Purification of envelope or membrane-bound biological particles

Abstract

This disclosure relates to chromatographic materials comprising a retaining material functionalized with a ligand containing a boronate moiety for the separation of envelopes or membrane-like biological particles from impurities, and to the use thereof. Also disclosed are chromatographic devices and methods for separating envelopes or membrane-like biological particles from impurities, comprising the steps of: adding a liquid sample containing envelopes or membrane-like biological particles and one or more impurities to a chromatographic material comprising a retaining material functionalized with a ligand containing a boronate moiety; and eluting the envelopes or membrane-like biological particles from the chromatographic material into at least one elution fraction using an elution buffer, wherein the elution buffer optionally contains a polyhydroxyl compound, such as sucrose or sorbitol.

Description

Technical Field

[0001] 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 present disclosure is directed to the use of chromatographic materials for the separation of enveloped or membranous biological particles from impurities, as well as methods for separating enveloped or membranous biological particles from impurities and chromatographic materials comprising retention materials functionalized with ligands comprising boronate moieties.

Background Art

[0002] Viral vectors commonly used in pharmaceuticals include enveloped virus particles, such as lentivirus (LV). Extracellular vesicles (EVs) produced and released by cells are another example of vectors with potential in cell and gene therapy.

[0003] Lentiviruses are classified as retroviruses and have a single-stranded RNA genome with reverse transcriptase. Lentiviruses have a viral envelope with glycosylated proteins that act as ligands with affinity for receptors in the outer cell membrane surface of host cells. When the virus enters a cell, it transcribes the viral genetic material. The benefit of using LV as a viral vector is that, unlike other retroviruses that only enter mitotic cells, it can enter the nuclear envelope in both dividing and non-dividing cells. Many cell types in an adult individual do not divide, and LV would be the only option to transfer genetic material to such cells.

[0004] To produce lentiviruses, several plasmids are transfected into so-called packaging cell lines. One or more plasmids, commonly referred to as packaging plasmids, encode virion proteins, such as the capsid and reverse transcriptase. Another plasmid contains the genetic material delivered by the vector. This is transcribed to produce a single-stranded RNA viral genome, characterized by the presence of a ψ (psi) sequence. This sequence is used to package the genome into virions. For lentiviruses to be used in gene therapy, after transfection, the virions must be purified from host cell proteins and DNA, as well as cellular impurities such as excess plasmids. Typically, the host cells harvested to produce lentiviruses are treated with nucleases, and the lentiviruses are purified using several filtration techniques, such as conventional flow microfiltration, ultrafiltration, and dialysis, to reduce the level of impurities to an acceptable level.

[0005] As noted in WO2023 / 274989 A1 (Cytiva Bioprocess R&D AB), lentiviruses are unstable, sensitive to shear forces and buffer components such as salts, and degrade rapidly at room temperature; therefore, current downstream purification processes for lentiviruses often result in low recovery rates of infectious viruses. WO2023 / 274989 addresses chromatographic media containing weak anion-exchange ligands that may be used for the purification of enveloped virus particles and exosomes.

[0006] However, in this field, there is a continuing need for novel chromatographic materials and purification methods that provide improved or at least alternative means for separating envelopes or membrane-like biological particles from impurities. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2023 / 274989 A1 [Patent Document 2] US6602990B1 [Patent Document 3] US7396467B2 [Patent Document 4] US8309709B2 [Patent Document 5] US 6,428,707 [Overview of the Initiative]

[0008] One object of this disclosure is to provide a chromatographic material for use in affinity chromatography for purifying envelopes or membrane-bound biological particles, which can be used to recover envelopes or membrane-bound biological particles on an industrial scale and under elution conditions mild enough to obtain commercially viable yields.

[0009] This disclosure provides the use of chromatographic materials comprising a retention material functionalized with a ligand containing a boronate moiety for the separation of envelope or membrane-like biological particles from impurities.

[0010] A method for separating envelopes or membranous biological particles from impurities is further provided, comprising the steps of: adding a liquid sample containing envelopes or membranous biological particles and one or more impurities to a chromatographic material containing a retaining material functionalized with a ligand containing a boronate portion; and eluting the envelopes or membranous biological particles from the chromatographic material into at least one elution fraction using an elution buffer.

[0011] In addition, the present disclosure relates to a chromatography material comprising a retaining material functionalized with a ligand containing a boronate moiety, wherein the ligand is of formula I

[0012] [ka]

[0013] (wherein 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 R0 is independently selected from H and alkyl, each Z1 is independently selected from 2-pyrrolidone, N-isopropylformamide, 1-methoxy-2,3-propanediol, N-[tris(hydroxymethyl)methyl]formamide, methylamide, N-alkylformamide, N,N'-dialkylformamide, and alkyl formate, m is an integer from 1 to 40, preferably 1 ≤ m ≤ 10, each R1 is independently selected from H and OH, each R2 is independently selected from H and C6H5, each Z2 is

[0014] [Chemical formula]

[0015] and 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)-, where u is an integer from 1 to 6, p is 0 or 1, q is an integer from 0 to 6, each R3 is H and

[0016] [Chemical formula]

[0017] is independently selected from each R4, R5, R6, and R7 is independently selected from NH2 or its salt, H, Br, F, Cl, alkyl, OH, O-alkyl, and CF3) It provides chromatography materials as defined by [the specified method].

[0018] This disclosure is defined by the attached independent claims. Non-limiting embodiments will become apparent from the dependent claims, the attached drawings and the following description. It should be noted that this disclosure relates to all possible combinations of the features enumerated in the claims.

[0019] These and other aspects of the present disclosure will be described in more detail with reference to the accompanying drawings illustrating embodiments of the present disclosure. [Brief explanation of the drawing]

[0020] [Figure 1] This figure illustrates a method for separating enveloped or membrane-like biological particles from impurities according to the present disclosure. [Figure 2] This figure shows the synthetic pathways for coupling boronate ligands to resin (Figure 2A) and fibrous material (Figure 2B). [Figure 3] This figure shows chromatograms from two consecutive runs (Figure 3A and Figure 3B, respectively) of lentivirus capture using a boronate resin prototype. [Figure 4] This graph shows the analysis of fractions from two consecutive runs (Figures 4A and 4B, respectively) of lentivirus capture using a boronate resin prototype. [Figure 5] This graph shows the physical titer of recovered viral particles (Figure 5A), as well as the total DNA (Figure 5B) and total protein (Figure 5C) removed, for lentiviral capture using a boronate fibro prototype. [Figure 6] This graph shows a comparison of the bonding capabilities between a boronate fibrous prototype and a boronate resin prototype. [Modes for carrying out the invention]

[0021] This disclosure provides improved or at least alternative chromatographic materials and uses thereof for the separation of envelopes or membrane-bound biological particles from impurities. More particularly, it provides the use of chromatographic materials comprising retention materials functionalized with a ligand containing a boronate moiety for the separation of envelopes or membrane-bound biological particles from impurities.

[0022] It is understood that the chromatographic materials referred to in the use of the chromatographic materials disclosed above are defined and illustrated in detail below in relation to the description of the chromatographic materials themselves, including but not limited to formulas I-V that define the ligands.

[0023] The main advantage of lentiviral capture using boronate ligands is the achievement of efficient impurity removal. The use of polyhydroxyl compounds, acting as stabilizers for the virus, allows for the application of milder elution conditions for boronate ligands. In contrast, anion exchange capture with salt elution, as described previously, required direct dilution to low-salt conditions with stabilizers (see WO2023 / 274989).

[0024] Functionalized retention materials are suitable for use as chromatographic materials in affinity capture chromatography. In the operation, the chromatographic material containing the functionalized retention material comes into contact with a mobile phase, such as a liquid sample or feed, containing the target compound, i.e., an envelope or membrane-like biological particle. The target compound is retained in the chromatographic material by the ligand, preferentially over other components also present in the mobile phase. Such other components in the mobile phase may include impurities, such as cell host fragments, proteins, genomic DNA, serum proteins, some elements of the medium, helper DNA, or helper viruses.

[0025] The term “chromatographic material” is used herein to describe a type of separation matrix. The term “separation matrix” is used herein to describe a material comprising a retaining material to which one or more ligands containing functional groups are coupled. The functional groups of the ligands are bound to the compounds herein, also called analytes, which are separated from a liquid sample and / or from other compounds present in the liquid sample. The separation matrix may further contain compounds that couple the ligands to the retaining material. The terms “linker,” “extender,” and “surface extender” may be used to describe compounds such as those described in more detail further below. In this specification, the term “retaining material” may be used interchangeably with the term “retainer.”

[0026] In this context, “ligand” is a molecule having known or unknown affinity to a given analyte, including any functional groups or scavengers immobilized on its surface, while “analyte” includes any specific binding partner to the ligand. The term “ligand” may be used interchangeably herein with the terms “specific binding molecule,” “specific binding partner,” “scavenger,” and “scavenger.” “Ligand” is intended to mean a ligand species, and it should be understood that the singular term may encompass a number of individual ligands.

[0027] In this specification, an analyte in a liquid sample that interacts with a ligand may be referred to as a “biological target compound,” “target compound,” or simply “target.” The target compound of interest according to this disclosure is an envelope or a membrane-like biological particle.

[0028] The term "envelope or membranous biological particle" includes enveloped viral particles, extracellular vesicles (e.g., exosomes), and virus-like particles. A particularly interesting example of an enveloped viral particle is the lentiviral particle.

[0029] Enveloped viruses generally have a size ranging from 20 nm to a maximum of 300 nm, depending on the type of virus. Lentiviruses can be 80–120 nm, often in the range of 100–120 nm. Enveloped viruses can be larger than non-enveloped viruses, such as adenoviruses, which are 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, can have a size ranging from 30–180 nm.

[0030] The term “viral particle” is used herein to describe a complete infectious viral particle. This particle contains a core containing the viral genome (i.e., viral genome) in either 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 encapsulated viral genome together constitute a so-called nucleocapsid. In enveloped viruses, the nucleocapsid is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of producing viral vectors for various applications such as therapeutics, the genome of a viral particle is modified to include a gene insertion containing the desired genetic material.

[0031] The term “vector” is used herein to describe viral particles, typically recombinant viral particles intended for use to achieve gene transfer for the modification of a particular cell type or tissue. Viral particles can be manipulated to provide, for example, a vector that expresses a therapeutic gene. Several viral species are currently being investigated for use in delivering genetic material (e.g., genes) to cells to provide either transient or persistent transgene expression. These include enveloped viruses, such as retroviruses (gamma-retroviruses and lentiviruses), poxviruses, baculoviruses, and herpes simplex viruses, as well as non-enveloped viruses, such as adenoviruses and adeno-associated viruses (AAVs). In this specification, the term “vector” may be used interchangeably with the term “viral particle.”

[0032] The term "virus-like particle" is intended to refer to a virus-derived structure composed of one or more different molecules that possesses the ability to self-assemble, mimics the morphology and size of a virus particle, but lacks genetic material and is incapable of infecting host cells.

[0033] In this specification, the term "impurity" is intended to mean any molecule or substance present in a liquid sample that is not the desired biological target compound. In the context of this invention, "impurity" primarily refers to host cell proteins (HCPs) and host cell DNA. However, the term "impurity" can also generally refer to aggregates, such as aggregates of the biological target compound, and fragments of the biological target compound.

[0034] In this specification, the term "surface" means all external surfaces, and in the case of a porous retainer, it includes the outer surface and the pore surface.

[0035] The ligands of the chromatography materials disclosed herein include a boronate moiety. As is well known in the art, boronate is a salt or ester of a boronic acid. Ligands containing a boronate moiety have binding affinity to enveloped biological particles and to membranous biological particles. Therefore, ligands can specifically bind to enveloped biological particles and membranous biological particles. In particular, ligands can bind to enveloped or membranous biological particles having an envelope / membrane containing glycosylated proteins that can reach the outer portion of the envelope or membrane, respectively.

[0036] The ligand may instead be described as comprising an affinity group having binding affinity to the envelope or membrane-like biological particle. The affinity group comprises a boronate moiety.

[0037] In this specification, the term “part” is used to describe the fact that boronate is part or a portion of a ligand. In addition to boronate, ligands may include aromatic structures, aliphatic structures and / or polymeric structures, as will be defined in more detail below.

[0038] More specifically, the present disclosure relates to a chromatographic material comprising a retaining material functionalized with a ligand containing a boronate moiety, wherein the ligand is of formula I

[0039] [ka]

[0040] (In the formula, 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, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, preferably 0 ≤ n ≤ 10. Each R0 is independently selected from H and alkyl groups. Each Z1 is independently selected from 2-pyrrolidone, N-isopropylformylamide, 1-methoxy-2,3-propanediol, N-[tris(hydroxymethyl)methyl]formylamide, methylamide, N-alkylformamide, N,N'-dialkylformamide, and alkylformate. m is an integer from 1 to 40, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, preferably 1 ≤ m ≤ 10. Each R1 is selected independently from H and OH. Each R2 is selected independently from H and C6H5. Each Z2 is

[0041] [ka]

[0042] And, Each Y is independently selected from CH2, S, NH, O, -C(O)NH-, -NHC(O)-, NH-(CH2)uC(O)-NH, and (CH2)u-NH-C(O), where u is an integer from 1 to 6, i.e., 1, 2, 3, 4, 5, or 6. p is either 0 or 1. q is an integer from 0 to 6, i.e., 0, 1, 2, 3, 4, 5, or 6. Each R3 is H and

[0043] [ka]

[0044] Selected independently from, Each of R4, R5, R6, and R7 is independently selected from NH2 or its salts, H, Br, F, Cl, alkyl, OH, O-alkyl, and CF3. It provides chromatography materials as defined by [the specified method].

[0045] wavy line (

[0046] [ka]

[0047] ) represents the retaining material to which the ligand binds, and if a linker, such as a surface extender, is included in the chromatography material for coupling the ligand to the retaining material, the dashed line represents both the retaining material and the linker.

[0048] If R4, R5, R6, and R7 are amines (NH2), then these are the charged parts, and in this case these are the counterions, such as Cl. - or SO4 2- It can bind to the amine, thereby forming an NH2 salt. The salt can be formed by adding an acid, such as hydrochloric acid or sulfuric acid, to the amine.

[0049] According to an unrestrictive embodiment of formula I (wherein X is CH2-CH(OH)-CH2-O, n=0, m=1, R1 is OH, R2 is H, p=1, and Z2 is defined by formula (VI)), the ligand is formula II

[0050] [ka]

[0051] (In the formula, Y is selected from S, NH, or O. q is an integer from 0 to 6. Each R3 is H and

[0052] [ka]

[0053] Selected independently from, Each of R4, R5, R6, and R7 is independently selected from NH2 or its salts, H, Br, F, and alkyl groups. It can be defined by:

[0054] According to a non-limiting embodiment of formula II (wherein R5, R6, and R7 are each H), the ligand is formula III

[0055] [ka]

[0056] (In the formula, Y is selected from S, NH, or O. q is an integer from 0 to 6. R4 is independently selected from NH2 or its salts, H, Br, F, and alkyl. It can be defined by:

[0057] According to the currently preferred non-limiting embodiment of Equation III, R4 is F, Y is NH, and q=1.

[0058] According to another currently preferred non-limiting embodiment of Equation III, R4 is H, Y is NH, and q=1.

[0059] According to an unrestrictive embodiment of formula I (wherein X is CH2-CH(OH)-CH2-O, n=0, m=1, R1 is OH, R2 is H, p=1, and Z2 is defined by formula (VII)), the ligand is formula IV

[0060] [ka]

[0061] (In the formula, Y is selected from NH, O, -C(O)NH- and -NHC(O)-, q is an integer from 1 to 6, Each R3 is H and

[0062] [ka]

[0063] (Selected independently of) It can be defined by:

[0064] According to the currently preferred non-limiting embodiment of Equation IV, Y is NH, R3 is H, and q = 3.

[0065] According to another currently preferred non-limiting embodiment of Equation IV, Y is O, R3 is H, and q = 4.

[0066] According to a non-limiting embodiment of formula I (wherein R1 is H), the ligand is formula V

[0067] [ka]

[0068] (In the formula, X is selected from CH2-CH(OH)-CH2-O-CH2-CH2-, CH2 and -O-CH2-CH2-SO2-CH2. n is an integer between 0 and 40, preferably 0 ≤ n ≤ 10. Each R0 is independently selected from H and alkyl groups. Each Z1 is independently selected from 2-pyrrolidone, N-isopropylformylamide, 1-methoxy-2,3-propanediol, N-[tris(hydroxymethyl)methyl]formylamide, methylamide, N-alkylformamide, N,N'-dialkylformamide, and alkylformate. m is an integer between 1 and 40, preferably 1 ≤ m ≤ 10. Each R2 is selected independently from H and C6H5. Each Z2 is

[0069] [ka]

[0070] And, Each Y is independently selected from CH2 and -C(O)NH-, p is either 0 or 1. q is an integer from 0 to 6. Each R3 is H and

[0071] [ka]

[0072] Selected independently from, Each of R4, R5, R6, and R7 is independently selected from NH2 or its salts, H, Br, F, Cl, alkyl, OH, O-alkyl, and CF3. It can be defined by:

[0073] According to a currently preferred non-limiting embodiment of formula V (wherein X is CH2-CH(OH)-CH2-O-CH2-CH2-, R0 is H, Z1 is 2-pyrrolidone, Z2 is defined by formula (VI), each of R2, R3, R4, R5, R6 and R7 is H, n is an integer from 1 to 40, p=0 and q=0), the ligand comprises a copolymer of vinylphenylboronic acid and vinylpyrrolidone.

[0074] According to another currently preferred non-limiting embodiment of formula V (wherein X is CH2-CH(OH)-CH2-O-CH2-CH2-, Z2 is defined by formula (VI), and each of R2, R3, R4, R5, R6 and R7 is H, n=0, p=0 and q=0), the ligand comprises a polymer of vinylphenylboronic acid.

[0075] The holding material for chromatography materials may include film-like structures, nanofibers, monoliths, porous particles, non-porous particles, magnetic particles, or an expanded bed medium.

[0076] In a non-limiting example, the membrane structure may be convection-based and may include a non-woven web of polymer nanofibers, where the polymer may be a cellulose polymer, such as cellulose acetate, or a synthetic polymer, or a combination thereof. In an even more non-limiting example, the convection-based membrane structure may include a single membrane, multiple membranes, or a filter.

[0077] A non-limiting example of a membrane-like structure is the Mustang® membrane (Cytiva).

[0078] Another non-limiting example of a membrane structure is Fibro® (Cytiva), which is also a non-limiting example of a nanofiber-containing retaining material. Fibro® is a convection-based membrane structure containing nanofibers made from cellulose or a cellulose derivative.

[0079] A non-restrictive example of a monolith is CIMmultus® (Sartorius, Germany).

[0080] Non-limiting examples of porous particles that may instead be called beads include porous particles based on polysaccharides, such as agarose. Polysaccharide beads, such as agarose beads, may be manufactured as described in US6602990B1, US7396467B2, or US8309709B2, which are incorporated herein. The beads may contain crosslinked agarose. Chromatographic materials containing particles or beads may be called resins.

[0081] A non-restrictive example of magnetic particles is Mag Sepharose (Cytiva).

[0082] A non-exclusive example of an extended bed medium is STREAMLINE resin (Cytiva).

[0083] According to currently preferred non-limiting embodiments of chromatography materials, the holding material comprises a convection-based membrane structure containing nanofibers, and the ligand is defined by formula II or formula III.

[0084] According to another currently preferred non-limiting embodiment of the chromatography material, the holding material comprises a convection-based membrane structure containing porous particles or nanofibers, and the ligand is defined by formula IV.

[0085] As described in the Experiments section of this specification, of the chromatography material prototypes prepared and used for the purification of lentiviral particles, the Fibro prototype contains ligands that are all reachable for binding to the viral particles. In contrast, the resin prototype contains ligands on the surface of the beads that can bind to viral particles, but also contains ligands inside the pores of the beads that are too large for the viral particles to enter the pores and therefore not available for binding to the viral particles.

[0086] As briefly mentioned above, the chromatographic materials referred to herein may include a linker that connects the ligand to the retainer; that is, the coupling of the ligand to the retainer is provided by introducing a linker between the retainer and the ligand. Coupling can be carried out according to any conventional covalent coupling methodology, for example by using epichlorohydrin; epibromohydrin; allyl-glycidyl ether; bis-epoxide, e.g., butanediol diglycidyl ether; halogen-substituted aliphatic substances, e.g., dichloro-propanol; and divinyl sulfone. Non-limiting examples of suitable linkers include vinyl sulfone derivatives, vinyl sulfone derivatives combined with glycidol derivatives, polyethylene glycol (PEG) having 2 to 6 carbon atoms, carbohydrates having 3 to 6 carbon atoms, or polyhydric alcohols having 3 to 6 carbon atoms. Alternatively, the ligand may be coupled to the retainer via a longer linker molecule also known as a "surface extender" or simply "extender." Extenders are well known in this art and are commonly used to sterically increase the distance between the ligand and the retainer. The extender may be described as a tentacle or flexible arm. For a more detailed description of possible chemical structures, see, for example, US 6,428,707, which is included herein by reference. In short, the extender may be in the form of a polymer, such as a homopolymer or copolymer. Hydrophilic polymer extenders may be of synthetic origin, i.e., have a synthetic skeleton, or they may be of biological origin, i.e., biopolymers with a naturally occurring skeleton. Typical synthetic polymers include polyvinyl alcohol, polyacrylic and polymethacrylamide, polyvinyl ether, etc. Typical biopolymers include polysaccharides, such as starch, cellulose, dextran, and agarose.

[0087] Non-limiting examples of chromatographic materials disclosed herein include surface extenders, such as dextran. According to non-limiting embodiments, the chromatographic material comprises a retaining material in the form of porous particles and dextran, which couples the ligand with the retaining material.

[0088] This disclosure further provides chromatography devices including holders containing chromatography materials as described in detail elsewhere herein. Not limited examples of suitable chromatography devices include (i) columns containing porous particles, non-porous particles or monoliths, (ii) cartridges or capsules containing membrane structures or nanofibers, and (iii) containers or vessels containing magnetic particles.

[0089] This disclosure provides a method 100 for separating envelope or membrane-like biological particles from one or more impurities, as illustrated in Figure 1. - Step 110 involves adding a liquid sample containing envelopes or membrane-like biological particles and one or more impurities to a chromatographic material containing a retaining material functionalized with a ligand containing a boronate portion. - Step 120 of eluting envelope or membrane-like biological particles from the chromatography material into at least one elution fraction using an elution buffer. Further methods are provided, including the following.

[0090] In some cases, the elution buffer may contain polyhydroxyl compounds, such as sorbitol or sucrose, or other additives conventionally used for elution in boronate affinity chromatography, provided that the additives do not interfere with the ability of any further chromatographic material applied after the affinity chromatography step to function as intended, for example, by binding impurities and allowing envelopes or membrane-like biological particles to flow through the further chromatographic material. Such additives may function, for example, by competing with envelopes or membrane-like biological particles for binding to boronate.

[0091] The term "polyhydroxyl compound" is intended to mean an organic compound containing multiple hydroxyl groups (i.e., -OH), for example, two, three, four, or more hydroxyl groups.

[0092] The term "eluate" is used in its conventional sense in this field, which refers to the portion of the liquid sample that elutes from the chromatographic material after the liquid sample has been loaded onto the chromatographic material.

[0093] The chromatographic materials referred to in Method 100 are understood to be as defined and illustrated in detail elsewhere herein, in relation to a description of the chromatographic materials themselves, including but not limited to formulas I-V that define the ligands.

[0094] Step 120 for eluting an envelope or membrane-like biological particle may include a first elution step 124 using a first elution buffer and a second elution step 128 using a second elution buffer, wherein the first elution buffer contains a first polyhydroxyl compound and the second elution buffer contains a second polyhydroxyl compound.

[0095] Depending on the circumstances, the method may further include a washing step 126 between the first elution step 124 and the second elution step 128.

[0096] Method 100 may include a step 130 of cleaning the chromatography material using a cleaning solution under acidic conditions, for example, at a pH of about 2 to about 5.

[0097] In some cases, the cleaning solution may contain acetic acid.

[0098] The cleaning step 130 preferably follows a step 120 in which envelopes or membrane-like biological particles are eluted from the chromatography material.

[0099] The cleaning step 130 may be followed by a step 140 to re-equilibrium the chromatographic material before reapplying the chromatographic material in the method 100 for separating envelopes or membrane-like biological particles from one or more impurities.

[0100] The buffer may have a pH of approximately 7–8 for both the addition (i.e., binding) step 110 and the elution step 120 of the biological target compound. The buffer is appropriately selected from buffers commonly recommended for affinity chromatography and suitable for the above pH range. Non-limiting examples include tris(hydroxymethyl)aminomethane (i.e., Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (i.e., bis-trispropane), triethanolamine, N-methyldiethanolamine, diethanolamine, 1,3-diaminopropane, ethanolamine, phosphate buffer, bis-Tris, imidazole, MOPS, and HEPES.

[0101] To achieve elution of the bound biological target compound, the buffer in elution step 120 contains additional components compared to the buffer used in step (a). Non-limiting examples of such additional components are carbohydrates, polyhydroxyls (referred to as polyols instead, and including diols in this specification), such as sucrose, sorbitol, and glycerol.

[0102] Those skilled in the art can select an appropriate concentration for any one of the buffers listed above.

[0103] A currently preferred non-limiting example of a suitable combination of buffers used for the isolation of lentiviral particles is the following buffer system: - Buffer A (i.e., binding buffer for step 110): 50 mM ammonium bicarbonate, pH 7.9, 130 mM NaCl, and - Buffer B (i.e., elution buffer for step 120): 50 mM ammonium bicarbonate, pH 7.9, 130 mM NaCl, and 146 mM (5%) sucrose or 300 mM (5.2%) sorbitol.

[0104] Another currently preferred non-restrictive example of a suitable combination of buffers is the following buffer system: - Buffer A: 50 mM sodium phosphate (NaPi) pH 7.0 or 7.4, 130 mM NaCl, and - Buffer B: 50 mM sodium phosphate (NaPi) pH 7.0 or 7.4, 130 mM NaCl, 500 mM (9.1%) sorbitol.

[0105] In method 100 described above, the residence time of the liquid sample in the chromatographic material can be adapted to the relevant binding affinity of the target compound and ligand for a particular separation method. For residence times relevant to the present invention (e.g., about 1 minute or less), the binding kinetics between the target compound and ligand may be considered for the purpose of process efficiency. In contrast, binding kinetics is not a typical consideration for affinity interactions in diffusion chromatography media, such as conventional resins containing porous particles. When resins are used, the rate-limiting step is usually time-consuming due to diffusion through the pores of porous beads. However, when porous particles are applied in this context, the target compound is too large to diffuse through the pores of the particles. Therefore, the use of porous particles is associated with residence times as short as those of membrane structures containing nanofibers.

[0106] For example, in a method according to some embodiments of the present invention for recovering lentivirus particles, the residence time may be about 0.25 minutes to about 1 minute, e.g., 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 minute, when porous beads are used. When a membrane structure containing nanofibers is used, the residence time may be about 0.2 minutes, e.g., 0.1, 0.15, 0.2, 0.25, or 0.3 minutes.

[0107] The liquid sample added in step 110 of Method 100 disclosed herein may be an unpurified liquid sample. Method 100 disclosed herein may include a step 105 containing an unpurified envelope or membranous biological particle, by separating the unpurified envelope or membranous biological particle from a cell culture sample containing the envelope or membranous biological particle, before adding the unpurified liquid sample to the chromatographic material according to step 110 of Method 100, thereby obtaining an unpurified liquid sample containing the envelope or membranous biological particle.

[0108] Such a pre-purification step 105, in the context of liquid chromatography, refers to the first step of the separation procedure. Most commonly, this includes clarification (e.g., by filtration, centrifugation, or precipitation) and usually also includes concentration and / or stabilization of the sample. Step 110 then provides significant purification from soluble impurities by applying chromatography, which is described in detail elsewhere in this specification.

[0109] In this specification, the term “cell culture” means a culture of cells or a group of cells, where cells may be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be unclarified, i.e., contain cells, or it may be cell-depleted, i.e., the culture may contain no or very few cells but contain biomolecules released from the cells before the cells were removed. Furthermore, an unclarified cell culture may contain intact cells, destroyed cells, cell homogenates, and / or cell solubilates.

[0110] As used herein, the term "cell culture sample" refers to a cell culture sample collected and removed from a container or apparatus in which cells were cultured.

[0111] Non-limiting examples of separation devices suitable for use in pre-purification step 105 as described herein include filtration devices, chromatography columns, and membrane devices.

[0112] After step 120 of method 100 disclosed herein, intermediate purification may follow to further reduce the amount of impurities, such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of cell culture media, such as antifoaming agents and antibiotics, as well as product-related impurities, such as aggregates and misfolded species.

[0113] This disclosure is not limited to the exemplary embodiments described below, and it should be understood that several conceivable modifications of this disclosure are possible within the following claims. Any reference numerals placed in parentheses within the claims should not be construed as limiting the claims. The use of the verb "includes" and its conjugations does not exclude the existence of elements or processes other than those described. The article "a" or "an" preceding an element does not exclude the existence of multiple such elements.

[0114] Experiment section [Examples]

[0115] Isolation of lentiviral particles on boronate prototype chromatography material 1A) Isolation of lentiviral particles using a boronate resin prototype A prototype of a chromatography material was prepared by functionalizing a retaining material containing porous beads, referred to herein as resin, with a boronate-containing ligand.

[0116] Preparation of Boronate resin prototypes Activation of the resin before ligand coupling: Alylation of cross-linked agarose gel beads using allyl glycidyl ether: A cross-linked agarose gel (220 g = 220 mL) was transferred to a frit glass filter and washed with distilled water (×10 GV). Meanwhile, the 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 was adjusted to 244.44 mL by adding distilled water (i.e., 24.44 mL of DW). A mechanical stirrer was then attached to the flask, and stirring was started at approximately 300 rpm. A solution of 50% NaOH (371.56 g, 245.25 mL) was added, and the flask was lowered into the water bath. Stirring was continued for 30 minutes, after which AGE (58.43 mL) was added. The reaction mixture was then stirred at 50°C for 17 hours. The flask was removed from the water bath, and after it reached 25 ± 5°C, the mixture was transferred to a glass filter. The allylated gel was washed in the following order: DW 2×GV, EtOH 1×GV, and DW 7×GV. The gel was almost completely drained, and a portion was saved for allyl titration.

[0117] Bromoization: A 20 mL allylated agarose gel, washed twice with DI water, was transferred to a 100 mL three-necked round-bottom flask containing 10 mL of distilled water and 0.89 g of NaOAc.3H2O. The reaction mixture was stirred with a mechanical stirrer for 20 minutes, then 0.33 mL of elemental bromine (excess amount) was added, and stirring was continued for 15 minutes. 6 mL of 3 M sodium formate solution was slowly added to the slurry until the orange solution was decolorized. The brominated gel was washed with distilled water (×8 GV).

[0118] Prototype prepared by direct coupling of ligand to resin (i.e., without the use of an additional linker): (i) Coupling of 4-methylaminophenylboronate The synthesis pathway is shown in Figure 2A.

[0119] 5 mL of newly activated agarose gel was added to 2 mL of DI water and 0.013 mL of 50% NaOH solution. 4-methylaminophenylboronate ligand (674 mg, 3.6 mmol, 3 equivalents) was added to the mixture, and the mixture was stirred at 50°C for 18 hours (pH was approximately 10.5). After cooling, the gel was filtered and washed with 2 GV of DI water. The ligand density was determined to be 84.8 μmol / mL.

[0120] (ii) Coupling of 3-fluoro-4-methylaminophenylboronate: 5 mL of newly activated agarose gel was added to 2 mL of DI water and 0.013 mL of 50% NaOH solution. 3-Fluoro-4-aminomethylphenylboronate ligand (740 mg, 3.6 mmol, 3.0 equivalents) was added to the mixture and stirred at 50°C for 18 hours (pH was approximately 10.5). After cooling, the gel was filtered and washed with 2 GV of DI water. The ligand density was determined to be 82.5 μmol / mL.

[0121] (iii) Coupling of 2-fluoro-5-methylaminophenylboronate: 5 mL of newly activated agarose gel was added to 2 mL of DI water and 0.013 mL of 50% NaOH solution. 2-Fluoro-5-aminomethylphenylboronate ligand (740 mg, 3.6 mmol, 3.0 equivalents) was added to the mixture and stirred at 50°C for 18 hours (pH was approximately 10.5). After cooling, the gel was filtered and washed with 2 GV of DI water. The ligand density was determined to be 52.7 μmol / mL.

[0122] Sodium hydroxide treatment was performed after each of the direct ligand-coupling reactions described above: In a three-necked flask, distilled water (2 mL) and 50% NaOH solution (0.24 mL) were added to the isolated gel. The mixture was stirred at 40°C for 5 hours. The gel was then washed with DI water (10 GV).

[0123] Ligand coupling by the use of linkers, and more specifically, surface extenders such as dextran: Epoxy activation: The cross-linked agarose gel was washed with 10 × 1 GV of distilled water on a P3 sintered glass funnel. 50.09 mL of the discharged gel (1 mL corresponds to 1 g) was transferred to a 100 mL round-bottom flask. 12.1 mL of distilled water and 8.0 mL of 50% NaOH were added. A glass stopper and a Mickey Mouse stirring bar were attached to the rbf and placed on a stirring motor on a 28°C water bath. The rbf was immersed in the bath and the stirring speed was set to 250 rpm. After 10 minutes, 12.5 mL of ECH was added via dosimat, and the reaction was allowed to proceed for a further 120 minutes. The gel was washed with distilled water at a rate of 10 × 1 GV. The gel was analyzed for epoxide content and dry weight determination.

[0124] Dextran coupling: Dextran 40 was dissolved in a 100 mL three-necked flask containing water with slow stirring for approximately 5 hours. The dextran solution was added to the epoxy-activated gel drained from the above, and the slurry was heated to 40°C and stirred for 60 minutes (300 rpm). Nitrogen gas was blown into the solution at 0.15 NL / min for 60 minutes. Next, 50% NaOH and NaBH4 were added to the flask, and the mixture was left to stir overnight at 40°C. The reaction mixture was diluted, and the gel was washed with distilled water at 10 × 1 GV. The dry weight of the dextran coupling gel was analyzed.

[0125] Alylation 1: Approximately 45 mL (g) of allylated dextran agarose gel (from the dextran coupling product described above) in distilled water was washed with 50% NaOH at 3 × GV. The gel was then dried by suction 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 in a 50°C water bath. After 20 minutes, 15 mL of allyl glycidyl ether (AGE) was added. The reaction was allowed to proceed for 17.5 hours at 230 rpm. The gel was washed with distilled water at 1 × GV, with ethanol at 5 × GV, and then with distilled water at 8 × GV.

[0126] Alylation 2: Approximately 43 mL (g) of allylated dextran agarose gel (from the above allylation product 1) in distilled water was washed with 50% NaOH at 3 × GV. The gel was then dried by suction 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 in a 50°C water bath. After 20 minutes, 15 mL of AGE was added. The reaction was allowed to proceed for 17.5 hours at 230 rpm. The gel was washed with distilled water at 1 × GV, with ethanol at 5 × GV, and then with distilled water at 8 × GV.

[0127] Bromoization: Furthermore, as described under the heading "Activation of the resin before ligand coupling" in section 1A above, the above allylated diproduct was brominated.

[0128] Ligand coupling reaction: The newly activated 5 mL agarose gel was added to DI water (2 mL) and 13 μL of 50% NaOH solution. 4-Methylaminol (aminol) phenylboronate ligand (1.43 g, 7.65 mmol, 3.0 equivalents) was added to the mixture, and the pH was adjusted 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 filtered and washed with 10 GV of DI water. The ligand density was determined to be 76.8 μmol / mL.

[0129] Sodium hydroxide treatment: In a three-necked flask, distilled water (2 mL) and 50% NaOH solution (0.24 mL) were added to the isolated gel (5 mL) from the above. The mixture was stirred at 40°C for 5 hours. The gel was then washed with DI water (10 GV).

[0130] Coupling using multiple allylations: Alylation of allylated cross-linked agarose, part 2: A 25 mL allylated agarose gel (244.1 μmol / mL) was washed with 50% NaOH at 3 × 1 GV and transferred to a 100 mL round-bottom flask. 52.8 mL of 50% NaOH was added to the gel along with 55.6 mg of NaBH4. The flask was immersed in a 50°C bath. After 30 minutes, 8.33 mL of AGE was added, and the reaction mixture was stirred at 50°C for 17 hours. The flask was removed from the bath and transferred to a glass filter after reaching 25 ± 5°C. The allylated gel was washed in the following order: 2 × GV distilled water, 1 × GV EtOH, and 7 × GV distilled water. Neutralization was not performed before washing. The sample was collected for allyl titration.

[0131] Activation of allyl groups using bromine: 22 mL of allylated agarose gel (532 μmol / mL) was transferred to a 100 mL three-necked round-bottom flask to which distilled water (10.56 mL) and NaOAc × 3H₂O (0.979 g) had been added. The reaction mixture was stirred with a mechanical stirrer for 15 minutes, then bromine water (excess amount = yellow to orange) was added, and stirring was continued for another 15 minutes. 3 M sodium formate (20 g per 100 mL) was slowly added to the slurry until the orange solution was decolorized. The brominated gel was washed with distilled water (× 10 GV).

[0132] Coupling of cysteamine hydrochloride: 22 g of the activated resin from the above was transferred to a 100 mL round-bottom flask. 15 mL of water was added along with the ligand, cysteamine hydrochloride (14.86 g, 11.2 equivalents). The pH was adjusted to >12 using 5 M / 17 M NaOH. The reaction mixture was left at room temperature overnight (17 h). The reaction mixture was washed with DV20 (3 × 1 GV), 1 M NaCl (3 × 1 GV), and DV20 (3 × 1 GV).

[0133] Cl - Titration - General Procedure: The resin was washed with 0.5 M HCl 3 × 1 GV and 1 mM HCl 3 × 1 GV. 1 mL of cube-shaped resin was added to a titration cup with 20 mL of distilled water. Two drops of 2 M HNO3 were added, followed by 3 mL of 20 w / w% PVA solution. Titration of Cl- and Br- using titration method nr08, 0.1 M AgNO3 was started, and the ion capacities were determined.

[0134] Coupling of 4-(bromomethyl)phenylboronic acid: 4-(bromomethyl)phenylboronic acid reagent (1.45 g, 6.73 mmol, 3.0 equivalents relative to the cysteamine groups on the resin) was added to 2.38 (1.88 + 0.5) mL of distilled water and 1.5 mL of toluene, and the mixture was stirred at 50°C until nearly dissolved (insufficient time). 7 mL of cysteamine gel (320 μmol / mL) was then added to the mixture, and the pH was adjusted to 9 using 5 M NaOH solution. The reaction mixture was then stirred at 50°C for 1 day to produce 4-methyl-phenylboronic acid ligand coupled to the resin. After cooling, the gel was filtered and washed with 5 × 1 GV methanol and 10 × GV water. The ligand density was determined to be 70.2 μmol / mL at 50°C.

[0135] Preparation of resin chromatography devices: A 4-methylaminophenyl boronate resin prototype (ligand density 84.8 μmol / mL) was placed in a Tricorn® 5 / 50 column (Cytiva, Sweden).

[0136] Separation process and analysis: 12 mL of clarified lentivirus supply (7 × 10 9 The lentiviral particles (VP / mL) were linked to an AKTA Pure25 chromatography system and applied to a Tricorn® 5 / 50 column containing 1 mL of 4-methylaminophenyl boronate resin prototype (ligand density 84.8 μmol / mL). Following the protocol in Table 1, after sample loading, the column was washed, eluted with a buffer containing sucrose and sorbitol, cleaned in place (CIP), and re-equilibriumized. The fractions were collected and analyzed for the physical titer of the recovered viral particles (VP) and the removed impurities (total DNA and total protein). The physical titer of lentiviral particles was determined by p24 ELISA (VP / mL), total DNA was analyzed by Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific), and total protein was analyzed using a Micro BCA assay. The residence time of the feed in the boronate resin prototype chromatography material was 0.25–1 minute.

[0137] [Table 1]

[0138] 1B) Isolation of lentiviral particles using the Boronate Fibro® prototype. A prototype of a chromatography material was prepared by functionalizing a holding material containing a nonwoven web of cellulose acetate nanofibers, referred to herein as Fibro® film, with a boronate-containing ligand.

[0139] Preparation of Boronate Fibro Prototype Coupling of 4-aminomethylphenylboronic acid onto Fibro® membrane: The synthesis pathway is shown in Figure 2B.

[0140] A Fibro® cellulose acetate (CA) sheet was washed in a food container on a vibrating table with MQ water (4 × 100 mL, 10 min). Twelve 32 mm diameter discs were punched out from a 10 × 15 cm membrane sheet. 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 was added to the center. A glass stopper was used to prevent the rolls from spinning during stirring. KOH (1 M, 172 mL) was added and the reaction mixture was stirred for 10 minutes. Allyl glycidyl ether (AGE) (28 mL) was added to the reaction mixture and the reaction mixture was stirred overnight at room temperature. The reaction solution was decanted. The rolls were washed with acetone / MQ water (1:1, 2 × 300 mL, 10 min), followed by washing with MQ water (3 × 300 mL, 10 min). A solution of Na2CO3 (0.28M, 7.5g) in 25% v / v MeCN in MQ water was added to a roll (250mL). Divinyl sulfone * (40 mL) was added, the reaction mixture was stirred for 18 hours, and then the reaction solution was decanted. The rolls were then washed with acetone / MQ water (1:1, 2 × 300 mL, 10 min), followed by MQ water (3 × 300 mL, 10 min). NBS (7 g) in MeCN (250 mL, 75% v / v) in MQ water was added to the beaker and stirred for 4 hours. The reaction solution was decanted. The rolls were then washed with MQ water (6 × 300 mL, 10 min). 4-aminomethylphenylboronate (29.99 g, 0.8 M) was dissolved in 200 mL of MQ water at 50°C. The pH was adjusted to 10.5 (±0.5). The ligand dissolved when added to the beaker with two rolls. The reaction mixture was stirred at 60°C for 17 hours. The next morning, the reaction solution had separated into two distinct phases. The reaction solution was decanted, and the roll was washed with MQ water (6 × 300 mL, 10 minutes). The roll was removed from the beaker and carefully rolled up. The disc was removed and placed in a 6-well plate with 20% EtOH. The phase containing the ligand was stored for solubility testing. It was soluble when the pH was lowered. It was insoluble in MeCN, and a small amount dissolved in acetone.

[0141] Analysis of pressure flow and ligand density yielded an estimated ligand density of approximately 30 μmol / mL.

[0142] Preparation of Fibro chromatography devices: The boronate Fibro prototype was collected in a HiTrap Fibro® unit (Cytiva) 0.4 mL type chromatography device, which is referred to herein as the Fibro unit.

[0143] Separation process and analysis: 10 mL of clarified lentivirus supply (2.5 × 10 10 The lentiviral compound (VP / mL) was applied to a 0.4 mL non-fluorinated boronate prototype unit (ligand density 30.0 μmol / mL) linked to an AKTA Pure25 chromatography system. Following the protocol in Table 2, after sample loading, the column was washed, eluted with a sorbitol-containing buffer, cleaned in place (CIP), and re-equilibriumized. Flow-through fraction (FT), wash (W), and sorbitol eluate fraction (E) were collected, and the physical titer of the recovered viral particles (VP) and the removed impurities (total DNA and total protein) were analyzed. The physical titer of lentiviral particles was determined by p24 ELISA (VP / mL), total DNA was analyzed by Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific), and total protein was analyzed using a Micro BCA assay. The residence time of the feed in the boronate Fibro prototype chromatography material was 0.2 minutes.

[0144] [Table 2]

[0145] Results and conclusions regarding boronate resin and boronate fibro prototypes Figure 3 shows chromatograms from two consecutive runs (Figures 3A and 3B, respectively) of lentivirus capture using a column containing 1 mL of boronate resin prototype. UV280 is shown by a solid line, and elution with elution buffer containing sucrose or sorbitol (referred to as B buffer) is shown by a dotted line. The cleansing-in-place (CIP) phase with acetic acid is also shown.

[0146] Figure 4 shows the analysis of fractions from two runs (Figures 4A and 4B, respectively) of lentivirus capture using a boronate resin prototype, based on the chromatograms in Figures 3A and 3B. Flow-through fractions (FT1-5) and sucrose (Esuc) and sorbitol (Esorb) eluate fractions were analyzed for physical titer (p24 ELISA), total protein, and total DNA.

[0147] Figure 5 shows the physical titer of recovered viral particles (Figure 5A), as well as the removed total DNA (Figure 5B) and total protein (Figure 5C), for lentivirus capture using 0.4 mL units of boronate fibrous with pH 7 or pH 7.4, following the protocol in Table 2.

[0148] Figure 6 shows a comparison of binding capacities between the boronate Fibro prototype and the boronate resin prototype. Binding capacity was determined as the number of virus particles that could be bound and eluted (p24 ELISA). For the resin, the 1 mL column was overloaded, and lentiviruses were detected in the flow-through. For the Fibro, no virus was detected in the flow-through, indicating a potentially higher binding capacity. The capacity of the Fibro was at least 2.5 × 10⁶. 11 It is VP, and the capacity of the resin is approximately 2.5 x 10 10 It had VP (10 times lower binding capacity).

[0149] The boronate elution yield using sucrose was approximately 40–60% VP for both boronate resin and boronate fibro. Elution with sorbitol or other carbohydrates is alternative. When the boronate column was cleaned in place with NaOH (CIP), lentiviral particles bound more strongly to boronate, and sorbitol was required for elution (data not shown). Instead, CIP in acetic acid allowed for milder elution in sucrose, and lentiviral elution was minimal in the second sorbitol elution step (Figures 4 and 5).

[0150] Impurity removal was similar in fibroboronate as in the resin boronate prototype (Figures 4 and 5), but binding ability was 10 times higher despite a lower ligand density (Figure 6). [Examples]

[0151] Preparation of polymer boronate prototypes 2A) Preparation of polymer boronate resin prototype: (i) comprising a copolymer of vinylpyrrolidone and vinylphenylboronic acid: A washed allylated agarose gel (20 mL, allyl content 59 μmol / 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 vinylphenylboronic acid (1 g) were mixed to adjust the pH to 8.5. N2 was blown into the reaction solution for 20 minutes. 2,2'-azobis-(2-amidopropane) hydrochloride (ADBA) (0.04 g), followed by the reaction mixture, was added to the round-bottom flask. The slurry was stirred at 250 rpm and heated to 50°C. A stream of N2 was placed over the reaction mixture. The reaction mixture was left at 50°C overnight. The slurry was transferred to a glass filter (P3) and washed with water (3 × GV), ethanol (3 × GV), and water (7 × GV).

[0152] (ii) A polymer containing 100% vinylphenylboronic acid: A washed allylated agarose gel (15 mL, allyl content 59 μmol / mL) was added to a 100 mL round-bottom flask. In a beaker, water / DMSO (1:2, 33 mL) and vinylphenylboronic acid (2.2 g) were mixed to adjust the pH to 8.5. N2 was blown into the reaction solution for 10 minutes. The reaction mixture was added to the round-bottom flask. The slurry was stirred at 250 rpm, heated to 50°C, and N2 was blown in. ADBA (0.05 g) was added. An N2 stream was set up over the reaction mixture. Before adding further ADBA (0.1 g), the reaction mixture was left at 50°C for 1 hour. The reaction mixture was left at 50°C for a further 3 hours. The slurry was transferred to a glass filter (P3) and washed with water (3 × GV), ethanol (3 × GV), and water (7 × GV).

[0153] 2B) Preparation of polymer boronate Fibro® prototype: Contains a copolymer of vinylpyrrolidone and vinylphenylboronic acid: A Fibro prototype was transplanted from a divinyl sulfone crosslinked membrane that did not have glycidol surface tension. The starting material was placed in a food container and washed with water (4 × 100 mL, 20 minutes, shaking at 70 rpm). Twelve discs with a diameter of 32 mm were punched out and placed between two chemically inert nets and rolled on a chemically inert plastic roll. Two rolls with five discs each were prepared. The rolls were placed in a spinner flask reactor (100 mL) and a magnetic stirrer was added to the center of the rolls. A glass stopper was used to prevent the rolls from spinning during stirring.

[0154] Partial inactivation: KOH (0.5M, 100mL) was added to one of the reactors. The reaction mixture was stirred for 30 minutes. The reaction solution was decanted, and the sheet was washed with MQ water (6 × 100mL).

[0155] Coupling: According to Table 3 below, the reaction solution was mixed in a beaker, degassed with N2, and the pH was adjusted to 8.

[0156] [Table 3]

[0157] The reaction mixture was added to the reactor, the lid was closed, the reactor was placed in a 50°C water bath, and N2 was blown in. The reaction mixture was left overnight. The reaction mixture polymerized to such a high degree that it was difficult to remove the discs without damage. However, three discs were recovered and washed with MQ water (4 × 100 mL), ethanol (3 × 100 mL), and MQ water (6 × 100 mL).

[0158] Conclusion: The resin containing polymer boronate ligand and the Fibro® prototype were successfully synthesized. [Examples]

[0159] Isolation of lentiviral particles under variable conditions The experimental design for separating lentiviral particles from impurities was carried out using the apparatus and samples as in Example 1 above, and further varying the boronate chromatography materials as in Examples 1 and 2. • Ligand density: >85 μmol / mL for resins, and >30 μmol / mL for fibrous materials. • Ligand chemistry: Polymer boronate ligand according to Example 2 • Lentivirus feed: Material filtered by tangential flow filtration instead of clarification feed, especially for separation on resin materials. • Buffer and elution conditions: • Different buffers A, e.g., Tris, Hepes, Mops <130mM NaCl • Additives in the buffer: e.g., MgCl2 • Elution buffer: Contains glycerol or other polyhydroxyl compounds instead of sucrose or sorbitol. ·CIP conditions: • <1M NaOH, e.g., 0.1~0.5M NaOH • Clean with NaOH, followed by acetic acid (e.g., 0.1-0.3M).

[0160] References WO2023 / 274989 A1 US6602990 B1 US7396467 B2 US8309709 B2

Claims

1. Use of chromatographic materials, including a retention material functionalized with a ligand containing a boronate moiety, for the separation of envelope or membrane-like biological particles from impurities.

2. The ligand is of formula I 【Chemistry 1】 (In the formula, X is O, CH 2 -CH(OH)-CH 2 -O, CH 2 -CH(OH)-CH 2 -O-CH 2 -CH 2 -, CH 2 and -O-CH 2 -CH 2 -SO 2 -CH 2 selected from n is an integer between 0 and 40, preferably 0 ≤ n ≤ 10. Each R 0 It is independently selected from H and alkyl, each Z 1 This is independently selected from 2-pyrrolidone, N-isopropylformylamide, 1-methoxy-2,3-propanediol, N-[tris(hydroxymethyl)methyl]formylamide, methylamide, N-alkylformamide, N,N'-dialkylformamide, and alkylformate. m is an integer between 1 and 40, preferably 1 ≤ m ≤ 10. Each R 1 It is selected independently from H and OH, Each R 2 H and C 6 H 5 Selected independently from, each Z 2 teeth 【Chemistry 2】 And, Each Y is CH 2 , S, NH, O, -C(O)NH-, -NHC(O)-, NH-(CH 2 )uC(O)-NH and (CH 2 ) is independently selected from u-NH-C(O), where u is an integer from 1 to 6. p is either 0 or 1. q is an integer from 0 to 6, Each R 3 H and 【Transformation 3】 Selected independently from, Each R 4 , R 5 , R 6 and R 7 NH 2 or their salts, H, Br, F, Cl, alkyl, OH, O-alkyl and CF 3 (Selected independently of) The use according to claim 1, as defined by...

3. The ligand is, Formula II 【Chemistry 4】 (In the formula, Y is selected from S, NH, or O. q is an integer from 0 to 6, Each R 3 H and 【Transformation 5】 Selected independently from, Each R 4 , R 5 , R 6 and R 7 NH 2 (or a salt thereof, independently selected from H, Br, F, and alkyl) The use according to claim 2, as defined by...

4. The ligand is, Formula III 【Transformation 6】 (In the formula, Y is selected from S, NH, or O. q is an integer from 0 to 6, R 4 NH 2 or a salt thereof, selected from H, Br, F, and alkyl, Preferably, (i)R 4 is F, Y is NH, and q=1, or (ii)R 4 (where is H, Y is NH, and q=1) The use according to claim 3, as defined by...

5. The ligand is, Formula IV 【Transformation 7】 (In the formula, Y is selected from NH, O, -C(O)NH- and -NHC(O)-, q is an integer from 1 to 6, Each R 3 H and 【Transformation 8】 Selected independently from, Preferably, (i) Y is NH, and R 3 H is H, and q=3, or (ii) Y is O, R 3 (where H is and q=4) The use according to claim 2, as defined by...

6. The ligand is, formula V 【Chemistry 9】 (In the formula, X is CH 2 -CH(OH)-CH 2 -O-CH 2 -CH 2 -, CH 2 and -O-CH 2 -CH 2 -SO 2 -CH 2 Selected from, n is an integer between 0 and 40, preferably 0 ≤ n ≤ 10. Each R 0 It is independently selected from H and alkyl, each Z 1 This is independently selected from 2-pyrrolidone, N-isopropylformylamide, 1-methoxy-2,3-propanediol, N-[tris(hydroxymethyl)methyl]formylamide, methylamide, N-alkylformamide, N,N'-dialkylformamide, and alkylformate. m is an integer between 1 and 40, preferably 1 ≤ m ≤ 10. Each R 2 H and C 6 H 5 Selected independently from, each Z 2 teeth 【Chemistry 10】 And, Each Y is CH 2 And selected independently from -C(O)NH-, p is either 0 or 1. q is an integer from 0 to 6, Each R 3 H and 【Chemistry 11】 Selected independently from, Each R 4 , R 5 , R 6 and R 7 NH 2 or their salts, H, Br, F, Cl, alkyl, OH, O-alkyl and CF 3 Selected independently from, Preferably, (i)X is CH 2 -CH(OH)-CH 2 -O-CH 2 -CH 2 - and R 0 H is Z 1 It is 2-pyrrolidone, and R 2 , R 3 , R 4 , R 5 , R 6 and R 7 Each of them is H, n is an integer from 1 to 40, p=0, q=0, or (ii) X is CH 2 -CH(OH)-CH 2 -O-CH 2 -CH 2 - and R 2 , R 3 , R 4 , R 5 , R 6 and R 7 Each of these is H, n=0, p=0, and q=0. The use according to claim 2, as defined by...

7. The use according to any one of claims 1 to 6, wherein the holding material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, magnetic particles, or an expanded bed medium.

8. The envelope or membrane particle is selected from enveloped virus particles, extracellular vesicles, and virus-like particles. In some cases, enveloped virus particles are lentiviral particles. In some cases, extracellular vesicles are exosomes. The use according to any one of claims 1 to 7.

9. A method (100) for separating envelope or membrane-like biological particles from impurities, A step (110) of adding a liquid sample containing an envelope or membrane-like biological particle and one or more impurities to a chromatographic material containing a retaining material functionalized with a ligand containing a boronate portion, Step (120) is to elute envelopes or membrane-like biological particles from a chromatographic material into at least one elution fraction using an elution buffer, wherein the elution buffer optionally contains a polyhydroxyl compound, such as sucrose or sorbitol. Method (100), including the method (100).

10. The method according to claim 9, wherein the step of eluting an envelope or membrane-like biological particle (120) comprises a first elution step (124) using a first elution buffer and a second elution step (128) using a second elution buffer, wherein the first elution buffer comprises a first polyhydroxyl compound and the second elution buffer comprises a second polyhydroxyl compound, and optionally the method further comprises a washing step (126) between the first elution step (124) and the second elution step (128).

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

12. The method according to any one of claims 9 to 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, and optionally 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 envelope or membrane particle is selected from enveloped virus particles, extracellular vesicles, and virus-like particles. In some cases, enveloped virus particles are lentiviral particles. In some cases, extracellular vesicles are exosomes. The method according to any one of claims 9 to 15.

17. A chromatography material comprising a retaining material functionalized with a ligand containing 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, and optionally 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 formula IV as defined in claim 5.

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

21. The chromatography material according to any one of claims 17 to 20, wherein the holding material comprises a film structure, nanofibers, monoliths, porous particles, non-porous particles, magnetic particles, or an expanded bed medium.

22. The chromatography material according to claim 21, wherein the retaining material comprises a convection-based membrane structure containing nanofibers, and the ligand is defined by formula II or formula III.

23. The chromatography material according to claim 21, wherein the retaining material comprises a convection-based membrane structure containing porous particles or nanofibers, and the ligand is defined by formula IV.

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

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