Purification of biological particles

Abstract

Chromatographic media comprising porous beads having an inner porous core and an outer porous shell for the chromatographic separation of enveloped or membrane-like biological particles from impurities such as contaminant DNA and / or proteins. The core is capable of binding molecules by hydrophobic interaction, however, the pore size of the shell does not allow particles having a size of 20 nm and greater to penetrate into the beads and interact with the core. The separation is carried out at a pH of less than 7.4. The coated or film-like bio-particles may be subjected to a pre-chromatographic capture step. When used to purify enveloped viral particles, the method of the invention was found to produce very high ratios of infectious viral particles.

Description

Technical Field

[0001] The present invention relates to a method for the chromatographic purification of membrane-encapsulated or membrane-like biological particles and to the use of a chromatographic medium comprising porous beads having an inner porous core and an outer porous shell for the purification of membrane-encapsulated or membrane-like biological particles. Background Art

[0002] Advanced Therapy Medicinal Products (ATMPs) form an emerging field with great promise for treating or preventing serious diseases. They include, for example, gene and cell therapies aimed at restoring or replacing defective genes, as well as other therapies based on recombinant nucleic acids such as DNA and RNA vaccines.

[0003] In order to deliver the recombinant nucleic acid to the recipient cell, a vector is generally required. Depending on the type of treatment, the target cell type and the nucleic acid to be delivered, different vectors can be used. Commonly used vectors include enveloped viral particles, such as lentivirus (LV). Extracellular vesicles (EVs) produced and released by cells are another example of potential vectors in cell and gene therapy.

[0004] Lentiviruses (LVs) are classified as retroviruses and have a single-stranded RNA genome with a reverse transcriptase enzyme. Lentiviruses consist of a viral envelope with glycosylated proteins that act as ligands and have affinity for host cell receptors on the outer cell membrane. Upon entry, the virus influences the transcription of the viral genetic material. The viral genome consists of RNA sequences that encode specific proteins that promote integration of the viral sequence into the host cell genome.

[0005] The virus infects the host cell by docking onto the CD4 glycoprotein on the host cell's surface. The virus then injects its material into the host cell's cytoplasm, where the reverse transcriptase enzyme reversely transcribes the viral RNA, producing a viral DNA genome that is transported to the host cell's nucleus and integrated into the host cell's genome. The host cell begins transcribing the viral RNA and expressing the viral proteins that form the capsid. The viral RNA and viral proteins then assemble, and the resulting new virion leaves the host cell.

[0006] When enveloped viruses (such as lentiviruses) are released from host cells, the virus buds from the host cell and therefore has an outer lipid bilayer derived from the cell membrane containing viral glycoproteins. Inside the enveloped virus particle is a protein capsid containing the viral genetic material. The envelope is critical for infection of the host cell because it binds to and fuses with the host cell membrane.

[0007] For use in gene therapy, viruses are modified to act as vectors to insert beneficial genes into cells. 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, which is different from other retroviruses that only penetrate cells undergoing mitosis. Many cell types in adult individuals do not divide, and LV may be the only option for transferring genetic material into such cells. Genetically modified LVs for cell and gene therapy have been shown to be promising candidates for curing diseases such as diabetes, prostate cancer, chronic granulomatous diseases, and vascular diseases. Therefore, it is important to modify the viral genome so that it cannot replicate itself and that it is permanently integrated into the cell genome. Most of the transduction of human cells by genetically modified lentiviruses is performed ex vivo by transfecting human T cells.

[0008] In order to produce lentivirus, 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 capsid and reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce a single-stranded RNA viral genome and is marked by the presence of a ψ (psi) sequence. This sequence is used to package the genome into virions. In order to use lentivirus in gene therapy, the virions must be purified from cellular impurities such as host cell proteins and DNA and excess plasmids after transfection. Typically, the harvested host cells producing lentivirus are treated with nucleases and the lentivirus is purified using several filtration techniques such as normal flow microfiltration, ultrafiltration, and diafiltration to reduce the impurity level to an approved level.

[0009] However, the current downstream purification methods of lentiviruses are generally synonymous with low recovery rates of infectious viruses (usually 10-20%), because lentiviruses are unstable and sensitive to shear forces, buffer components such as salts, and rapidly degrade at room temperature. Time-consuming multi-step methods are also considered unhelpful. It is reported that lentiviruses are stable in a very narrow pH range of the treatment solution, 7.0-7.4 (Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology 280, 124-131 (2001)) and conductivity window, <0.2M NaCl (Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016), which makes downstream purification methods challenging.

[0010] Therefore, there is a need for improved or at least alternative methods of purifying encapsulated viral particles (such as lentiviral particles) and other sensitive biological particles for use as vehicles for cell or gene therapy or for the preparation of ATMPs. Summary of the Invention

[0011] It is an object of the present invention to overcome or at least partially alleviate the disadvantages of the prior art.

[0012] It was therefore an object of the present invention to provide an improved purification method by which enveloped or membrane-like biological particles, such as enveloped viruses, can be purified.

[0013] On the one hand, the chromatographic medium that comprises the porous beads with inner porous core and outer porous shell is used for chromatographic separation of membrane or membranous biological particles and impurities such as pollutant DNA and / or protein to realize this and other purposes.Described core can be by hydrophobic interaction binding molecule, and the pore size of described shell does not allow the particle with 20nm and larger size to penetrate into described pearl and interact with described core.Described separation is less than pH 7.4, as 6.0 to less than pH under the scope of 7.4, as 6.5 to 7.2, as 6.1 to 7.1.Isolated membrane or membranous biological particles are obtained in flow-through usually.Before being applied to the described chromatographic medium that comprises porous beads, membrane or membranous biological particles can be carried out to the chromatographic capture step in advance, produce the product of the partial purification that contains membrane or membranous biological particles and pollutant DNA.

[0014] Chromatographic separation using the porous shell / bead chromatography media described herein at the indicated pH values (and alternatively referred to herein as the polishing step) was found to produce unexpectedly high rates of infectious viral particles. This chromatography step itself is expected to provide infectious recoveries of 70-100% (relative to the input material) while also being very effective in removing small-sized impurities such as residual host cell proteins and DNA. The high infectious recoveries achieved when used to purify enveloped lentiviral particles are also an indication that the method is mild enough to be suitable for purifying similarly sensitive enveloped or membranous bioparticles, such as other enveloped viruses or extracellular vesicles.

[0015] In another aspect, the present invention provides a method for purifying membrane-encapsulated or membrane-like bioparticles from a feed, the method comprising:

[0016] a) adding a feed comprising an envelope or membrane-like bioparticle and one or more impurities to a first chromatography medium comprising a support material functionalized with a ligand that captures the envelope or membrane-like bioparticle at a pH of 6-10, preferably 6-8,

[0017] b) eluting the envelope or membrane-like biological particles from the first chromatographic medium in at least one elution fraction containing the envelope or membrane-like biological particles,

[0018] c) if necessary, adjusting the pH of the eluted fraction of step b) to a pH of less than 7.4,

[0019] d) adding the eluted fraction to a second chromatography medium comprising porous beads having an inner porous core and an outer porous shell at a pH in the range of 6.0 to less than 7.4, wherein the core is capable of binding molecules by hydrophobic interactions, and wherein the pore size of the shell prevents particles having a size of 20 nm and larger from contacting the core, and

[0020] e) obtaining at least one flow-through fraction containing the purified envelope or membrane-like bioparticles from the chromatography medium of step d.

[0021] The method of the present invention is advantageous for the purification of sensitive biological particles in several aspects. As demonstrated herein, carrying out step d at a pH lower than 7.4 significantly increases the amount of infectious viral particles recovered compared to pH 7.4. For example, for enveloped viral particles, the method of the present invention (all of steps ae) can enable an infectivity recovery of at least 30%, such as at least 50%, relative to the infectious viral particle content in the untreated feed. This represents a substantial improvement over currently available methods, which typically produce an infectivity recovery of 10-20%. Using a feed that has been clarified prior to step a, the method steps ae of the present invention can produce an infectivity recovery of at least 50%, such as at least 60%, such as 70%, relative to the clarified feed.

[0022] Looking at the individual chromatography steps individually, the first capture (steps ab) can provide an infectivity recovery of enveloped viral particles of at least 60%, such as at least 70%, relative to the feed. The polishing using a porous shell / bead resin (step de) can, by itself, independently produce an infectivity recovery of at least 70% and up to 100%, relative to the content of infectious viral particles provided by the previous step.

[0023] Furthermore, as described herein, combining chromatographic capture with a polishing step, wherein purified enveloped or membrane-like bioparticles are obtained in the flow-through, provides for shorter processing times, which is believed to be beneficial for stability. Furthermore, it is also believed that the bioparticles experience relatively low shear forces during the flow-through step, which also helps maintain integrity and / or infectivity properties.

[0024] The enveloped or membranous biological particle may be selected from the group consisting of an enveloped viral particle and an extracellular vesicle, such as an exosome. The enveloped viral particle may be, for example, a lentiviral particle.

[0025] The term "virion" is used herein to refer to a fully infectious viral particle. It includes a core comprising a viral genome (i.e., viral genome) in the form of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the core is surrounded by a morphologically defined shell. The shell is referred to as a capsid. The capsid and the encapsulated viral genome together constitute a so-called nucleocapsid. The nucleocapsid of an enveloped virus is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, in order to produce viral vectors for various applications such as treatment, the genome of the viral particle is modified to include a genetic insert comprising the genetic material of interest.

[0026] The term "impurity" is intended herein to mean any molecule or substance present in a liquid sample that is not the desired target entity. In the context of the present invention, "impurities" primarily include host cell proteins (HCPs) and host cell DNA. However, the term "impurity" generally also includes aggregates, such as aggregates of the target entity, and fragments of the target entity. In the context of the present invention, the target entity is an envelope or membranous bioparticle.

[0027] As used herein, the expression "functionalized with a ligand" means that a ligand is chemically coupled, optionally covalently coupled, to an entity functionalized according to conventional methods. The coupling may involve the use of linkers, spacers or surface extensions. In this context, a "ligand" is a molecule capable of binding to a given analyte or binding partner. The binding may be specific, as in the case of affinity ligands that bind to a specific target entity, or non-specific, as in the case of hydrophobic interaction ligands or ion exchange ligands that are capable of binding to any entity having the desired properties (e.g., charge). It will be understood that "ligand" is intended to mean a ligand substance, and that the singular form of the term may include a large number of individual ligands.

[0028] The support material of the first chromatographic medium may be selected from a monolith, a membrane, porous beads, non-porous beads, magnetic beads or an expanded bed medium.

[0029] The ligand of the first chromatographic medium for capturing the envelope or membranous bioparticles can be selected from affinity ligands and anion exchange ligands. The ligand can be an anion exchange ligand comprising a diamine functional group that produces at least one weak anion exchange group, achieving an ion capacity of 10-500 μmol / mL. The anion exchange group is positively charged or partially positively charged at a pH of 6-10.

[0030] Suitable anion exchange ligands can be described by formula (I):

[0031]

[0032] wherein X is independently selected at each occurrence from H, OH or C 1-3group, and R1, R2, R3 and R4 are independently selected from H and C 1-3 Group,

[0033] Wherein the C3 group is straight chain or branched,

[0034] Among them C 1-3 The group comprises independently selected from OH, OC 1-2 , SC 1-2 , NH, NHR, NR2, wherein R is selected from H and C 1-3 group.

[0035] For example, suitable AIEX ligands may be selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propylenediamine and N,N-diethylethylenediamine. N,N-diethylethylenediamine may be preferred.

[0036] In the method of the invention, elution in step b) may be provided by contacting the first chromatography medium with an elution buffer having a salt concentration of at most 0.65 M. Elution may be provided using gradually increasing salt concentrations.

[0037] The eluted fraction may be added to the chromatography medium in step d) at a pH of 6.5 to 7.2, such as 6.5 to 7.1, or 6.8 to 7.1.

[0038] In an embodiment, the at least one flow-through fraction obtained in step e) containing the purified enveloped or membranous bioparticles contains no detectable DNA.

[0039] In the methods or uses of the present invention, the porosity or pore size of the shell can be such that it does not allow molecules larger than 700 kDa to contact the core. For purification of relatively small encapsulated or membranous bioparticles, the porosity or pore size of the shell can be such that it does not allow molecules larger than 400 kDa to contact the core. In terms of particle size, the porosity or pore size of the shell can exclude particles having a size of 20 nm or more, or particles having a size of 30 nm or more, or particles having a size of 60 nm or more, such as 100 nm or more, from contacting the core.

[0040] The porous beads may comprise a hydrophilic polymer, such as a polysaccharide, such as agarose. Preferably, the core and shell may comprise agarose.

[0041] The porous core can be functionalized with a hydrophobic interaction ligand. The hydrophobic interaction ligand can comprise an aliphatic or aromatic C4 to C16 hydrocarbon, such as an aliphatic C4 to C16 hydrocarbon. The hydrophobic interaction ligand is a C4 to C16 alkylamine, such as octylamine.

[0042] In the method or use of the present invention, the residence time of the membrane-coated or membrane-like bioparticles in the chromatographic medium comprising porous beads having an inner porous core and an outer porous shell may be 0.5 to 10 minutes. The flow rate may be 0.1 mL / min to 3 mL / min.

[0043] Optionally, the feed may be subjected to a nuclease treatment prior to the chromatography step of the present invention.In the method of the present invention, the nuclease treatment may be performed prior to step a).

[0044] Preferred aspects of the disclosure are described in the following detailed description and in the dependent claims.It is noted that the invention relates to all possible combinations of features recited in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] This and other aspects of the present invention will now be described in more detail with reference to the accompanying drawings, in which:

[0046] Figure 1 is a flow chart outlining the steps of a method according to the present invention.

[0047] Figure 2 An exemplary capture chromatography device for purifying enveloped or membrane-like bioparticles is shown schematically in cross-section.

[0048] Figure 3 Exemplary chromatography beads for purifying, for example, lentiviruses (LVs) as used in the present invention are shown schematically in cross section.

[0049] Figure 4 As described in the experimental section, Capto TM Chromatogram of lentivirus capture by DEAE anion exchange.

[0050] Figure 5 Figure 2 shows the Capto TM Graph showing the % total lentiviral recovery (p24) and infectious lentiviral recovery after DEAE capture, as well as the reduction of impurities (DNA, HCP). On the x-axis, "FT" represents flow-through, and E1-5 represents elution fractions 1-5. High levels of infectious lentiviral particles were obtained, particularly in fraction E3, and coeluted with DNA.

[0051] Figure 6 are shown respectively as described in the experimental section using Capto TM Figure 2: Graph of total recovery (p24) and infectivity recovery of lentivirus purified at different pH values using Core 700. pH 7.0 resulted in significant improvement compared to pH 8.0 and pH 7.4.

[0052] As shown in the drawings, some features may be exaggerated for illustrative purposes and therefore are provided to illustrate the general structure of an embodiment of the invention. DETAILED DESCRIPTION

[0053] The present invention can be used to purify membrane-encapsulated or membranous bioparticles that are typically sensitive to shear forces, salts, pH, and detergents. The membrane-encapsulated or membranous bioparticles can be of natural or synthetic origin and can be obtained from a biological sample, or produced in vivo by recombinant expression in prokaryotic or eukaryotic cells, or produced in vitro. The membrane of the membrane-encapsulated bioparticle can comprise a lipid bilayer. The membrane of the membranous bioparticle can comprise a lipid bilayer.

[0054] The enveloped bioparticles may be enveloped viruses. Examples of enveloped viruses are DNA viruses such as herpesviruses, poxviruses, hepadnaviruses, and asfarviridae; RNA viruses such as flaviviruses, alphaviruses, enveloped viruses, coronaviruses, hepatitis D virus, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and filoviruses; and retroviruses such as lentiviruses. Membranous bioparticles may be extracellular vesicles such as exosomes. In addition, virus-like particles (VLPs) are included in the envelope or membranous bioparticles.

[0055] The envelope or membranous bioparticles can contain, for example, genetic cargo intended for gene therapy. The genetic cargo can be one or more exogenous nucleic acid sequences contained together with the natural nucleic acid content of the particle. In the case of enveloped viral particles, the viral genome can contain exogenous DNA or RNA inserts. Envelope or membranous bioparticles, such as viral particles containing exogenous genetic inserts, can be recombinantly produced according to known methods. The presence of large genetic inserts (exogenous DNA or RNA) may make the viral particles more sensitive and unstable.

[0056] The present invention provides a gentle yet effective purification method that can be used to purify sensitive enveloped or membrane-like bioparticles. As demonstrated herein, the present invention is particularly useful for purifying enveloped retroviruses, such as lentiviruses, with very high infectivity recoveries.

[0057] Figure 1 A method 100 for purifying enveloped or membrane-like biological particles is schematically shown, and Figure 2 The principle of the employed AIEX chromatography method is shown.

[0058] Envelope or membranous bioparticles 3 are present in feed 1 together with one or more impurities 2. For example, feed comprising enveloped viral particles such as lentiviral particles or exosomes can be produced by cell lines such as HEK 293 cells (human embryonic kidney cells). Before being applied to the first chromatographic medium, the feed can be clarified or otherwise pretreated according to known methods. In the clarified harvest, the amount of solids has been reduced, and the one or more impurities can be soluble impurities, such as host cell proteins (HCP) and DNA. For example, the feed can be treated with nuclease to partially degrade contaminant polynucleotides and reduce their size. In the case of purified RNA viruses, the treatment with DNA enzyme can be applied.

[0059] In the first step 101 of the method 100, a feed 1 is added to a first chromatography medium 4 for capturing an envelope or membrane-like bioparticle at a pH of 6-10. To capture the envelope or membrane-like bioparticle, the chromatography medium 4 comprises a support material functionalized with a ligand capable of reversibly binding to the target envelope or membrane-like bioparticle.

[0060] The ligand may be an affinity ligand capable of binding to the relevant envelope or membranous bioparticle. Alternatively, the ligand may be an anion exchange (AIEX) ligand, and preferably a weak AIEX ligand.

[0061] Feed 1 is typically loaded onto the first chromatographic medium 4 using a loading buffer having a pH in the range of 6-10, such as 6-8. Prior to loading, the medium 4 can be equilibrated using the loading buffer or another buffer having an appropriate pH of 6-10, such as 6-8. Under the loading conditions, the membrane-coated or membranous bioparticles bind to the ligand, while at least some impurities 2, particularly host cell proteins, are washed through the column. The feed can be loaded at a flow rate of 1 to 10 mL / min.

[0062] After loading the feed and optionally additional washing, there is a step 102 of eluting the envelope or membrane-like bioparticles. Elution can be achieved by changing the ionic strength. For example, an elution buffer providing an increasing concentration of salt can be used.

[0063] The salt may comprise (i) a compound selected from the group consisting of CO3 2- 、SO4 2- 、S2O3 2- 、H2PO4 - 、HPO4 2- , acetate - , citrate- and Cl - anion, and (ii) selected from NH 4+ , K + 、Na + He Li +In some embodiments, the present invention provides the cation of the present invention. For example, the salt can include sodium acetate (NaOAc) or sodium chloride (NaCl). However, it should be understood that other salts composed of a combination of an anion as listed in (i) and a cationic as listed in (ii) can alternatively be used to elute the capsid. The limiting examples of this type of other salts are LiCl, KCl or other equivalent metal salts suitable for salt elution as known in the art. The limiting examples of a suitable concentration of NaCl include about 5mM to about 1M, such as about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000mM. Non-limiting examples of suitable concentrations of NaOAc include about 5 mM to about 500 mM, such as about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

[0064] During elution (step 102), the salt concentration can be increased stepwise or continuously. A linear gradient of NaCl ranging from about 100 mM (e.g., 130 mM) to about 650 mM can be used. This relatively low salt concentration is beneficial because enveloped viruses and exosomes have reduced stability at higher conductivities, especially at concentrations above 0.65 M. Optionally, the salt concentration during elution can be up to 450 mM.

[0065] Elution can be performed at a flow rate of 1 to 10 mL / min. The flow rate can be the same as the flow rate during loading.

[0066] Typically, elution of the membrane-coated or film-like bioparticles from a first chromatography medium having a support material that is a convection-based fibrous substrate (e.g., a fibrous nonwoven polymer matrix) can be performed at flow rates as low as 60 MV / min with a residence time of a few seconds, and as high as 0.2 MV / min with a residence time of several minutes (up to 6 minutes) if desired. The optimal residence time for such fibrous support materials is 5-20 MV / min. For resins, typical residence times are 1-8 minutes, and sufficient residence times are often achieved after 4 minutes.

[0067] Optionally, the elution buffer may contain arginine or another additive conventionally used for elution in affinity or anion exchange chromatography, provided that the additive does not interfere with the ability of the second chromatography medium to bind impurities and allow the membrane-coated or membrane-like bioparticles to flow through.

[0068] Thus at least one fraction of eluted enveloped or membranous bioparticles is provided. Optionally, more than one fraction may be corrected and optionally combined before proceeding to the next step of the method.

[0069] In methods for purifying enveloped viruses such as lentiviruses, the elution fraction(s) containing the virus typically contain a high level of recovered infectious viral particles. For example, an infectivity recovery of at least 50%, such as at least 60%, at least 70%, or at least 80%, can be achieved after step 102.

[0070] During the elution step, direct dilution in a buffer containing sucrose to reduce conductivity can stabilize the virus and thus improve recovery.

[0071] The first chromatographic medium can be functionalized with a ligand to achieve an ionic capacity (number of charged functional groups per ml of medium (μmol / ml)) of 10-500 μmol / mL, or in the range of 50-300 μmol / mL or 100-300 μmol / mL. The ligand can have a diamine functional group that produces at least one weak anion exchange group, and the ionic capacity (number of charged functional groups per ml of medium (μmol / mL)) is 10-500 μmol / mL, or in the range of 50-300 μmol / mL or 100-300 μmol / mL. The weak anion exchange group can include a multimodal weak anion exchange group, that is, the anion exchange group provides at least two different but synergistic sites that interact with the compound to be bound (i.e., the envelope or membranous bioparticle). For example, one of these sites can produce an attractive charge-charge interaction between the ligand and the binding target. The other site can promote binding by introducing a second local charge or by increasing the local amount of solvated water that affects the binding capacity.

[0072] Weak anion exchange groups can be positively charged or partially positively charged at a pH of 6-10. Such positively charged or partially positively charged weak anion exchange groups can attract enveloped or membranous bioparticles that are negatively charged at neutral pH, such as lentiviral particles. Weak anion exchange groups can be positively charged or partially positively charged at a pH of 6-10, 6-9.5, 6-9, or 6-8.

[0073] Weak ion exchange groups are those that have a gradient from fully charged to uncharged depending on pH, with a neutral charge (equal amounts of + and -) at the PI (isoelectric point). In contrast, strong anion exchange groups based on quaternary amines are always charged. Almost all other anion exchange groups (not based on quaternary amines) are weak anion exchange groups, i.e., the charge varies (and can be zero) within the reasonable pH range of use (e.g., pH 2-11).

[0074] The ligand or a portion of the ligand can be described by formula (I):

[0075]

[0076] wherein X is independently selected at each occurrence from H, OH or C 1-3 group, and

[0077] R1, R2, R3 and R4 are independently selected from H and C 1-3 Group,

[0078] Wherein the C3 group is straight chain or branched,

[0079] Among them C 1-3 The group comprises independently selected from OH, OC 1-2 , SC 1-2 , NH, NHR, NR2, wherein R is selected from H and C 1-3 group.

[0080] The ligand or a portion of the ligand may be described by formula (I) above. This means that the diamine ligand may form part of a larger structure such as a polymer. The ligand or a portion of the ligand may be part of a larger structure produced, for example, by reaction of a solid support with a lower molecular weight amine chemical containing a leaving group, such as 2-chloro-N,N-diethylethylamine (DEAE), 2-chloro-N,N-diethylethylamine, 2-chloroethylamine, 3-chloropropylamine, 2-chloro-N,N-dimethylethylamine, 3-chloro-N-methylprop-1-amine. In the ligand or portion of the ligand described by the formula above comprising a diamine functional group producing at least one weak anion exchange group, the two amines may be separated by 2-4 carbon atoms and each amine group may be substituted by two R groups, which may be selected from H and C 1-4 Alkyl groups, and may be branched and / or may also be substituted with other groups such as hydroxyl, amine, ether and thioether, these other groups are then limited to 3-8 atoms.

[0081] The ligand described by the above formula may be selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propylenediamine and N,N-diethylethylenediamine.

[0082] The support material of the chromatography medium 4 may be selected from a monolith, a membrane, a filter, porous beads, non-porous beads, magnetic beads or an expanded bed medium.

[0083] Beads of different bead sizes may be used, such as beads having a diameter of 1-120 μm or 10-120 μm. The beads may comprise a polysaccharide, such as agarose, which may be cross-linked. The beads may have a uniform porosity.

[0084] A monolith is a single piece of porous material characterized by a highly interconnected network of channels with diameters in the range of 10-4000 nm.

[0085] The membrane material can be inorganic-organic materials (eg, alkoxysilane coated on glass fiber), alumina membranes, and organic materials (ie, cellulose and its derivatives, regenerated cellulose, nylon, polyethersulfone, polypropylene, polyvinylidene fluoride, etc.).

[0086] The support material may be a woven material. Alternatively, the support material may be a nonwoven material having a mean flow pore size of 0.1-2.0 μm, for example comprising fibers, such as cellulose fibers. The mean flow pore (MFP) size is an indicator of the flow properties of a material and is measured by capillary flow porometry, based on the displacement of a wetting liquid with a known surface tension from a sample pore by applying a gas under gradually increasing pressure. The larger the MFP size, the greater the liquid flow through the material at a given pressure. The mean flow pore size is calculated from the point at which 50% of the flow is through the sample. Therefore, the mean flow pore size corresponds to the pore size calculated at the pressure at which the wet curve and the semi-dry curve meet. In an alternative definition, the mean flow pore size of the support material can be viewed as the effective pore size defined as the size of the largest sphere that can pass through the pore.

[0087] The mean flow pore size of the support material may be 0.1-1.8 μm, 0.1-1.6 μm, 0.1-1.4 μm, 0.1-1.2 μm, 0.1-1.0 μm, 0.1-0.8 μm, 0.1-0.6 μm, 0.1-0.4 μm, 0.1-0.2 μm, 0.2-2.0 μm, 0.4-2.0 μm, 0.6-2.0 μm, 0.8-2.0 μm, 1.0-2.0 μm, 1.2-2.0 μm, 1.4-2.0 μm, 1.6-2.0 μm, 1.8-2.0 μm, or 0.5-1.5 μm.

[0088] Chromatography support material can comprise the chromatography matrix based on convection. The described chromatography matrix based on convection can be a fiber substrate. The described fiber substrate can be based on electrostatic spinning polymer fiber or cellulose fiber, optionally non-woven fiber. Therefore, the fiber substrate can be a fiber non-woven polymer matrix. The fiber included in the described fiber substrate has a cross-sectional diameter of 10-1000nm, such as 200-800nm, 200-400nm or 300-400nm. This fiber substrate can be found in the HiTrap Fibro department of Cytiva, Sweden. Usually, from the chromatography medium eluting envelope or membranous bioparticles with the support material based on the fiber substrate (such as fiber non-woven polymer matrix) of convection, it can be carried out with a flow velocity of 60MV / min, i.e., a residence time of several seconds, and up to a residence time of several minutes (up to 6min) 0.2MV / min, as low as a few seconds. The optimal residence time of this type of fiber support material is 5-20MV / min. For resin, typical residence time is 1-8 minute, and enough residence time is often obtained after 4 minutes.

[0089] The ligand of the first chromatographic medium 4 can be connected to the support material by an extension group, which can be selected from a polysaccharide structure and a polymer structure. The extension group can be, for example, dextran, acrylamide or polyglycidol. If dextran is used as an extension, it can have a molecular weight within the range of 5,000 to 2M daltons. The extension group (if present) can be selected according to the type of support material and the ligand to be immobilized / connected to the support material.

[0090] The elution fraction containing the envelope or membranous bioparticles obtained in step 102 typically also contains one or more types of impurities, although a significant amount of impurities may have been removed during capture chromatography. Examples of impurities that can be bound and eluted together with the envelope or membranous bioparticles include host cell DNA (hcDNA). For example, the eluate can contain at least 50% of the hcDNA present in the feed. DNA is negatively charged and strongly binds to anion exchangers, and longer DNA sequences can bind more strongly than shorter DNA fragments. It has been previously found that the higher the elution conductivity of the viral particles, the higher the risk of co-elution of the viral particles with the DNA. Therefore, a lower elution conductivity can be preferred to reduce the hcDNA in the target elution fraction.

[0091] The elution fraction obtained in step 102, containing the enveloped or membranous bioparticles and usually some remaining impurities such as host cell DNA (preferably fragmented by prior nuclease treatment), is added to a second chromatography medium comprising porous beads having a core-shell structure, wherein the core is functionalized with a hydrophobic interaction ligand. Figure 1In the embodiment of the present invention, this is represented by step 104. The impurities are retained by the porous beads and coated or membrane-like bioparticles are obtained 105 in the flow-through.

[0092] The chromatography step 104 is carried out at a pH in the range of 6.0 to less than 7.4, such as 6.5 to 7.2, such as 6.5 to 7.1 or 6.8 to 7.1. Therefore, if the elution fraction obtained in step 102 has a pH outside the desired range, for example a pH of 7.4 or higher, the pH is appropriately adjusted in step 103 before being added to the second chromatography medium. The pH adjustment step 103 can be achieved by diluting the elution fraction with a buffer having a lower pH (if the pH needs to be lowered) until the desired pH is reached in the diluted elution fraction. Of course, if it is necessary to increase the pH, for example from a value below 7.0, a dilution buffer having a pH higher than that of the elution fraction can be used. The buffer used for dilution can be the same buffer as the buffer used as loading buffer in step 104. Figure 6 As shown, comparative examples using pH values of 7.4 and 8, respectively, for this step resulted in low infectivity recoveries of lentiviral particles, while performing step 104 at a pH of 7.0 resulted in the most significant increase in infectivity recoveries. It is believed that satisfactory recoveries also occur at pH values slightly above 7.0. Without wishing to be bound by any particular theory, it is believed that enveloped or membranous bioparticles may be slightly more tolerant of pH values below 7.0 than values above 7.0.

[0093] For the sake of clarity, if the pH of the eluted fraction is in the range of 6.0 to less than 7.4, then step 103 of adjusting the pH is optional. However, as indicated above, a pH above 6.0, such as at least 6.5 or at least 6.8, may still be preferred. Therefore, in the case where the pH is at least 6.0 but lower than the preferred value, the pH can be appropriately adjusted in step 103. Similarly, when the pH is lower than 7.4 but higher than the preferred value, the pH can be appropriately adjusted by step 103.

[0094] Figure 3 The chromatography beads 5 of the second chromatography medium of step 104 are schematically shown. The beads 5 are porous beads comprising a porous core 52 surrounded by a porous shell 51. The porosity of the core and the shell can be the same or different. However, at least the porosity of the shell 51 prevents large entities such as envelopes or membranous bioparticles (in Figure 3 The molecule (denoted by "LV" in FIG) penetrates through the shell and contacts the core.

[0095] The shell 51 is typically hydrophilic. Thus, the surface of the porous bead 5 that is accessible to larger entities such as envelopes or membranous bioparticles is hydrophilic and does not irreversibly adsorb or denature proteins. The shell can be formed from a hydrophilic material that exposes a plurality of polar groups, such as those containing oxygen and / or nitrogen atoms. Examples of such polar groups are hydroxyl, amino, carboxyl, sulfonate (S and SP ligands), lower alkyl ethers (such as (-CH2CH2O-), n H, where n is an integer 2, 3, 4 and higher).

[0096] The core 52 binds strongly to biomolecules such as proteins and DNA through hydrophobic interactions. The core can be hydrophobic. Preferably, the hydrophobic core 52 itself is hydrophilic and is based on a hydrophilic material, such as a hydrophilic polymer, and is functionalized with a hydrophobic interaction ligand to provide the desired hydrophobicity. Alternatively, however, the core can be hydrophobic based on a hydrophobic polymer. For example, styrene / ethylstyrene / DVB, vinyl ethers, and acrylates containing hydrophobic substituents and polymers containing fluoroalkanes are contemplated.

[0097] The hydrophobic interaction ligand may comprise an aliphatic hydrocarbon, such as a C1-C30 alkyl group, preferably a C4-C16 alkyl group, and / or an aromatic hydrocarbon, such as phenyl, anthracene, naphthalene.

[0098] Preferably, the ligand density may be greater than 90 μmole / ml core entity, which represents a ligand density higher than levels typically used for HIC resins. Advantageously, the hydrophobicity provided by the ligand may allow proteins to interact or be adsorbed at both very low and very high ionic strengths.

[0099] The hydrophilic polymer on which the shell and optionally the core are based is a polysaccharide, such as agarose. For example, both the core and the shell may comprise cross-linked agarose. The porous core-shell beads may be formed as WO

[0100] 2009 / 131526. In particular, the core and shell can be made of agarose, and the core can be functionalized with a hydrocarbon interaction ligand containing 4-16 carbons, preferably an octyl ligand. Useful chromatography media are available under the trade names Capto TM Core 400 and Capto TM Core 700 was purchased from Cytiva, Sweden.

[0101] The shell covers or surrounds the core. Due to the limited porosity of the shell 51, only entities, such as molecules, that are small enough to penetrate the shell can interact with the hydrophobic core. Therefore, the porous beads 5 have size exclusion properties. In the context of the present invention, impurities such as residual host cell proteins and DNA (preferably fragmented DNA) can enter the porous beads and bind, while target envelopes or membranous bioparticles cannot enter and are instead captured in the flow-through fraction.

[0102] The hydrophobic core is able to strongly bind any impurities that penetrate into the bead 5. Therefore, under the treatment conditions of the present invention, the impurities are not released from the core, but remain bound to the core.

[0103] The porosity of the shell can be defined as not allowing particles having a size (diameter) of 20 nm to enter the bead, such that particles of this size and larger are not retained in the chromatographic medium but are allowed to flow through the chromatographic medium, while smaller particles can enter the bead to be adsorbed or bound by the hydrophobic core. For example, the shell can have a porosity small enough to prevent particles having a size of at least 30 nm, such as 80 nm and larger.

[0104] Enveloped viruses typically have a size ranging from 20 nm to up to 300 nm, depending on the type of virus. Lentiviruses can have a size in the range of 80-120 nm, typically 100-120 nm. Enveloped viruses can be larger than non-enveloped viruses, such as adenoviruses, which are only packaged in the capsid. Enveloped viruses are generally larger than adeno-associated viruses, which are typically about 25 nm in size. Extracellular vesicles, such as exosomes, can have a size in the range of 30 to 180 nm.

[0105] In an alternative definition, the porosity of the shell can be described as excluding molecules with a molecular weight of 400kDa and higher, while molecules with a molecular weight lower than 400kDa can enter the beads and interact with the hydrophobic core. In an embodiment, the porosity of the shell can exclude molecules with a molecular weight of 700kDa and higher, or 1,000kDa and higher, or 2,000kDa and higher. On the contrary, molecules with a molecular weight lower than the corresponding limit can enter the beads and interact with the hydrophobic core. Low cutoff values such as 400kDa can be used to purify relatively small particles, such as particles with a size within the range of 20-100nm. When the target particle to be purified is larger, for example 150-300nm, a higher cutoff value such as 1,000 or 2,000kDa can be used.

[0106] The shell 51 may be inert or unfunctionalized. For example, the shell is not functionalized with the HIC ligand of the core, nor with any other HIC ligand. The porous shell may be hydrophilic.

[0107] For a 1 mL column, chromatography step 104 can be performed at a flow rate of 0.1 mL / min to 3 mL / min, preferably 0.3 mL / min to 2 mL / min, such as 0.3 mL / min to 1.4 mL / min. The flow rate of step 104 can be lower than the flow rate used in the previous steps 101 and / or 102.

[0108] In step 104, the residence time of the sample (eluate obtained and optionally diluted in step 102) in the second chromatographic medium can be 0.5 to 10 minutes, such as 0.5 to 3.5 minutes. For example, the residence time can be 1 minute or shorter, such as 0.5 to 0.7 minutes. Longer residence time (slower flow velocity) can provide higher purity, because impurities will have more time to penetrate into the porous beads and interact with core 52. Shorter residence time (faster flow velocity) can be conducive to processing efficiency and economy. In addition, for sensitive biological particles such as enveloped viruses, short processing time can be preferably used to minimize exposure to conditions (such as room temperature) that may have a negative impact on viral stability.

[0109] Advantageously, the methods described herein can be performed on large sample volumes relative to the volume of the chromatographic medium. Therefore, a relatively large feed volume can be used, particularly when the support material is a fibrous material, relative to the volume of the first chromatographic medium that can have a high dynamic binding capacity. In addition, in step 104, a relatively large volume of sample (obtained from the capture chromatography step, optionally diluted to adjust the pH eluate as described above) can be applied to the second chromatographic medium comprising porous beads 5. For example, the sample volume applied can represent 15-30 times of column volumes (CV), such as 25-30 times of CV, where a column volume is the volume of the chromatographic medium comprising porous beads 5.

[0110] As demonstrated herein, performing step d at a pH below 7.4 can provide a significant increase in recovered infectious viral particles compared to pH 7.4. For example, for enveloped viral particles, the present method 100 can enable an infectivity recovery of at least 30%, such as at least 50%, relative to the content of infectious viral particles in the untreated feed. This represents a substantial improvement over currently available methods, which typically yield infectivity recoveries of 10-20%.

[0111] Pretreatment, such as clarifying the cell culture harvest to provide a clarified feed to be used in step 101, can result in a small loss of infectious virus. Typically, the infectivity recovery after clarification can be at least 80%. Using feed that has been clarified prior to step 101, the present method 100 can produce an infectivity recovery of at least 50%, such as at least 60%, such as 70%.

[0112] Looking at the individual chromatography steps individually, the first capture (steps 101 and 102) can provide an infectivity recovery of enveloped virus particles of at least 60%, such as at least 70%, relative to the feed. The polishing using the porous shell / bead resin (steps 104 and 105) can itself independently produce an infectivity recovery of at least 70% and up to 100% relative to the content of infectious virus particles provided by the previous step.

[0113] Typically, the addition of stabilizers such as sucrose can benefit the stability of the membrane-coated or membrane-like bioparticles. Stabilizers can be added to all mobile phases and formulation solutions used in the methods of the present invention.

[0114] Although the present invention is described herein with respect to exemplary embodiments, skilled artisans will appreciate that the invention is not limited thereto.In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0115] The use of the verb "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. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0116] experiment

[0117] Determination of ion capacity

[0118] The dynamic small ion binding capacity (DBC) of the chromatography materials functionalized with AIEX ligands, such as diethylethanolamine (DEAE), quaternary amine (Q) or N,N-diethylethylenediamine (DAX) is referred to below as the nanofiber chromatography material (Fibro TM The method is described in detail in the literature (Membrane Adsorber, Cytiva, Sweden) and should be generally applicable to any AIEX ligand. The method uses a conductometric titration in which added HCl protonates deprotonated weak AIEX ligands or neutralizes and displaces OH- bound to strong AIEX ligands. In contrast to protein DBC methods, The system monitors the conductivity signal of the permeate instead of the UV signal.

[0119] A membrane disk with a diameter of 25 mm was attached in a membrane holder device suitable for allowing chromatography of the membrane and was placed in a vacuum chamber equipped with a sample pump. The method was run on an Explorer 10 system (Cytiva, Sweden). The method consisted of the following steps:

[0120] 1. Rinse System and membrane (loaded in PEEK device).

[0121] 2. Load the membrane with excess NaOH

[0122] 3. Rinse the membrane with MQ water

[0123] 4. Flush HCl solution in the bypass

[0124] 5. Loading the Membrane with HCl and Monitoring Conductivity Breakthrough

[0125] 6. Reload the membrane with excess NaOH (optionally, to prepare for weight normalization)

[0126] 7. Rinse the membrane with MQ water

[0127] Each batch of membranes was analyzed using triplicate plates. Normalization was performed by plate volume and optionally also by plate dry weight.

[0128] Determination of mean flow pore size

[0129] The mean flow pore size of porous materials can be measured using capillary flow analysis using commercially available equipment. In this example, the equipment used was a POROLUX™ 100 porosimeter (IB-FT GmbH, Berlin, Germany) according to the manufacturer's manual and the method given in Table 1.

[0130] Table 1: Capillary flow porometry

[0131]

[0132] Experimental example

[0133] Lentivirus production and clarification

[0134] Lentivirus with a green fluorescent protein (GFP) insert was produced in HEK293T suspension cell culture following transfection with four plasmids according to conventional methods. At harvest, the feed was treated with 10 U / mL of Denarase with 2 mM MgCl2 and, after cell sedimentation, clarified using 10 μm, 5 μm, 0.5 μm, and 0.2 μm filters. Titers of the clarified feed lentivirus were ≥E9 VP / mL and ≥E6 TU / mL.

[0135] Lentivirus capture using weak AIEX chromatography

[0136] Apply the clarified feed to the Pure 25 (Cytiva, Sweden) connected to a 5 ml HiTrap equilibrated in 50 mM Tris-HCl, pH 8.0 (buffer A) TMA Capto™ DEAE column (Cytiva, Sweden) was used to elute the lentivirus using a short linear NaCl gradient ranging from 130 to 650 mM. The protocol can be found in Table 2. "CV" indicates column volume. Directly after elution, the eluate was diluted 1:5 with 5% sucrose to stabilize the virus and maintain infectivity.

[0137] The flow rate for all steps was 5 ml / min (1 min residence time).All runs were performed in duplicate.

[0138] Table 2.

[0139]

[0140] Impurity removal using layered bead resins with HIC ligands at different pH and flow rates

[0141] The eluate from the weak AIEX capture step was applied to Pure 25 (Cytiva, Sweden) connected to 1 mL HiTrap equilibrated in 50 mM Tris-HCl at pH 8.0, 7.4 or 7.0 TM Capto TM Core700 column (Cytiva, Sweden). The lentiviral load was approximately E11 VP in 50mM Tris-HCl (pH 7.0, 7.4 or 8.0), 4% sucrose and 130mM NaCl. For pH 7.0, the flow rates evaluated were 0.3mL / min, 0.9mL / min, 1.4mL / min and 1.9mL / min (corresponding to 3.3, 1.1, 0.7 and 0.5 minute residence times). All runs were performed in duplicate.

[0142] Fractions were analyzed for physical / total viral particles (p24 ELISA, VP / mL), infectious virus (cell-based transduction assay, measuring GFP-producing cells after transduction, TU / mL), total protein (micro BCA kit, μg / mL), and total DNA (picoGreen assay, ng / mL).

[0143] result

[0144] The capture chromatogram obtained using weak anion exchange with a short NaCl gradient is shown in Figure 4 . Use Capto TM Capture by DEAE resulted in >60% recovery of infectious lentivirus in the combined gradients ( Figure 5The gradient was divided into 5 fractions, and most of the lentivirus eluted in elution fractions 2 and 3. Most of the host cell proteins (HCP) (98% of the total protein) were obtained in the flow-through and wash, but about 50% of the DNA co-eluted with the lentivirus ( Figure 5 ).

[0145] Capto TM The DEAE eluate was diluted with buffer at pH 7.0, 7.4 or 8 and applied to a Capto TM Core 700 column. The flow-through fractions were collected and analyzed. It was found that the buffer with a pH of 7.0 resulted in an 80% recovery of infectivity, which was a very significant improvement over the recovery obtained with buffers of pH 8.0 or 7.4 (which was approximately 35%). Figure 6 All pH buffers produced similar levels of p24 recovery. Impurity levels were below the detection limits (BCA, picogreen) for both HCP and DNA for all pH values (data not shown).

[0146] Finally, if different flow rates are used as described above, the evaluation showed that flow rates up to 1.4 mL / min with a residence time of ≥0.7 min could be used with excellent slow infectious recovery (nearly 100%). Similarly, at pH 7.0, 1.9 mL / min or a residence time of 0.5 min could be used with an infectious recovery that was greatly improved (70%) relative to prior art methods. For all flow rates, impurity levels were below the detection limits (BCA, picogreen) for both HCPs and DNA.

[0147] References

[0148] Kinetic Analyses of Stability of Simple and Complex RetroviralVectors,F.Higashikawa et al.,Virology 280,124-131(2001)

[0149] Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016

[0150] WO 2009 / 131526(GE HEALTHCARE BIO SCIENCES AB)

Claims

1. A method (100) for purifying membrane-coated or membrane-like bioparticles (3) from a feed (1), the method comprising: a) adding (101) a feed (1) comprising an envelope or membrane-like bioparticle and one or more impurities to a first chromatography medium (4) at a pH of 6-10, preferably 6-8, said first chromatography medium comprising a support material functionalized with a ligand that captures said envelope or membrane-like bioparticle (3), b) eluting (102) the envelope or membrane-like biological particles (3) from the first chromatography medium (4) in at least one elution fraction containing the envelope or membrane-like biological particles, c) if necessary, adjusting (103) the pH of the eluted fraction of step b) to a pH of less than 7.4, d) adding (104) the eluted fraction to a second chromatography medium comprising porous beads (5) having an inner porous core (52) and an outer porous shell (51) at a pH in the range of 6.0 to less than 7.4, wherein the core is capable of binding molecules by hydrophobic interactions, and wherein the pore size of the shell prevents particles having a size of 20 nm and larger from contacting the core, and e) obtaining (105) at least one flow-through fraction containing the purified envelope or membrane-like bioparticles from the chromatography medium of step d).

2. The method according to claim 1, wherein the enveloped or membranous biological particle is selected from the group consisting of enveloped viral particles and exosomes.

3. The method according to claim 1 or 2, wherein the ligand that captures the envelope or membrane-like bioparticle is selected from affinity ligands and anion exchange ligands.

4. The method according to claim 2, wherein the support material of the first chromatographic medium (4) is functionalized with an anion exchange ligand comprising a diamine function generating at least one weak anion exchange group, achieving an ion capacity of 10-500 μmol / mL.

5. The method according to claim 4, wherein the anion exchange groups are positively charged or partially positively charged at a pH of 6-10.

6. The method according to claim 4 or 5, wherein the ligand is described by formula (I): wherein X is independently selected at each occurrence from H, OH or C 1-3 group, and R1, R2, R3 and R4 are independently selected from H and C 1-3 Group, Wherein the C3 group is a straight chain or a branched chain, Among them C 1-3 The group comprises independently selected from OH, OC 1-2 , SC 1-2 、NH、NHR、NR2 groups, Wherein R is selected from H and C 1-3 group.

7. The method according to claim 6, wherein the anion exchange ligand is selected from N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 2-methyl-1,3-propylenediamine and N,N-diethylethylenediamine, and is preferably N,N-diethylethylenediamine.

8. The method according to any one of the preceding claims, wherein the support material of the first chromatography medium (4) is selected from a monolith, a membrane, porous beads, non-porous beads, magnetic beads or an expanded bed medium.

9. The method according to any one of the preceding claims, wherein the support material of the first chromatography medium (4) is a non-woven fibrous material having a mean flow pore size of 0.1-2.0 μm.

10. The method according to any one of the preceding claims, wherein the ligand that captures the membrane or membranous bioparticle is attached to the support material via an extension group selected from polysaccharide structures and polymer structures.

11. The method according to any one of the preceding claims, wherein the elution in step b) is provided using gradually increasing salt concentrations.

12. The method according to any one of the preceding claims, wherein the elution in step b) is provided by contacting the first chromatography medium with an elution buffer having a salt concentration of at most 0.65M.

13. The method according to any one of the preceding claims, wherein the pore size of the shell (51) does not allow molecules larger than 700 kDa to contact the core.

14. The method according to claim 13, wherein the pore size of the shell (51) does not allow molecules larger than 400 kDa to contact the core.

15. The method according to any of the preceding claims, wherein the pore size of the shell (51) does not allow particles having a size of 20 nm and above, such as 30 nm and above, such as 60 nm and above, such as 100 nm and above to contact the core.

16. The method according to any one of the preceding claims, wherein the porous beads (5) comprise a hydrophilic polymer, such as a polysaccharide, such as agarose.

17. The method according to any one of the preceding claims, wherein the porous core (52) is functionalized with a hydrophobic interaction ligand, preferably wherein the hydrophobic interaction ligand comprises an aliphatic or aromatic C4 to C16 hydrocarbon, such as an aliphatic C4 to C16 hydrocarbon.

18. The method of claim 17, wherein the hydrophobic interaction ligand is a C4 to C16 alkylamine, such as octylamine.

19. The method according to any one of the preceding claims, wherein the eluted fraction is added to the chromatography medium in step d) at a pH of 6.5 to 7.2, such as 6.5 to 7.1, or 6.8 to 7.

1.

20. The method according to any one of the preceding claims, wherein the residence time of the eluted fraction in the chromatography medium of step d) is from 0.5 to 10 minutes.

21. The method according to any one of the preceding claims, wherein the eluted fraction is added to the chromatography medium of step d) at a flow rate of 0.1 mL / min to 3 mL / min.

22. The method according to any one of the preceding claims, wherein the feed (1) has been subjected to a nuclease treatment before step a).

23. The method according to any one of the preceding claims, wherein the at least one flow-through fraction containing purified enveloped or membrane-like bioparticles obtained in step e) contains no detectable DNA.

24. A chromatographic medium comprising porous beads (5) having an inner porous core (52) and an outer porous shell (51) for the chromatographic separation of enveloped or membranous bioparticles from impurities such as contaminant DNA at a pH of less than 7.4, wherein the core is capable of binding molecules by hydrophobic interactions and wherein the pore size of the shell does not allow particles having a size of 20 nm and larger to penetrate into the beads and interact with the core.

25. Use according to claim 24, wherein the pore size of the shell (51) does not allow molecules with a molecular weight of 400 kDa and above to penetrate into the bead and interact with the ligands of the core.

26. Use according to claim 24 or 25, wherein the pore size of the shell (51) does not allow particles having a size of 20 nm and above, such as 30 nm and above, such as 60 nm and above, such as 100 nm and above to penetrate into the bead and interact with the ligands of the core.

27. Use according to any one of claims 24 to 26, wherein the core is functionalized with a hydrophobic interaction ligand as defined in any one of claims 17 and 18.

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