl, and CF3.

[0044] The squiggly line ( / vww’ ) symbolises the support material to which the ligand is bound, and in cases where a linker, such as a surface extender, is included in the chromatography material for coupling of the ligand to the support material, the squiggly line symbolises both the support material and said linker.

[0045] Where R4, Rs, Rs, and R7is an amine (NH2), it may be a charged moiety, in which case it may be bound to a counter ion, such as Cl’ or SO42’, thereby forming a salt of NH2. The salt may be formed by adding an acid, such as hydrochloric acid or sulfuric acid, to the amine.

[0046] According to non-limiting embodiments of Formula I, wherein X is CH2-CH(OH)-CH2-O, n = 0, m

[0047] = 1, Ri is OH, R2 is H, p = 1, and Z2 is defined by Formula (VI), the ligand may be defined by Formula II: wherein

[0048] Y is selected from S, NH, or O; q is an integer from 0 to 6; each R3 is independently selected from H and and each R4, RS, RS, and R7is independently selected from NHj or a salt thereof, H, Br, F, and alkyl.

[0049] According to non-limiting embodiments of Formula II, wherein each of R5, Rs, and R7is H, the ligand may be defined by Formula III: wherein

[0050] Y is selected from S, NH, or O; q is an integer from 0 to 6; and

[0051] R4is selected from NH2 or a salt thereof, H, Br, F, and alkyl.

[0052] According to a currently preferred, non-limiting embodiment of Formula III, R4is F, Y is NH, and q = l.

[0053] According to another currently preferred, non-limiting embodiment of Formula III, R4is H, Y is NH, and q = 1.

[0054] According to non-limiting embodiments of Formula I, wherein X is CH2-CH(OH)-CH2-O, n = 0, m = 1, Ri is OH, R2 is H, p = 1, and Z2 is defined by Formula (VII), the ligand may be defined by Formula IV: wherein

[0055] Y is selected from NH, O, -C(O)NH-, and -NHC(O)-; q is an integer from 1 to 6; and each R3 is independently selected from H and According to a currently preferred, non-limiting embodiment of Formula IV, Y is NH, R3 is H, and q = 3.

[0056] According to another currently preferred, non-limiting embodiment of Formula IV, Y is O, R3 is H, and q = 4.

[0057] According to non-limiting embodiments of Formula I, wherein Ri is H, the ligand may be defined by Formula V:

[0058] (V) wherein

[0059] X is selected from CH2-CH(OH)-CH2-O-CH2-CH2-, CH2, and -O-CH2-CH2-SO2-CH2; n is an integer from 0 to 40, preferably 0 < n < 10; each Ro is independently selected from H and alkyl; each Zi is independently selected from 2-pyrrolidone, / V-isopropylformylamide, l-methoxy-2,3- propanediol, / V-[tris(hydroxymethyl)methyl] formylamide, methylamide, / V-alkylformamide, N,N'- dialkylformamide, and alkyl formate; m is an integer from 1 to 40, preferably 1 < m < 10; each R2is independently selected from H and CsH5; each Z2is: each Y is independently selected from CH2and -C(O)NH-; p is 0 or 1; q is an integer from 0 to 6; each R3 is independently selected from H and anc| each R4, RS, RS, and R7is independently selected from NH2or a salt thereof, H, Br, F, Cl, alkyl, OH, O- alkyl, and CF3.

[0060] According to a currently preferred, non-limiting embodiment of Formula V, wherein X is CH2- CH(OH)-CH2-O-CH2-CH2-, RO is H, Zi is 2-pyrrolidone, Z2is defined by Formula (VI), each of R2, R3, R4, Rs, Rs, and R7is H, n is an integer from 1 to 40, p = 0, and q = 0, the ligand comprises a copolymer of vinylphenylboronic acid and vinylpyrrolidone. According to another currently preferred, non-limiting embodiment of Formula V, wherein X is

[0061] CH2-CH(OH)-CH2-O-CH2-CH2-, Z2 is defined by Formula (VI), each of R2, R3, R4, Rs, Rs, and R7is H, n = 0, p

[0062] = 0, and q = 0, the ligand comprises a polymer of vinylphenylboronic acid.

[0063] The support material of the chromatography material may comprise a membranous structure, nanofibres, a monolith, porous particles, non-porous particles, magnetic particles, or expanded bed media.

[0064] According to a non-limiting example, the membranous structure may be convection-based and may comprise a non-woven web of polymer nanofibres, optionally wherein the polymer is a cellulosic polymer, such as cellulose acetate, or a synthetic polymer, or a combination thereof. According to further non-limiting examples, the convection-based membranous structure may comprise a single membrane, a pile of membranes or a filter.

[0065] A non-limiting example of a membranous structure is a Mustang® membrane (Cytiva).

[0066] Another non-limiting example of a membranous structure is Fibro™ (Cytiva), which is at the same time a non-limiting example of a support material comprising nanofibres. Fibro™ is a convectionbased membranous structure comprising nanofibres made of cellulose or a cellulose derivative.

[0067] A non-limiting example of a monolith is CIMmultus® (Sartorius, Germany).

[0068] Non-limiting examples of porous particles, which may alternatively be called beads, include porous particles based on polysaccharide, such as agarose. Polysaccharide beads, such as agarose beads, may be produced e.g. as described in US6602990B1, US7396467B2 or US8309709B2, which are incorporated herein. The beads may comprise cross-linked agarose. A chromatography material comprising particles or beads may be called a resin.

[0069] A non-limiting example of magnetic particles is Mag Sepharose (Cytiva).

[0070] A non-limiting example of expanded bed media is STREAMLINE resins (Cytiva).

[0071] According to a currently preferred, non-limiting embodiment of the chromatography material, the support material comprises a convection-based membranous structure comprising nanofibres and the ligand is defined by Formula II or Formula III.

[0072] According to another currently preferred, non-limiting embodiment of the chromatography material, the support material comprises porous particles or a convection-based membranous structure comprising nanofibres, and the ligand is defined by Formula IV.

[0073] Among the chromatography material prototypes prepared and used for purification of lentivirus particles, as described in the experimental section herein, the Fibro prototype contains ligands which are all accessible for binding to virus particles. In contrast, the resin prototype contains ligands on the surface of the beads, which ligands can bind to the virus particles, but also contains ligands inside pores of the beads, which ligands are not available for binding of the virus particles since the virus particles are too large to enter the pores.

[0074] As mentioned briefly above, the chromatography materials referred to herein may comprise a linker connecting the ligand to the support, i.e., the coupling of the ligand to the support is provided by introducing a linker between the support and ligand. The coupling may be carried out following any conventional covalent coupling methodology such as by use of epichlorohydrin; epibromohydrin; allyl- glycidylether; bis-epoxides such as butanedioldiglycidylether; halogen-substituted aliphatic substances such as di-chloro- propanol; and divinyl sulfone. Non-limiting examples of suitable linkers comprise vinyl sulfone derivatives, vinyl sulfone derivatives in combination with glycidol derivatives, polyethylene glycol (PEG) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, or polyalcohols having 3-6 carbon atoms. Alternatively, the ligand may be coupled to the support via a longer linker molecule, also known as a "surface extender", or simply "extender". Extenders are well known in this field, and commonly used to sterically increase the distance between ligand and support. Extenders are sometimes denoted tentacles or flexible arms. For a more detailed description of possible chemical structures, see for example US 6,428,707, which is hereby included herein by reference. In brief, the extender may be in the form of a polymer such as a homo- or a copolymer. Hydrophilic polymeric extenders may be of synthetic origin, i.e., with a synthetic skeleton, or of biological origin, i.e., a biopolymer with a naturally occurring skeleton. Typical synthetic polymers are polyvinyl alcohols, polyacryl- and polymethacrylamides, polyvinyl ethers etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.

[0075] A non-limiting example of the chromatography material as disclosed herein comprises a surface extender, such as dextran. According to a non-limiting embodiment, the chromatography material comprises a support material in the form of porous particles and dextran, which couples the ligand to the support material.

[0076] The present disclosure further provides a chromatography device comprising a holder comprising chromatography material as described in detail elsewhere herein. Non-limiting examples of suitable chromatography devices are: (i) columns comprising porous particles, non-porous particles, or monoliths, (ii) cartridges or capsules comprising membranous structures or nanofibres, and (iii) containers or vessels comprising magnetic particles.

[0077] The present disclosure additionally provides, as illustrated in Fig. 1, a method 100 for separating enveloped or membranous biological particles from one or more impurities, the method comprising: Adding 110 a liquid sample comprising enveloped or membranous biological particles and one or more impurities, to a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety; and

[0078] Eluting 120 the enveloped or membranous biological particles from the chromatography material in at least one eluate fraction using an elution buffer.

[0079] Optionally, the elution buffer may contain a poly-hydroxyl compound, such as sorbitol or sucrose, or another additive conventionally used for elution in boronate affinity chromatography, provided that the additive does not interfere with the ability of any further chromatography materials, applied after the affinity chromatography step, to function as intended, e.g., their ability to bind impurities and allow the enveloped or membranous biological particles to flow through the further chromatography material. Such an additive may for example function by competing with the enveloped or membranous biological particles for the binding to boronate.

[0080] The term "poly-hydroxyl compound" is intended to mean an organic compound containing multiple hydroxyl groups (i.e., -OH), such as two, three, four or more hydroxyl groups.

[0081] The term "eluate" is used in its conventional meaning in this field, i.e., the part(s) of a liquid sample which are eluted from a chromatography material after having loaded the liquid sample onto the chromatography material.

[0082] It is to be understood that the chromatography material referred to in the method 100 is as defined and exemplified in detail elsewhere herein in connection with the description of the chromatography material as such, including but not limited to the Formulas l-V defining the ligand.

[0083] The step of eluting 120 the enveloped or membranous biological particles may comprise a first step of eluting 124 using a first elution buffer, and a second step of eluting 128 using a second elution buffer, wherein the first elution buffer comprises a first poly-hydroxyl compound and the second elution buffer comprises a second poly-hydroxyl compound.

[0084] Optionally, the method may further comprise a step of washing 126 between the first step of eluting 124 and the second step of eluting 128.

[0085] The method 100 may comprise a step of cleaning 130 the chromatography material using a cleaning solution under acidic conditions, such as at a pH of from about 2 to about 5.

[0086] Optionally, the cleaning solution may comprise acetic acid.

[0087] The step of cleaning 130 may suitably follow eluting 120 the enveloped or membranous biological particles from the chromatography material.

[0088] The cleaning 130 may be followed by re-equilibrating 140 the chromatography material, before applying the chromatography material once again in a method 100 for separating enveloped or membranous biological particles from one or more impurities. The buffer may have a pH around 7-8 for both the step of adding 110 (i.e., for binding) and the step of eluting 120 the biological target compounds. Said buffer is suitably selected from buffers generally recommended for affinity chromatography, and which are suitable for the above-mentioned pH range. Non-limiting examples include tris(hydroxymethyl)amino-methane (i.e., Tris), 1,3- bis(tris(hydroxymethyl)methylamino) propane (i.e., bis-Tris propane), triethanolamine, N- methyldiethanolamine, diethanolamine, 1,3-diaminopropane, ethanolamine, phosphate buffer, bisTris, Imidazole, MOPS, and HEPES.

[0089] To achieve elution of the bound biological target compounds, the buffer in the step of eluting 120 comprises additional components compared to the buffer used in step (a). Non-limiting examples of such additional components are carbohydrates, poly-hydroxyls (alternatively called polyols, herein including diols), such as sucrose, sorbitol, glycerol etc.

[0090] A person skilled in the art is able to choose a suitable concentration for any one of the abovelisted buffers.

[0091] A currently preferred, non-limiting example of a suitable combination of buffers used for separation of lentivirus particles is the following buffer system:

[0092] Buffer A (i.e. binding buffer for step 110): 50 mM ammonium bicarbonate pH 7.9, 130 mM

[0093] NaCI, and

[0094] Buffer B (i.e., elution buffer for step 120): 50 mM ammonium bicarbonate pH 7.9, 130 mM

[0095] NaCI, and either 146 mM (5%) sucrose or 300 mM (5.2%) sorbitol.

[0096] Another currently preferred, non-limiting example of a suitable combination of buffers is the following buffer system:

[0097] Buffer A: 50 mM sodium phosphate (NaPi) pH 7.0 or 7.4, 130 mM NaCI, and

[0098] Buffer B: 50 mM sodium phosphate (NaPi) pH 7.0 or 7.4, 130 mM NaCI, 500 mM (9.1%) sorbitol.

[0099] In said method 100, the residence time of the liquid sample within the chromatography material may be adapted to the target compound of a particular separation method and the associated binding affinity of the ligand. It is believed that at residence times relevant to the present invention (such as residence time of about 1 minute or less than 1 minute), the binding kinetics between the target compound and the ligand may be considered for the purpose of process efficiency. In contrast, the binding kinetics is not a typical consideration for affinity interactions on diffusive chromatography media, such as conventional resins, which comprise porous particles. With resins, the rate limiting step is usually the time required for diffusion through the pores of the porous beads. However, when applying porous particles in the present context, the target compounds are too big to diffuse through the pores of the particles. Accordingly, use of porous particles will be associated with residence times almost as short as when using a membranous structure comprising nanofibres.

[0100] For example, in a method according to embodiments of the invention for recovering a lentivirus particle, the residence time may be from about 0.25 min to about 1 min when using porous beads, such as 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 min. When using a membranous structure comprising nanofibres, the residence time may be about 0.2 min, such as 0.1, 0.15, 0.2, 0.25, or 0.3 min.

[0101] The liquid sample added in step 110 of the herein disclosed method 100 may advantageously be a pre-purified liquid sample. The herein disclosed method 100 may comprise a step 105 which comprises pre-purifying enveloped or membranous biological particles by separating them from a cell culture harvest containing enveloped or membranous biological particles, thereby obtaining a prepurified liquid sample comprising enveloped or membranous biological particles, before adding said pre-purified liquid sample to the chromatography material according to step 110 of the method 100.

[0102] Such a pre-purifying step 105 refers in the context of liquid chromatography to the initial step(s) of a separation procedure. Most commonly, it includes clarification (e.g., by filtration, centrifugation, or precipitation), and normally also concentration and / or stabilisation of the sample. The subsequent step 110 then provides significant purification from soluble impurities by applying chromatography as described in detail elsewhere herein.

[0103] Herein, the term "cell culture" refers to a culture of cells or a group of cells being cultivated, wherein the cells may be any type of cells, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be unclarified, i.e., comprising cells, or may be cell-depleted, i.e., a culture comprising no or few cells but comprising biomolecules released from the cells before removing the cells. Further, an unclarified cell culture may comprise intact cells, disrupted cells, a cell homogenate, and / or a cell lysate.

[0104] The term "cell culture harvest" is used herein to denote a cell culture which has been harvested and removed from the vessel or equipment, in which the cells have been cultivated.

[0105] Non-limiting examples of separation devices suitable for use in a pre-purification step 105, as described herein, are filtration apparatuses, chromatography columns and membrane devices.

[0106] After step 120 of the herein disclosed method 100, an intermediate purification may follow, which further reduces remaining amounts of impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of a cell culture medium, such as antifoam agents and antibiotics, and product-related impurities, such as aggregates, and misfolded species.

[0107] It is to be understood that the present disclosure is not restricted to the below-described exemplifying embodiments thereof and that several conceivable modifications of the present disclosure are possible within the scope of the following claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0108] EXPERIMENTAL SECTION

[0109] Example 1: Separation of lentivirus particles on boronate prototype chromatography materials

[0110] 1A) Separation of lentivirus particles by use of boronate resin prototype

[0111] A prototype of chromatography material was prepared by functionalising a support material with a ligand comprising boronate, wherein the support material comprised porous beads, herein alternatively referred to as a resin.

[0112] Preparation of boronate resin prototypes

[0113] Activation of resin before coupling of ligand:

[0114] Allylation of cross-linked agarose gel beads with allyl glycidyl ether:

[0115] Cross-linked agarose gel (220 g = 220 mL) was transferred to a frit glass filter and washed with distilled water (xlO GV). Meanwhile, a water bath was heated to 50 °C. The drained gel was then transferred to a 500 mL three-necked round bottom flask (rbf) and the total volume adjusted to 244.44 mL with distilled water ( / .e. adding 24.44 mL of DW). The flask was then equipped with mechanical stirrer and stirring started at around 300 rpm. A solution of NaOH 50% (371.56 g, 245.25 mL) was added and the flask was lowered into the warm bath. Stirring continued for 30 min after which AGE (58.43 mL) was added. The reaction was then stirred for 17 h at 50 °C. The flask was taken out of the warm bath and transferred to a glass filter after reaching 25 ± 5 °C. The allylated gel was washed in the following order: DW 2xGV, EtOH lxGV, and DW 7xGV. The gel was almost completely drained and a part of it was saved for allyl titration.

[0116] Bromination:

[0117] Allylated agarose gel (20 mL), washed twice with DI water, was transferred to a 100 mL 3-neck round bottom flask to which distilled water (10 mL) and NaOAc.SHjO (0.89 g) were added. The reaction was stirred with the mechanical stirrer for 20 min after which elemental bromine (0.33 mL, in excess amount) was added and the stirring continued for 15 min. Sodium formate 3 M solution (6 mL) was added slowly to the slurry until the orange solution was decoloured. The brominated gel was washed with distilled water (x8 GV).

[0118] Prototypes prepared by direct coupling of the ligand to the resin (i.e., no additional linker used):

[0119] (i) Coupling of 4-methylaminophenylboronic acid hydrochloride

[0120] The synthetic pathway is shown in Fig. 2A.

[0121] 5 mL of freshly activated agarose gel was added to DI water (2 mL) and 0.013 mL of 50% NaOH solution. The 4-methylaminophenylboronic acid hydrochloride ligand (674 mg, 3.6 mmol, 3 eq.) was added to the mixture, which was then stirred at 50°C for 18 hours (pH was around 10.5). After cooling, the gel was then filtered off and washed with 2 GV of DI water. Ligand density was determined to be 84.8 pmol / mL.

[0122] (ii) Coupling of 3-fluoro-4-methylaminophenylboronic acid hydrochloride:

[0123] 5 mL of freshly activated agarose gel was added to DI water (2 mL) and 0.013 mL of 50% NaOH solution. The 3-fluoro-4-aminomethylphenylboronic acid hydrochloride ligand (740 mg, 3.6 mmol, 3.0 eq.) was added to the mixture, which was then stirred at 50°C for 18 hours (pH was around 10.5). After cooling, the gel was then filtered off and washed with 2 GV of DI water. Ligand density was determined to be 82.5 pmol / mL.

[0124] (Hi) Coupling of 2-fluoro-5-methylaminophenylboronic acid hydrochloride:

[0125] 5 mL of freshly activated agarose gel was added to DI water (2 mL) and 0.013 mL of 50% NaOH solution. The 2-fluoro-5-aminomethylphenylboronic acid hydrochloride ligand (740 mg, 3.6 mmol, 3.0 eq.) was added to the mixture, which was then stirred at 50°C for 18 hours (pH was around 10.5). After cooling, the gel was then filtered off and washed with 2 GV of DI water. Ligand density was determined to be 52.7 pmol / mL.

[0126] Sodium hydroxide treatment - performed after each of the above-described direct ligand-coupling reactions:

[0127] To the isolated gel was added distilled water (2 mL) and 50% NaOH solution (0.24 mL) in a 3- neck flask. The mixture was stirred at 40°C for 5 hours. The gel was then washed with DI water (10 GV). Coupling of ligand by use of a linker, more particularly the surface extender dextran:

[0128] Epoxy Activation:

[0129] Cross-linked agarose gel was washed with 10 x 1 GV portions of distilled water on a P3 sintered glass funnel. 50.09 mL drained gel (1 mL corresponds to lg) was transferred into a 100 mL roundbottom flask. 12.1 mL of distilled water and 8.0 mL of 50% NaOH were added. The rbf was fitted with glass stoppers and a Mickey Mouse stirrer and mounted to a stirring motor above a 28°C water bath. The rbf was immersed in the bath and the stirring was set to 250 rpm. After 10 min, 12.5 mL of ECH was added by a dosimat and the reaction then progressed for another 120 min. The gel was washed lOxlGV with distilled water. The gel was analyzed for epoxide content and dry weight determination. Dextran Coupling:

[0130] Dextran 40 was dissolved in a 100 mL three-necked flask with water during slow stirring for approximately 5 hours. The dextran solution was added to the drained epoxy activated gel from above and the slurry was heated to 40°C and stirred (300 rpm) for 60 minutes. Nitrogen gas was bubbled through the solution, 0.15 NL / min for 60 min. To the flask was then added 50% NaOH and NaBH4and the mixture was then left stirring at 40°C overnight. The reaction mixture was diluted, and the gel was washed lOxlGV with distilled water. The dry weight of the dextran coupled gel was analysed. Allylation 1:

[0131] ~45 mL (g) of al lylated dextran agarose gel (from the above-described dextran coupled product), in distilled water, was washed 3xGV with 50% NaOH. The gel was then sucked dry and transferred to a 250 mL round bottom flask. 95 mL of 50% NaOH and 100 mg of sodium borohydride were added and mechanical propeller stirring was applied (200 rpm). The flask was immersed into a water bath at 50°C. After 20 minutes 15 mL of allyl glycidyl ether (AGE) was added. The reaction progressed for 17.5 h at 230 rpm. The gel was washed lxGV with distilled water, 5xGV with ethanol and then 8xGV with distilled water.

[0132] Allylation 2:

[0133] ~43 mL (g) of al lylated dextran agarose gel (from the above-described allylation 1 product), in distilled water, was washed 3xGV with 50% NaOH. The gel was then sucked dry and transferred to a 250 mL round bottom flask. 95 mL of 50% NaOH and 100 mg of sodium borohydride were added and mechanical propeller stirring was applied (200 rpm). The flask was immersed into a water bath at 50 °C. After 20 minutes 15 mL of AGE was added. The reaction progressed for 17.5 h at 230 rpm. The gel was washed lxGV with distilled water, 5xGV with ethanol and then 8xGV with distilled water. Bromination:

[0134] The above-described Allylation 2 product was brominated, as described further above under the heading "Activation of resin before coupling of ligand" of section 1A. Ligand Coupling Reaction:

[0135] 5 mL of such freshly activated agarose gel was added to DI water (2 mL) and 13 pL of 50% NaOH solution. The 4-methylaminolphenylboronic acid hydrochloride ligand (1.43 g, 7.65 mmol, 3.0 eq.) was added to the mixture and the pH was corrected to 10.5-11 using 50% NaOH solution. The reaction mixture was then stirred at 50°C for 18 hours. After cooling, the gel was then filtered off and washed with 10 GV of DI water. Ligand density was determined to be 76.8 pmol / mL.

[0136] Sodium hydroxide treatment:

[0137] To the isolated gel from above (5 mL) was added distilled water (2 mL) and 50% NaOH solution (0.24 mL) in a 3-neck flask. The mixture was stirred at 40°C for 5 hours. The gel was then washed with DI water (10 GV).

[0138] Coupling using multiple allylations:

[0139] Allylation no. 2 of allylated cross-linked agarose:

[0140] 25 mL of allylated agarose gel (244.1 pmol / mL) was washed 3xlGV with 50% NaOH and placed drained in a 100 mL round bottom flask. 52.8 mL 50% NaOH was added to the gel together with 55.6 mg NaBH4. The flask was immersed in a 50 °C warm bath. After 30 minutes, 8.33 mL of AGE was added, and the reaction mixture was stirred for 17 hours at 50°C. The flask was taken out of the warm bath and transferred to a glass filter after reaching 25 ± 5 °C. The allylated gel was washed in the following order: distilled water 2xGV, EtOH lxGV, and distilled water 7xGV. No neutralization was performed before the washing. A sample was withdrawn for allyl titration.

[0141] Activation of allyl groups using bromine:

[0142] Allylated agarose gel (532 pmol / mL) (22 mL) was transferred to a 100 mL 3-neck round bottom flask to which distilled water (10.56 mL) and NaOAcx3HjO (0.979 g) were added. The reaction was stirred with the mechanical stirrer for 15 min after which bromine water (in excess amount=yellow to orange in color) was added and the stirring continued for 15 min. 3IVI (20 g in 100 mL) Sodium formate was added slowly to the slurry until the orange solution became decolored. The brominated gel was washed with distilled water (xlO GV).

[0143] Coupling of cysteamine hydrochloride:

[0144] 22 g of activated resin from above was transferred to a 100 mL round bottom flask. 15 mL of water was added together with the ligand cysteamine hydrochloride (14.86 g, 11.2 eq). pH was adjusted to >12 using 5 M / 17 M NaOH. The reaction was left over night (17 h) at room temperature. The reaction was washed with DV20 (3xlGV), 1 M NaCI (3xlGV), DV20 (3xlGV).

[0145] Cl' Titration - general procedure: The resin was washed with 0.5 M HCI 3xlGV and ImM HCI 3xlGV. ImL cubed resin was added into a titration cup together with 20 mL distilled water. 2 drops of 2 M HNO3 was added and 3 mL of 20 w / w% PVA-solution, the titration method nr 08 titration of Cl- and Br- with 0.1M AgNOa, was started and the ion capacity was determined.

[0146] Coupling of 4-(bromomethyl)phenylboronic acid:

[0147] A 4-(bromomethyl)phenylboronic acid reagent (1.45 g, 6.73 mmol, 3.0 eq. to cysteamine groups on the resin) was added to 2.38 (1.88 + 0.5) mL distilled water and 1.5 mL toluene and stirred at 50°C until almost dissolved (short of time). 7 mL of the cysteamine gel (320 pmol / mL) was then added to the mixture and the pH was corrected to 9 using 5M NaOH solution. The reaction mixture was then stirred at 50°C for 1 day, resulting in a 4-methyl-phenylboronic acid ligand being coupled to the resin. After cooling, the gel was then filtered off and washed with 5xlGV methanol and lOxGV aq. The ligand density was determined to be 70.2 pmol / mL at 50 °C.

[0148] Preparation of resin chromatography device:

[0149] The 4-methylaminophenyl boronate resin prototype (ligand density 84.8 pmol / mL) was packed into a Tricorn™ 5 / 50 column (Cytiva, Sweden).

[0150] Separation process and analysis:

[0151] 12 mL clarified lentivirus feed (7 x 109VP / mL) was applied to Tricorn™ 5 / 50 column packed with 1 mL of 4-methylaminophenyl boronate resin prototype (ligand density 84.8 pmol / mL) connected to an AKTA Pure 25 chromatography system. After sample load the column was washed, eluted with sucrose and sorbitol containing buffer, cleaned in place (abbreviated CIP) and re-equilibrated according to the protocol in Table 1. The fractions were collected and analysed for physical titer of virus particles (VP) recovered and impurities (total DNA and total protein) removed. Physical titer of lentivirus particles was determined by p24 ELISA (VP / mL), total DNA was analysed by Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific), and total protein was analysed using a Micro BCA assay. The residence time of the feed within the boronate resin prototype chromatography material was 0.25-1 min.

[0152] Table 1. Protocol for Lentivirus capture using boronate resin.

[0153] IB) Separation of lentivirus particles by use of boronate Fibro™ prototype

[0154] A prototype of chromatography material was prepared by functionalising a support material with a ligand comprising boronate, wherein the support material comprised a non-woven web of cellulose acetate nanofibres, herein alternatively referred to as Fibro™ membrane.

[0155] Preparation of boronate Fibro prototype

[0156] Coupling of 4-aminomethylphenylboronic acid onto Fibro™ membrane:

[0157] The synthetic pathway is shown in Fig. 2B.

[0158] A Fibro™ cellulose acetate (CA) sheet was washed with MQ-water (4 x 100 mL, 10 min) in a food box on a shaking table. 12 discs with 32 mm diameter were punched out of the 10 x 15 cm membrane sheets. The discs were placed between two chemically inert nets and rolled on a chemically inert plastic roll. The two rolls were placed in a beaker (600 mL) and a magnetic stirrer added in the middle. A glass stopper was used to stop the rolls from spinning during stirring. KOH (1 M, 172 mL) was added, and the reaction stirred for 10 min. To the reaction, allyl glycidyl ether (AGE) (28 mL) was added and the reaction was stirred overnight at room temperature. The reaction solution was decanted. The rolls were washed with acetone / MQ-water (1:1, 2 x 300 mL, 10 min) followed by wash with MQ-water (3 x 300 mL, 10 min). A solution of NajCOs (0.28 M, 7.5 g) in 25 % v / v MeCN in MQ- water was added to the rolls (250 mL). Divinyl sulfone* (40 mL) was added and the reactions stirred for 18 h after which the reaction solution was decanted. The rolls were then washed with acetone / MQ- water (1:1, 2 x 300 mL, 10 min) followed by a wash with MQ-water (3 x 300 mL, 10 min). NBS (7 g) in MeCN in MQ-water (250 mL, 75% v / v) was added to the beaker and left stirring for 4 hours. The reaction solution was decanted. The rolls were then washed with MQ-water (6 x 300 mL, 10 min). 4- aminomethylphenylboronic acid hydrochloride (29.99 g, 0.8 M) was dissolved in 200 mL MQ-water at 50°C. The pH was adjusted to 10.5 (±0.5). The ligand was dissolved when it was added to the beaker with the two rolls. The reaction was stirred for 17 h at 60°C. The morning after, the reaction solution had separated in two different phases. The reaction solution was decanted, and the rolls were washed with MQ-water (6 x 300 mL, 10 min). The rolls were removed from the beakers and carefully rolled up. The discs were removed and put in 20% EtOH in 6-hole plates. The phase containing the ligand was kept for solubility investigations. It was soluble when the pH was lowered. It was insoluble in MeCN, and a small portion was dissolved in acetone.

[0159] Analysis of pressure flow and ligand density gave an estimated ligand density of ~30 pmol / mL.

[0160] Preparation of Fibro chromatography device:

[0161] The boronate Fibro prototype was assembled into a chromatography device of the type HiTrap Fibro™ unit (Cytiva) 0.4 mL, herein alternatively called a Fibro unit.

[0162] Separation process and analysis:

[0163] 10 mL clarified lentivirus feed (2.5 x 1010VP / mL) was applied to the Fibro 0.4 mL nonfluorinated boronate prototype unit (ligand density 30.0 pmol / mL) connected to an AKTA Pure 25 chromatography system. After sample load the column was washed, eluted with sorbitol containing buffer, cleaned in place (CIP) and re-equilibrated according to the protocol in Table 2. The flowthrough fractions (FT), wash (W) and sorbitol eluate fractions (E) were collected and analysed for physical titer of virus particles (VP) recovered and impurities (total DNA and total protein) removed. Physical titer of lentivirus particles was determined by p24 ELISA (VP / mL), total DNA was analysed by Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific), and total protein was analysed using a Micro BCA assay. The residence time of the feed within the boronate Fibro prototype chromatography material was 0.2 min.

[0164] Table 2. Protocol for Lentivirus capture using boronate Fibro prototype.

[0165] Results and conclusions regarding boronate resin and boronate Fibro prototypes

[0166] Fig. 3 shows chromatograms from duplicate runs (Fig. 3A and Fig. 3B, respectively) for Lentivirus capture using a column packed with 1 mL of boronate resin prototype. The UV280 is shown by the solid line and elution with elution buffers (alternatively called B buffers) containing sucrose or sorbitol are indicated by a dotted line. The cleaning-in-place (CIP) phase with acetic acid is also indicated.

[0167] Fig. 4 shows an analysis of fractions from the duplicate runs (Fig. 4A and Fig. 4B, respectively) for the Lentivirus capture by boronate resin prototype, according to the chromatograms of Fig. 3A-B. The flowthrough fractions (FT 1-5) and sucrose (Esuc) and sorbitol (Esorb) eluate fractions were analysed for physical titer (p24 ELISA), total protein, and total DNA.

[0168] Fig. 5 shows the physical titer of virus particles recovered (Fig. 5A), as well as total DNA (Fig. 5B), and total protein (Fig. 5C) removed, for Lentivirus capture using a boronate Fibro 0.4 mL unit at pH 7 or pH 7.4 and using protocol in Table 2.

[0169] Fig. 6 shows a comparison of binding capacity between the boronate Fibro prototype and the boronate resin prototype. The binding capacity was determined as number of viral particles (p24 ELISA) that could be bound and eluted. For resin, the 1 mL column was overloaded, and lentivirus was detected in the flowthrough. For Fibro, no virus was detected in the flowthrough, so the binding capacity was potentially higher. Fibro capacity was at least 2.5 x 1011VP and for resin the capacity was ~2.5 x IO10VP (10-fold lower binding capacity).

[0170] The boronate elution yields using sucrose were approximately 40-60 % VP for both boronate resin and boronate Fibro. Elution in sorbitol or other carbohydrates are an alternative. If the boronate column was cleaned in place (CIP) with NaOH, the lentivirus particles bound stronger to boronate, and sorbitol was required for elution (data not shown). CIP in acetic acid instead allowed milder elution in sucrose with only little lentivirus elution in a second sorbitol elution step (Figs. 4 and 5).

[0171] The impurity removal was similar with fibro boronate as with the resin boronate prototypes (Figs. 4 and 5), but the binding capacity was 10-fold higher despite lower ligand density (Fig. 6).

[0172] Example 2: Preparation of polymeric boronate prototypes

[0173] 2A) Preparation of polymeric boronate resin prototypes:

[0174] (i) Including a copolymer of vinylpyrrolidone and vinylphenylboronic acid:

[0175] Washed allylated agarose gel (20 mL, allyl content 59 pmol / mL) was added to a 100 mL round bottom flask. In a beaker water / DMSO (2:1.5, 50 mL), vinylpyrrolidone (0.74 mL) and vinyl phenyl boronic acid (1 g) was mixed and the pH adjusted to 8.5. The reaction solution was bubbled with Nj for 20 min. 2,2'- Azobis-(2-amidopropane)hydrochloride (ADBA) (0.04 g) was added to the round bottomed flask followed by the reaction mixture. The slurry was stirred at 250 rpm and heated to 50°C. An Nj flow was set over the reaction. The reaction was left at 50°C over night. The slurry was transferred to a glass filter (P3) and washed with water (3 x GV), ethanol (3 x GV) and water (7 x GV).

[0176] (ii) Including a polymer containing 100% vinylphenylboronic acid:

[0177] Washed allylated agarose gel (15 mL, allyl content 59 pmol / mL) was added to a 100 mL round bottom flask. In a beaker water / DMSO (1:2, 33 mL) and vinylphenyl boronic acid (2.2 g) was mixed and the pH adjusted to 8.5. The reaction solution was bubbled with Nj for 10 min. The reaction mixture was added to the round bottomed flask. The slurry was stirred at 250 rpm, heated to 50 °C and bubbled with Nj. ADBA (0.05 g) was added. An Nj flow was set over the reaction. The reaction was left at 50 °C for 1 h before more ADBA (0.1 g) was added. The reaction was left at 50 °C for an additional 3 h. The slurry was transferred to a glass filter (P3) and washed with water (3 x GV), ethanol (3 x GV) and water (7 x GV).

[0178] 2B) Preparation of polymeric boronate Fibro™ prototype:

[0179] Including a copolymer of vinylpyrrolidone and vinylphenylboronic acid:

[0180] The Fibro prototype was grafted from divinyl sulfone crosslinked membranes without glycidol surface extension. The starting material was placed in a food box and washed with water (4 x 100 mL, 20 min, 70 rpm shaking). 12 discs with 32 mm diameter were punched out and placed between two chemically inert nets and rolled on a chemically inert plastic roll. Two rolls with 5 discs were made. The rolls were placed in spinner flask reactors (100 mL) and magnetic stirrers were added in the middle of the rolls. A glass stopper was used to stop the rolls from spinning during stirring.

[0181] Partial deactivation:

[0182] To one of the reactors, KOH (0.5 M, 100 mL) was added. The reaction was left stirring for 30 min. The reaction solution was decanted, and the sheets washed with MQ-water (6 x 100 mL). Coupling:

[0183] In a beaker, the reaction solution was mixed, degassed with N2and the pH adjusted to 8, according to Table 3 below.

[0184] Table 3. Reaction mixture for preparation of polymeric boronate Fibro prototype

[0185] Scale 100 g pH 8

[0186] Temperature 48 °C

[0187] 4-Vinylphenylboronic acid 5.2 g

[0188] Vinylpyrrolidone 3.9 g

[0189] 1:2 DMSO / H2O 91 mL

[0190] Initiator ADBA 0.19 g

[0191] The reaction mixture was added to the reactor, the lids fastened, and the reactor was put in 50°C water bath and N2bubbled through. The reaction was left overnight. The reaction mixture polymerized to a very high degree, making it difficult to extract the discs without damage. However, three discs were recovered, and were washed with MQ-water (4 x 100 mL), ethanol (3 x 100 mL) and MQ-water (6 x 100 mL).

[0192] Conclusions:

[0193] Resin and Fibro™ prototypes comprising polymeric boronate ligands were successfully synthesised.

[0194] Example 3: Separation of lentivirus particles under variable conditions

[0195] Experimental designs for separation of lentivirus particles from impurities are performed with equipment and samples as in Example 1 above, and further by use of boronate chromatography material as in Example 1 and Example 2, with the following variations:

[0196] • Ligand density: >85 pmol / mL for resin and >30 pmol / mL for Fibro

[0197] • Ligand chemistry: Polymeric boronate ligands, according to Example 2

[0198] • Lentivirus feed: Material filtered by tangential flow filtration instead of clarified feed, particularly for separation on resin material.

[0199] Buffers and elution conditions:

[0200] • Different buffer A, e.g., Tris, HEPES, MOPS • <130 mM NaCI

[0201] • Additives in buffer: e.g., MgCL

[0202] • Elution buffer: Include glycerol or other poly-hydroxyl compound instead of sucrose or sorbitol • CIP conditions:

[0203] • <1M NaOH, e.g., 0.1-0.5 M NaOH.

[0204] • NaOH followed by acetic acid (e.g., 0.1-0.3 M) cleaning-in-place. REFERENCES

[0205] WO2023 / 274989 Al

[0206] US6602990 Bl

[0207] US7396467 B2 US8309709 B2

Claims

CLAIMS1. Use of a chromatography material, comprising a support material functionalised with a ligand comprising a boronate moiety, for the separation of enveloped or membranous biological particles from impurities.

2. The use according to claim 1, wherein the ligand is defined by Formula I:whereinX is selected from O, CH2-CH(OH)-CH2-O, CH2-CH(OH)-CH2-O-CH2-CH2-, CH2, and -O-CH2-CH2-SO2-CH2; n is an integer from 0 to 40, preferably 0 < n < 10; each Ro is independently selected from H and alkyl; each Zi is independently selected from 2-pyrrolidone, / V-isopropylformylamide, l-methoxy-2,3- propanediol, / V-[tris(hydroxymethyl)methyl] formylamide, methylamide, / V-alkylformamide, N,N'- dialkylformamide, and alkyl formate; m is an integer from 1 to 40, preferably 1 < m < 10; each Ri is independently selected from H and OH; each R2is independently selected from H and CsH5; each Z2is:each Y is independently selected from CH2, S, NH, O, -C(O)NH-, -NHC(O)-, NH-(CH2)u-C(O)-NH, and (CH2)u-NH-C(O), wherein u is an integer from 1 to 6; p is 0 or 1; q is an integer from 0 to 6; each R3 is independently selected from H andeach R4, RS, RS, and R7is independently selected from NH2or a salt thereof, H, Br, F, Cl, alkyl, OH, O- alkyl, and CF3.

3. The use according to claim 2, wherein the ligand is defined by Formula II:whereinY is selected from S, NH, or O; q is an integer from 0 to 6; each R3 is independently selected from H andeach R4, RS, RS, and R7is independently selected from NH? or a salt thereof, H, Br, F, and alkyl.

4. The use according to claim 3, wherein the ligand is defined by Formula III:whereinY is selected from S, NH, or O; q is an integer from 0 to 6; andR4is selected from NH? or a salt thereof, H, Br, F, and alkyl; preferably wherein(i) R4is F; Y is NH; and q = 1; or(ii) R4is H; Y is NH; and q = 1.

5. The use according to claim 2, wherein the ligand is defined by Formula IV:whereinY is selected from NH, O, -C(O)NH-, and -NHC(O)-; q is an integer from 1 to 6; and each R3 is independently selected from H andpreferably wherein(i) Y is NH; R3is H; and q = 3; or(ii) Y is O; R3is H; and q = 4.

6. The use according to claim 2, wherein the ligand is defined by Formula V:(V) whereinX is selected from CH2-CH(OH)-CH2-O-CH2-CH2-, CH2, and -O-CH2-CH2-SO2-CH2; n is an integer from 0 to 40, preferably 0 < n < 10; each Ro is independently selected from H and alkyl; each Zi is independently selected from 2-pyrrolidone, / V-isopropylformylamide, l-methoxy-2,3- propanediol, / V-[tris(hydroxymethyl)methyl] formylamide, methylamide, / V-alkylformamide, N,N'- dialkylformamide, and alkyl formate; m is an integer from 1 to 40, preferably 1 < m < 10; each R2is independently selected from H and CsH5; each Z2is:each Y is independently selected from CH2and -C(O)NH-; p is 0 or 1; q is an integer from 0 to 6; each R3is independently selected from H andand each R4, RS, RS, and R7is independently selected from NH2or a salt thereof, H, Br, F, Cl, alkyl, OH, O- alkyl, and CF3; preferably wherein(i) X is CH2-CH(OH)-CH2-O-CH2-CH2-; RO is H; Zi is 2-pyrrolidone; each of R2, R3, R4, Rs, Rs, and R7is H; n is an integer from 1 to 40; p = 0; and q = 0; or(ii) X is CH2-CH(OH)-CH2-O-CH2-CH2-; each of R2, R3, R4, R5, Rs, and R7is H; n = 0; p = 0; and q = 0.

7. The use according to any one of the preceding claims, wherein the support material comprises a membranous structure, nanofibres, a monolith, porous particles, non-porous particles, magnetic particles, or expanded bed media.

8. The use according to any one of the preceding claims, wherein the enveloped or membranous particles are selected from enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles; optionally wherein the extracellular vesicles are exosomes.

9. A method (100) for separating enveloped or membranous biological particles from impurities, comprising:Adding (110) a liquid sample comprising enveloped or membranous biological particles and one or more impurities, to a chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety;Eluting (120) the enveloped or membranous biological particles from the chromatography material in at least one eluate fraction using an elution buffer, the elution buffer optionally comprising a polyhydroxyl compound, such as sucrose or sorbitol.

10. The method according to claim 9, wherein the step of eluting (120) the enveloped or membranous biological particles comprises a first step of eluting (124) using a first elution buffer, and a second step of eluting (128) using a second elution buffer, wherein the first elution buffer comprises a first poly-hydroxyl compound and the second elution buffer comprises a second poly-hydroxyl compound, optionally wherein the method further comprises a step of washing (126) between the first step of eluting (124) and the second step of eluting (128).

11. The method according to claim 9 or 10, further comprising a step of cleaning (130) the chromatography material using a cleaning solution under acidic conditions, such as at a pH of from about 2 to about 5, optionally wherein the cleaning solution comprises acetic acid.

12. The method according to any one of claims 9-11, wherein the ligand is defined by Formula I as defined in claim 2.

13. The method according to claim 12, wherein the ligand is defined by Formula II as defined in claim 3, optionally wherein the ligand is defined by Formula III as defined in claim 4.

14. The method according to claim 12, wherein the ligand is defined by Formula IV as defined in claim 5.

15. The method according to claim 12, wherein the ligand is defined by Formula V as defined in claim 6.

16. The method according to any one of claims 9-15, wherein the enveloped or membranous particles are selected from enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles; optionally wherein the extracellular vesicles are exosomes.

17. A chromatography material comprising a support material functionalised with a ligand comprising a boronate moiety, wherein the ligand is defined by Formula I as defined in claim 2.

18. The chromatography material according to claim 17, wherein the ligand is defined by Formula II as defined in claim 3, optionally wherein the ligand is defined by Formula III as defined in claim 4.

19. The chromatography material according to claim 17, wherein the ligand is defined by FormulaIV as defined in claim 5.

20. The chromatography material according to claim 17, wherein the ligand is defined by FormulaV as defined in claim 6.

21. The chromatography material according to any one of claims 17-20, wherein the support material comprises a membranous structure, nanofibres, a monolith, porous particles, non-porous particles, magnetic particles, or expanded bed media.

22. The chromatography material according to claim 21, wherein the support material comprises a convection-based membranous structure comprising nanofibres and the ligand is defined by Formula II or Formula III.

23. The chromatography material according to claim 21, wherein the support material comprises porous particles or a convection-based membranous structure comprising nanofibres, and the ligand is defined by Formula IV.

24. A chromatography device comprising a holder comprising the chromatography material according to any one of claims 17-23.

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