Biological particle purification

Abstract

Chromatographic media comprising porous beads having an inner porous core and an outer porous shell are used for the chromatographic separation of enveloped or membrane-bearing biological particles from impurities such as contaminating DNA and / or proteins. The core is capable of binding molecules through hydrophobic interactions; however, the pore size of the shell prevents particles with a size of 20 nm or larger from penetrating into the beads and interacting with the core. Separation is performed at a pH below 7.4. The enveloped or membrane-bearing biological particles may have been subjected to a prior chromatographic capture step. When used for the purification of enveloped viral particles, the method of the present invention has been found to result in a significantly higher rate of infectious viral particles.

Description

[Technical Field]

[0001] The present invention relates to a method for the chromatographic purification of biological particles having an envelope or membrane, 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 biological particles having an envelope or membrane. [Background technology]

[0002] Advanced therapeutic medicines (ATMPs) form a highly promising emerging field for the treatment or prevention of serious diseases and 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] To deliver recombinant nucleic acids to recipient cells, a vector is typically required. Different vectors can be used depending on the type of therapy, the type of target cell, and the nucleic acid to be delivered. Commonly used vectors include enveloped viral particles such as lentiviruses (LVs). Extracellular vesicles (EVs), which are produced and released by cells, are another example of a promising vector for cell therapy and gene therapy.

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

[0005] The virus infects a host cell by docking onto the host cell surface CD4 glycoprotein. The virus then injects viral material into the host cell cytoplasm, where reverse transcriptase reverse-transcribes the viral RNA, resulting in the production of a viral DNA genome, which is then transported to the host cell nucleus and integrated into the host cell genome. The host cell begins to transcribe the viral RNA and express viral proteins that form the capsid. The viral RNA and viral proteins then assemble, and the new virions thus formed exit the host cell.

[0006] When enveloped viruses, such as lentiviruses, are released from host cells, they bud off the host cell and thus have an outer lipid bilayer derived from the cell membrane that contains viral glycoproteins. Inside the enveloped virus particle is a protein capsid that contains the viral genetic material. The envelope is important for host cell infection because it binds and fuses with the host cell membrane.

[0007] For use in gene therapy, viruses are modified to act as vectors for inserting useful genes into cells. The advantage of using LV as a viral vector is that, unlike other retroviruses that only penetrate cells undergoing mitosis, LV can penetrate the nuclear membrane of dividing as well as non-dividing cells. 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 used in cell and gene therapy have proven to be promising candidates for curing diseases such as diabetes, prostate cancer, chronic granulomatous disease, and vascular disease. Therefore, it is important to modify the viral genome so that it cannot replicate itself and so that it can be permanently integrated into the cellular genome. Transduction of human cells with genetically modified lentiviruses is most often performed by transfecting human T cells ex vivo.

[0008] To produce lentivirus, several plasmids are transfected into a so-called packaging cell line. One or more plasmids, generally referred to as packaging plasmids, encode virion proteins such as capsid and reverse transcriptase. Another plasmid contains genetic material that can be delivered by the vector. It is transcribed to produce a single-stranded RNA viral genome, characterized by the presence of a psi (psi) sequence. This sequence is used to package the genome into virions. To use lentivirus in gene therapy, it is necessary to purify the virions from cellular impurities such as host cell proteins and DNA, as well as excess plasmids after transfection. Typically, harvested host cells producing lentivirus are treated with nucleases, and the lentivirus is purified by several filtration techniques, such as conventional flow microfiltration, ultrafiltration, and diafiltration, to reduce the level of impurities to approved levels.

[0009] However, current downstream purification processes for lentiviruses often result in low recoveries of infectious virus (typically 10-20%) because lentiviruses are unstable and sensitive to shear stress, salt, and other buffer components, and they rapidly degrade at room temperature. Time-consuming, multi-step processes are also unlikely to be beneficial. Lentiviruses have been reported to be stable within a very narrow pH range of 7.0-7.4 (Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology 280, pp. 124-131 (2001)) and conductivity window of <0.2M NaCl (Process Development of Lentiviral Vector Expression, Purification, and Formulation for Gene Therapy Applications, Doctoral Thesis, Sara Nilsson, UCL, 2016), making downstream purification challenging.

[0010] Thus, there is a need for improved or at least alternative methods of purifying enveloped viral particles, such as lentiviral particles, and other sensitive biological particles for cell or gene therapy or to be used as vehicles in the preparation of ATMPs. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2009 / 131526 [Non-patent literature]

[0012] [Non-Patent Document 1] Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors, F. Higashikawa et al., Virology 280, pp. 124-131 (2001) [Non-patent document 2] Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016 Summary of the Invention [Means for solving the problem]

[0013] It is an object of the present invention to overcome or at least partially alleviate the disadvantages of the prior art. It is therefore an object of the present invention to provide an improved purification method by which enveloped or membrane-bearing biological particles, such as enveloped viruses, may be purified.

[0014] In one embodiment, this and other objects are achieved by the use of a chromatography medium comprising porous beads having an inner porous core and an outer porous shell for the chromatographic separation of enveloped or membrane-bearing biological particles from impurities such as contaminating DNA and / or proteins. The core is capable of binding molecules through hydrophobic interactions, and the pore size of the shell prevents particles 20 nm or larger in size from penetrating into the beads and interacting with the core. Separation is performed at a pH below 7.4, e.g., a pH within the range of 6.0 to less than 7.4, e.g., 6.5 to 7.2, e.g., 6.1 to 7.1. The separated enveloped or membrane-bearing biological particles are typically obtained in the flow-through. Before being applied to the chromatography medium comprising porous beads, the enveloped or membrane-bearing biological particles may be subjected to a prior chromatographic capture step, resulting in a partially purified product containing the enveloped or membrane-bearing biological particles and contaminating DNA.

[0015] It has been found that performing chromatographic separations (alternatively referred to herein as polishing steps) using porous shell / bead chromatography media as described herein at the indicated pH values ​​results in surprisingly high yields of infectious viral particles. It is contemplated that such a chromatographic step can itself provide 70-100% infectious recovery (relative to the input material) while also being highly effective in removing small-sized impurities such as residual host cell proteins and DNA. The high infectious recovery obtained when used for the purification of enveloped lentiviral particles also indicates that the method is sufficiently gentle to be suitable for the purification of similarly sensitive enveloped or membrane-bearing biological particles, such as other enveloped viruses or extracellular vesicles.

[0016] In another aspect, the present invention provides a method for purifying enveloped or membrane-bearing biological particles from a feed, the method comprising: a) applying a feed comprising enveloped or membrane-bearing biological particles and one or more impurities to a first chromatographic medium at a pH of 6 to 10, preferably 6 to 8, the first chromatographic medium comprising a support material functionalized with a ligand that captures the enveloped or membrane-bearing biological particles; b) eluting the enveloped or membrane-bearing biological particles from the first chromatographic medium into at least one elution fraction containing the enveloped or membrane-bearing biological particles; c) adjusting the pH of the eluted fraction of step b) to a pH below 7.4, if required; d) applying the elution fraction at a pH in the range of 6.0 to less than 7.4 to a second chromatographic medium comprising porous beads having an inner porous core and an outer porous shell, wherein the core is capable of binding molecules via hydrophobic interactions and the pore size of the shell prevents particles having a size of 20 nm or greater from contacting the core; and e) obtaining at least one flow-through fraction containing purified enveloped or membrane-bearing biological particles from the chromatography medium of step d).

[0017] The present method is advantageous in several aspects for the purification of sensitive biological particles. As demonstrated herein, performing step d at a pH below 7.4 allows for a significant increase in the recovered infectious viral particles compared to a pH of 7.4. For example, for enveloped viral particles, the present method (the entirety of steps a-e) can allow for an infectious recovery of at least 30%, e.g., 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 result in an infectious recovery of 10-20%. When using a feed that has been clarified prior to step a, steps a-e of the present method can result in an infectious recovery of at least 50%, e.g., at least 60%, e.g., 70%, relative to the clarified feed.

[0018] Viewed separately, the individual chromatography steps, the initial capture (steps a-b) may provide an infectious recovery of enveloped virus particles of at least 60%, e.g., at least 70%, relative to the feed. The polishing step (steps d-e) using a porous shell / bead resin may itself independently result in an infectious recovery of at least 70%, and up to 100%, relative to the infectious virus particle content provided by the preceding step.

[0019] Furthermore, combining chromatographic capture with a polishing step as described herein (resulting in purified enveloped or membrane-bearing biological particles in the flow-through) results in short process times, which may be beneficial for stability. Furthermore, biological particles may be subjected to relatively low shear stresses during the flow-through step, which may also contribute to maintaining their integrity and / or infectivity properties.

[0020] The enveloped or membrane-bearing biological particle may be selected from enveloped viral particles and extracellular vesicles such as exosomes. The enveloped viral particle may, for example, be a lentiviral particle.

[0021] The term "virion" is used herein to refer to a complete infectious virus particle. It comprises a core containing the viral genome (i.e., the viral genome) in the form of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), surrounded by a morphologically defined shell. The shell is called a capsid. The capsid and the enclosed viral genome together constitute the so-called nucleocapsid. The nucleocapsid of enveloped viruses is surrounded by a lipoprotein bilayer envelope. In the field of bioprocessing, for the purpose of generating viral vectors for various applications, such as therapeutics, the genome of the viral particle is modified to contain a gene insert containing the genetic material of interest.

[0022] 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 "impurities" also generally includes aggregates, such as aggregates of target entities, and fragments of target entities. In the context of the present invention, target entities are biological particles with an envelope or membrane.

[0023] As used herein, the expression "functionalized with a ligand" means that the ligand is chemically linked, optionally covalently, to an entity that has been functionalized by conventional methods. Linking may include the use of a linker, spacer, or surface extender. In this context, a "ligand" is a molecule capable of binding to a given analyte or binding partner. Binding may be specific, as in the case of affinity ligands that bind to a particular target entity, or non-specific, as in the case of hydrophobic interaction or ion-exchange ligands that are capable of binding to any entity with the required properties (e.g., charge). It should be understood that "ligand" is intended to mean a ligand species, and that the singular form of the term may encompass a number of individual ligands.

[0024] The support material of the first chromatography medium may be selected from a monolith, a membrane, porous beads, non-porous beads, magnetic beads, or an expanded bed medium.

[0025] The ligand of the first chromatography medium for capturing the enveloped or membrane-bearing biological particles can be selected from affinity ligands and anion exchange ligands. The ligand can be an anion exchange ligand containing a diamine functional group resulting in at least one weak anion exchange group with an ionic capacity of 10 to 500 μmol / mL. The anion exchange group is positively or partially positively charged at a pH of 6 to 10.

[0026] Suitable anion exchange ligands have the following formula (I):

[0027] [ka]

[0028] wherein X is, independently at each occurrence, H, OH, or C 1~3 groups, and R1, R2, R3, and R4 are selected from H and C 1~3 are independently selected from the group The C3 group is linear or branched; C 1~3 The groups are OH, OC 1~2 , SC 1~2 , NH, NHR, NR2, R is H and C 1~3 The group may be selected from the group

[0029] 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-propanediamine, and N,N-diethylethylenediamine. N,N-diethylethylenediamine may be preferred.

[0030] In this method, the eluting step of step b) may be effected by contacting the first chromatographic medium with an elution buffer having a salt concentration of at most 0.65 M. Elution may be effected using increasing salt concentrations.

[0031] The eluted fraction may be applied to the chromatography medium in step d) at a pH of from 6.5 to 7.2, such as from 6.5 to 7.1 or from 6.8 to 7.1.

[0032] In an embodiment, at least one flow-through fraction containing purified enveloped or membrane-bearing biological particles obtained in step e) does not contain detectable DNA.

[0033] In this method or use, the porosity or pore size of the shell may be such that it prevents molecules larger than 700 kDa from contacting the core. For the purpose of purifying biological particles with relatively small envelopes or membranes, the porosity or pore size of the shell may be such that it prevents molecules larger than 400 kDa from contacting the core. With respect to particle size, the porosity or pore size of the shell may exclude particles having a size of 20 nm or larger, or particles having a size of 30 nm or larger, or particles having a size of 60 nm or larger, such as 100 nm or larger, from contacting the core.

[0034] The porous beads may comprise a hydrophilic polymer, for example a polysaccharide such as agarose. Preferably, the core and shell may comprise agarose.

[0035] The porous core can be functionalized with a hydrophobic interaction ligand. The hydrophobic interaction ligand can include an aliphatic or aromatic C4-C16 hydrocarbon, for example, an aliphatic C4-C16 hydrocarbon. The hydrophobic interaction ligand can be a C4-C16 alkylamine, for example, octylamine.

[0036] In this method or use, the residence time of the enveloped or membrane-bearing biological particles in the chromatography medium comprising porous beads having an inner porous core and an outer porous shell may be between 0.5 and 10 minutes, and the flow rate may be from 0.1 mL / min to 3 mL / min.

[0037] Optionally, the feed may be subjected to a nuclease treatment prior to this chromatography step. In this method, a nuclease treatment may be carried out prior to step a).

[0038] Preferred aspects of the present disclosure are set out in the following detailed description and dependent claims, it being noted that the invention relates to all possible combinations of the features recited in the claims.

[0039] This and other aspects of the invention will now be described in more detail with reference to the accompanying drawings. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a flow chart outlining the steps of a method according to the present invention. [Figure 2] FIG. 1 is a schematic diagram illustrating a cross section of an exemplary capture chromatography device used for the purification of enveloped or membrane-bearing biological particles. [Figure 3] FIG. 1 is a schematic representation of the cross section of an exemplary chromatography bead as used in the present invention, for example, for lentivirus (LV) purification. [Figure 4] Chromatogram of lentiviral capture using Capto™ DEAE anion exchange, performed as described in the experimental section. [Figure 5] Figure 1 shows graphs showing % total lentiviral recovery (p24) and infectious lentiviral recovery and impurity (DNA, HCP) reduction after Capto™ DEAE capture, performed as described in the Experimental section. On the x-axis, "FT" indicates flow-through, and E1-5 represent elution fractions 1-5. High levels of infectious lentiviral particles were obtained, especially in fraction E3, which co-eluted with DNA. [Figure 6] 1 is a graph showing the total recovery (p24) and infectious recovery, respectively, of lentivirus purified using the Capto™ Core 700 as set out in the experimental section at different pH values, with a pH of 7.0 resulting in a significant improvement compared to pH 8.0 and pH 7.4. DETAILED DESCRIPTION OF THE INVENTION

[0041] As shown in the figures, some features may be exaggerated for illustrative purposes and, thus, are provided to illustrate the general structure of embodiments of the present invention.

[0042] The present invention is useful for purifying enveloped or membrane-bearing biological particles, which are typically sensitive to shear, salt, pH, and detergents. Envelope- or membrane-bearing biological particles can be of natural or synthetic origin, obtained from biological samples, or produced in vivo by recombinant expression in prokaryotic or eukaryotic cells, or produced in vitro. The envelope of an enveloped biological particle can comprise a lipid bilayer. The membrane of a membrane-bearing biological particle can comprise a lipid bilayer.

[0043] The enveloped biological particle can be an enveloped virus. Examples of enveloped viruses include DNA viruses such as herpesviruses, poxviruses, hepadnaviruses, and asfarviridae; RNA viruses such as flaviviruses, alphaviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and filoviruses; and retroviruses such as lentiviruses. The membrane-containing biological particle can be an extracellular vesicle such as an exosome. Additionally, virus-like particles (VLPs) are included in the enveloped or membrane-containing biological particles.

[0044] Enveloped or membrane-bearing biological particles may contain genetic cargo, e.g., for gene therapy. The genetic cargo may be one or more exogenous nucleic acid sequences contained along with the particle's natural nucleic acid content. In the case of enveloped viral particles, the viral genome may contain exogenous DNA or RNA inserts. Enveloped or membrane-bearing biological particles, such as viral particles containing exogenous gene inserts, can be recombinantly produced by known methods. The presence of large genetic inserts (exogenous DNA or RNA) can make the viral particle more sensitive and unstable.

[0045] The present invention provides a gentle, yet effective purification method that is useful for purifying sensitive enveloped or membrane-bearing biological particles. As demonstrated herein, the present invention is particularly useful for purifying enveloped retroviruses, such as lentiviruses, with very high infection recoveries.

[0046] FIG. 1 shows a schematic representation of a method 100 for purifying biological particles with an envelope or membrane, and FIG. 2 shows the AIEX chromatography principle used.

[0047] Enveloped or membrane-bearing biological particles 3 are present in feed 1 along with one or more impurities 2. For example, a feed containing enveloped viral particles, such as lentiviral particles, or exosomes can be produced by a cell line, such as HEK 293 cells (human embryonic kidney cells). The feed may be clarified or otherwise pretreated by known methods before being applied to the first chromatography medium. In the clarified harvest, the amount of solids is reduced, and the one or more impurities may be soluble impurities, such as host cell proteins (HCPs) and DNA. For example, the feed may be subjected to treatment with a nuclease to partially degrade contaminating polynucleotides and reduce their size. In the case of purification of RNA viruses, treatment with a deoxyribonuclease may be applied.

[0048] In a first step 101 of the method 100, a feed 1 is applied to a first chromatography medium 4 at a pH between 6 and 10 for capture of enveloped or membrane-bearing biological particles. For the purpose of capturing enveloped or membrane-bearing biological particles, the chromatography medium 4 comprises a support material that is functionalized with a ligand capable of reversibly binding to target enveloped or membrane-bearing biological particles.

[0049] The ligand may be an affinity ligand capable of binding to a biological particle with an associated envelope or membrane, or may be an anion exchange (AIEX) ligand, preferably a weak AIEX ligand.

[0050] The feed 1 is typically loaded onto the first chromatographic medium 4 using a loading buffer having a pH in the range of 6-10, e.g., 6-8. Prior to loading, the medium 4 may be equilibrated with the loading buffer or another buffer having a suitable pH of 6-10, e.g., 6-8. Under loading conditions, enveloped or membrane-bearing biological particles bind to the ligand, while at least some impurities 2, particularly host cell proteins, pass through the column and are washed. The feed may be loaded at a flow rate of 1 mL / min to 10 mL / min.

[0051] Loading of the feed and any additional washing is followed by step 102 of eluting the enveloped or membrane-bearing biological particles. Elution can be achieved by changing the ionic strength. By way of example, an elution buffer providing an increased concentration of salt can be used.

[0052] Salt is (i) CO3 2- , SO4 2- , S2O3 2- , H2PO4 - , HPO4 2- , acetate, citrate, and Cl - and (ii) an anion selected from the group consisting of NH + , K. + , Na+ , and Li + For example, the salt may include sodium acetate (NaOAc) or sodium chloride (NaCl). However, it should be understood that other salts consisting of a combination of anions such as those listed in (i) and cations such as those listed in (ii) may alternatively be used to elute capsids. Non-limiting examples of such other salts are LiCl, KCl, or other equivalent metal salts suitable for use in salt elution, as are well known in the art. Non-limiting examples of suitable concentrations of NaCl include from about 5 mM to about 1 M, e.g., about 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. Non-limiting examples of suitable concentrations of NaOAc include about 5 mM to about 500 mM, e.g., about 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.

[0053] 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. Such a relatively low salt concentration is beneficial because enveloped viruses and exosomes become less stable at higher conductivities, particularly at concentrations above 0.65 M. Optionally, the salt concentration during elution can be at most 450 mM.

[0054] Elution can be performed at a flow rate of 1-10 mL / min. The flow rate can be the same as during loading.

[0055] Typically, elution of enveloped or membrane-bearing biological particles from a first chromatographic medium having a support material that is a convection-based fibrous substrate, such as a fibrous nonwoven polymer matrix, can be carried out at flow rates down to residence times of a few seconds, i.e., 60 MV / min, or, if necessary, at a flow rate of 0.2 MV / min, up to residence times of several minutes (up to 6 minutes). The optimal residence time for such fibrous support materials is 5-20 MV / min. For resins, typical residence times are 1-8 minutes, with sufficient residence time often being achieved after 4 minutes.

[0056] Optionally, the elution buffer may contain arginine or another additive typically used for elution in affinity or anion exchange chromatography, provided that the additive does not interfere with the ability of the second chromatographic medium to bind impurities and allow enveloped or membrane-bearing biological particles to pass through.

[0057] Thus, at least one fraction of eluted enveloped or membrane-bearing biological particles is produced. Optionally, more than one fraction may be collected and optionally pooled before being subjected to the next step of the method.

[0058] In methods of purifying enveloped viruses, such as lentiviruses, the virus-containing elution fractions typically contain high levels of recovered infectious viral particles, e.g., an infectious recovery of at least 50%, e.g., at least 60%, at least 70%, or at least 80%, can be achieved after step 102.

[0059] In the elution step, direct dilution to reduce conductivity in a sucrose-containing buffer can stabilize the virus, thereby improving recovery.

[0060] The first chromatographic medium can be functionalized with a ligand to an ionic capacity (number of charged functional groups per ml of medium (µmol / ml)) within the range of 10-500 µmol / mL, or 50-300 µmol / mL, or 100-300 µmol / mL. The ligand can have a diamine functional group resulting in at least one weak anion exchange group, to an ionic capacity (number of charged functional groups per ml of medium (µmol / ml)) within the range of 10-500 µmol / mL, or 50-300 µmol / mL, or 100-300 µmol / mL. The weak anion exchange group can include a multimodal weak anion exchange group (i.e., the anion exchange group provides at least two distinct but cooperative sites for interaction with a compound to be bound (i.e., a biological particle with an envelope or membrane)). For example, one of these sites can provide an attractive type of charge-charge interaction between the ligand and the binding target. Other sites may contribute to binding by introducing a second local charge or increasing the local amount of water of hydration, affecting binding capacity.

[0061] The weak anion exchange group may be positively or partially positively charged at a pH of 6 to 10. Such positively or partially positively charged weak anion exchange groups may attract biological particles with envelopes or membranes that are negatively charged at neutral pH, such as lentiviral particles. The weak anion exchange group may be positively or partially positively charged at a pH of 6 to 10, or 6-9.5, 6-9, or 6-8.

[0062] Weak anion exchange groups mean that they have a pH gradient from fully charged to uncharged and have a neutral charge (equal amounts of + and -) in the PI. Strong anion exchange groups based on quaternary amines, on the other hand, always have a charge. Almost all other anion exchange groups not based on quaternary amines are weak, i.e., the charge varies (and can be zero) within a reasonable range of pH (e.g., pH 2-11) that can be used.

[0063] The ligand or a portion of the ligand has the following formula (I):

[0064] [ka]

[0065] wherein X is, independently at each occurrence, H, OH, or C 1~3 groups, and R1, R2, R3, and R4 are selected from H and C 1~3 are independently selected from the group The C3 group is linear or branched; C 1~3 The groups are OH, OC 1~2 , SC 1~2 , NH, NHR, NR2, R is H and C 1~3 The group may be selected from the group

[0066] The ligand or a portion of the ligand can be described by formula (I) above. This means that the diamine ligand can be a component part of a larger structure, such as a polymer. The ligand or a portion of the ligand can be part of a larger structure produced, for example, by reaction of a solid support with a lower molecular weight amine chemical containing a free radical, 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-methylpropan-1-amine, etc. In the ligand or portion of the ligand containing a diamine functional group that produces at least one weak anion exchange group as described by the formula above, the two amines may be separated by 2 to 4 carbon atoms, and each amine group may be substituted with two R groups, which may be selected from H and C1-4 alkyl groups, and the C1-4 alkyl groups may be branched and / or substituted with other groups such as hydroxyl, amine, ether, and thioether, the latter being limited to 3 to 8 atoms.

[0067] 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-propanediamine, and N,N-diethylethylenediamine.

[0068] 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.

[0069] Beads of different sizes may be used, for example, beads having diameters 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 be of uniform porosity.

[0070] A monolith is a single piece of porous material characterized by a highly interconnected network of channels with diameters in the range of 10 to 4000 nm.

[0071] Membrane materials can be inorganic-organic (eg, alkoxysilane coated on glass fiber), alumina membranes, and organic materials (ie, cellulose and its derivatives, regenerated cellulose, nylon, polyethersulfone, polypropylene, polyvinylidene, etc.).

[0072] The support material can be a woven material. Alternatively, the support material can be a nonwoven material containing fibers, e.g., made from cellulose, having a mean flow pore size of, e.g., 0.1 to 2.0 μm. Mean flow pore (MFP) size is a measure of a material's flow characteristics and is measured by capillary flow porometry based on the displacement of a wetting liquid with known surface tension from sample pores by applying gas at increasing pressures. The larger the MFP size, the greater the liquid flow rate through the material at a given pressure. The mean flow pore size is calculated from the point at which 50% of the flow rate passes through the sample. Thus, the mean flow pore size corresponds to the pore size calculated at the pressure where the wetting curve and the semi-dry curve intersect. In an alternative definition, the mean flow pore size of a support material can be viewed as its effective pore size, defined as the size of the largest sphere that can pass through the pore.

[0073] The average flow pore size of the support material can be 0.1 to 1.8 μm, 0.1 to 1.6 μm, 0.1 to 1.4 μm, 0.1 to 1.2 μm, 0.1 to 1.0 μm, 0.1 to 0.8 μm, 0.1 to 0.6 μm, 0.1 to 0.4 μm, 0.1 to 0.2 μm, 0.2 to 2.0 μm, 0.4 to 2.0 μm, 0.6 to 2.0 μm, 0.8 to 2.0 μm, 1.0 to 2.0 μm, 1.2 to 2.0 μm, 1.4 to 2.0 μm, 1.6 to 2.0 μm, 1.8 to 2.0 μm, or 0.5 to 1.5 μm.

[0074] The chromatography support material can include a convection-based chromatography matrix. The convection-based chromatography matrix can be a fibrous substrate. The fibrous substrate can be based on electrospun polymer fibers or cellulose fibers, optionally nonwoven fibers. Thus, the fibrous substrate can be a fibrous nonwoven polymer matrix. The fibers contained in the fibrous substrate have a cross-sectional diameter of 10 to 1000 nm, e.g., 200 to 800 nm, 200 to 400 nm, or 300 to 400 nm. Such a fibrous substrate can be found in a HiTrap Fibro unit manufactured by Cytiva, Sweden. Typically, elution of enveloped or membrane-bearing biological particles from a chromatography medium having a support material that is a convection-based fibrous substrate, such as a fibrous nonwoven polymer matrix, can be carried out at flow rates down to a residence time of a few seconds, i.e., 60 MV / min, or, if necessary, at a flow rate of 0.2 MV / min, up to a residence time of several minutes (up to 6 minutes). The optimal residence time for such a fibrous support material is 5 to 20 MV / min. For resins, typical residence times are 1 to 8 minutes, with sufficient residence time often being obtained after 4 minutes.

[0075] The ligand of the first chromatography medium 4 may be connected to the support material through an extender group, which may be selected from polysaccharide and polymer structures. The extender group may be, for example, dextran, acrylamide, or polyglycidol. When dextran is used as the extender, it may have a molecular weight in the range of 5000 Daltons to 2 M Daltons. The extender group, if present, may be selected in relation to the support material and the type of ligand to be immobilized / connected to the support material.

[0076] The elution fraction obtained in step 102 containing enveloped or membrane-bearing biological particles may have had a significant amount of impurities removed during capture chromatography, but typically still contains one or more types of impurities. An example of an impurity that can bind and elute with enveloped or membrane-bearing biological particles includes host cell DNA (hcDNA). For example, the eluate may contain at least 50% of the hcDNA present in the feed. DNA is negatively charged and binds strongly to anion exchange groups, and longer DNA sequences may bind more strongly than shorter DNA fragments. It has previously been found that the higher the elution conductivity for viral particles, the greater the risk of co-elution of viral particles with DNA. Therefore, a lower elution conductivity may be preferred to reduce hcDNA in the target elution fraction.

[0077] The elution fraction obtained in step 102, which contains the enveloped or membrane-bearing biological particles and typically also some remaining impurities such as host cell DNA (preferably fragmented by prior nuclease treatment), is applied to a second chromatographic medium comprising porous beads with a core-shell structure, the core of which is functionalized with hydrophobic interaction ligands. In Figure 1, this is represented by step 104. The impurities are retained by the porous beads, and the enveloped or membrane-bearing biological particles are obtained in the flow-through 105.

[0078] Chromatography step 104 is performed at a pH within the range of 6.0 to less than 7.4, e.g., 6.5 to 7.2, such as 6.5-7.1 or 6.8 to 7.1. Thus, if the elution fraction obtained in step 102 has a pH outside the desired range, e.g., a pH above 7.4, the pH is suitably adjusted in step 103 prior to addition to the second chromatography medium. pH adjustment step 103 can be achieved by diluting the elution fraction with a buffer having a lower pH (if a decrease is desired) until the desired pH is achieved in the diluted elution fraction. Of course, if a pH increase is desired, e.g., from a value below 7.0, a dilution buffer having a higher pH than the elution fraction can be used. The buffer used for dilution can be the same buffer used as the loading buffer in step 104. As shown in Figure 6, comparative examples for this step using pH values ​​of 7.4 and 8, respectively, resulted in low infectious recovery of lentiviral particles, while performing step 104 at a pH of 7.0 resulted in the most significant increase in infectious recovery. It is believed that satisfactory recovery occurs slightly above 7.0. Without intending to be bound by any particular theory, it is believed that enveloped or membraned biological particles may be slightly more tolerant to pH values ​​below 7.0 compared to values ​​above 7.0.

[0079] For clarity, if the pH of the eluted fraction is within the range of 6.0 to less than 7.4, then step 103 of adjusting the pH is optional. However, as noted above, a pH higher than 6.0 may still be preferred, such as at least 6.5 or at least 6.8. Thus, if the pH is at least 6.0 but lower than the preferred value, then the pH may suitably be adjusted in step 103. Similarly, if the pH is lower than 7.4 but higher than the preferred value, then the pH may suitably be adjusted by step 103.

[0080] Figure 3 shows a schematic of the chromatography beads 5 of the second chromatography medium of step 104. The beads 5 are porous beads comprising a porous core 52 surrounded by a porous shell 51. The porosity of the core and shell may be the same or different. However, the porosity of at least the shell 51 prevents larger entities, such as biological particles with envelopes or membranes (represented by "LV" in Figure 3), from penetrating the shell and accessing the core.

[0081] The shell 51 is typically hydrophilic. Thus, the surface of the porous bead 5 that is accessible to larger entities, such as biological particles with envelopes or membranes, is hydrophilic and does not irreversibly adsorb or denature proteins. The shell may be formed of a hydrophilic material that exposes a plurality of polar groups, including, for example, oxygen and / or nitrogen atoms. Examples of such polar groups include hydroxyl, amino, carboxy, sulfonic acid (S and SP ligands), esters, ethers of lower alkyls (e.g., (-CH2CHO-) n H, where n is an integer 2, 3, 4, and higher.

[0082] 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 is itself hydrophilic and is based on a hydrophilic material, such as a hydrophilic polymer, and functionalized with hydrophobic interaction ligands to impart the desired hydrophobicity. However, alternatively, the core can be based on a hydrophobic polymer and is itself hydrophobic. For example, styrene / ethylstyrene / DVB, vinyl ethers, and acrylates containing hydrophobic substituents, as well as fluoroalkane-containing polymers, are contemplated.

[0083] The hydrophobic interaction ligands may comprise aliphatic hydrocarbons such as C1-C30 alkyl, preferably C4-C16 alkyl, and / or aromatic hydrocarbons such as phenyl, anthracene, naphthalene, etc. Preferably, the ligand density may be greater than 90 μmol / ml of core entity, representing a higher ligand density than is typically used for HIC resins. Advantageously, the hydrophobicity provided by the ligand may be such that the protein can interact or adsorb at both very low and high ionic strength.

[0084] The hydrophilic polymer on which the shell and, optionally, the core may be based is a polysaccharide such as agarose. For example, both the core and shell may comprise cross-linked agarose. Porous core-shell beads may be prepared as described in WO 2009 / 131526. In particular, the core and shell may be made of agarose, and the core may be functionalized with hydrocarbon interaction ligands containing 4 to 16 carbons, preferably octyl ligands. Useful chromatographic media are available under the trade names Capto™ Core 400 and Capto™ Core 700, respectively, available from Cytiva, Sweden.

[0085] The shell covers or surrounds the core. Due to the limited porosity of the shell 51, only entities, such as molecules, small enough to penetrate the shell will be able to interact with the hydrophobic core. Thus, the porous beads 5 have size-exclusion properties. In this context, impurities, such as remaining host cell proteins and DNA (preferably fragmented DNA), can enter and bind to the porous beads, while biological particles bearing the target envelope or membrane cannot and are obtained in the flow-through fraction.

[0086] The hydrophobic core is capable of strongly binding any impurities that permeate into the beads 5. Therefore, under the present treatment conditions, the impurities are not released from the core but remain bound to it.

[0087] The porosity of the shell may be defined as preventing particles having a size (diameter) of 20 nm from entering the bead, such that particles of this size or larger are not retained within the chromatographic medium but are allowed to pass through it, while smaller particles can enter the bead and be adsorbed or bound by the hydrophobic core. For example, the shell may have a porosity small enough to prevent particles having a size of at least 30 nm, e.g., 80 nm or larger.

[0088] Enveloped viruses generally range in size from 20 nm to 300 nm, depending on the virus type. Lentiviruses can range in size from 80 to 120 nm, often from 100 to 120 nm. Enveloped viruses may be larger than non-enveloped viruses, such as adenoviruses, which are packaged only in capsids. Enveloped viruses are often larger than adeno-associated viruses, which are typically about 25 nm in size. Extracellular vesicles, such as exosomes, can range in size from 30 nm to 180 nm.

[0089] In an alternative definition, the shell porosity may be described as excluding molecules with a molecular weight of 400 kDa or greater, while molecules with a molecular weight less than 400 kDa can enter the beads and interact with the hydrophobic core. In embodiments, the shell porosity may exclude molecules with a molecular weight of 700 kDa or greater, or 1,000 kDa or greater, or 2,000 kDa or greater. In contrast, molecules with molecular weights below the respective limits can enter the beads and interact with the hydrophobic core. A lower cutoff value, such as 400 kDa, may be useful for purifying relatively small particles, such as those having a size in the 20-100 nm range. A higher cutoff value, such as 1,000 kDa or 2,000 kDa, may be used when the target particles to be purified are large, e.g., 150-300 nm.

[0090] Shell 51 can be inert or unfunctionalized, e.g., the shell is not functionalized with the HIC ligands of the core or any other HIC ligands. The porous shell can be hydrophilic.

[0091] 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, e.g., 0.3 mL / min to 1.4 mL / min, for a 1 mL column. The flow rate for step 104 may be lower than the flow rate used in the previous steps 101 and / or 102.

[0092] The residence time of the sample (the optionally diluted eluate obtained in step 102) in the second chromatography medium in step 104 can be from 0.5 to 10 minutes, e.g., from 0.5 to 3.5 minutes. For example, the retention time can be 1 minute or less, e.g., from 0.5 to 0.7 minutes. A longer residence time (slower flow rate) can provide higher purity because impurities will have more time to penetrate into the porous beads and interact with the core 52. A shorter retention time (faster flow rate) can favor process efficiency and economics. Also, for sensitive biological particles such as enveloped viruses, a short process time can be preferable to minimize exposure to conditions (e.g., room temperature) that can negatively affect virus stability.

[0093] Advantageously, the methods described herein can be performed on large sample volumes relative to the volume of the chromatography medium. Thus, a relatively large feed volume can be used relative to the volume of the first chromatography medium, which can have a high dynamic binding capacity, particularly if the support material is a fibrous material. Furthermore, a relatively large volume of sample (the eluate obtained from the capture chromatography step, optionally diluted to adjust the pH as described above) can be applied to the second chromatography medium containing porous beads 5 in step 104. For example, the applied sample volume can be expressed as 15-30 column volumes (CV), e.g., 25-30 CV, where the column volume is the volume of the chromatography medium containing porous beads 5.

[0094] As demonstrated herein, performing step d at a pH below 7.4 can provide a significant increase in recovered infectious viral particles compared to a pH of 7.4. For example, for enveloped viral particles, the present method 100 can enable an infectious recovery of at least 30%, e.g., at least 50%, relative to the infectious viral particle content in the raw feed. This represents a substantial improvement over currently available methods, which typically result in an infectious recovery of 10-20%.

[0095] Pre-treatment, such as clarification, of the cell culture harvest to provide the clarified feed used in step 101 may result in a small loss of infectious virus. Typically, the infection recovery rate after clarification may be at least 80%. Using a feed that is clarified prior to step 101, the method 100 may result in an infection recovery rate of at least 60%, such as at least 50%, e.g., 70%.

[0096] Viewed separately, the individual chromatography steps, the initial capture (steps 101 and 102) may provide an infectious recovery of enveloped viral particles of at least 60%, e.g., at least 70%, relative to the feed. The polishing step (steps 104 and 105) using a porous shell / bead resin may itself, independently, result in an infectious recovery of at least 70% and up to 100% relative to the infectious viral particle content provided by the preceding step.

[0097] In general, the addition of stabilizers such as sucrose can be beneficial to the stability of biological particles with envelopes or membranes. Stabilizers can be added to all mobile phases and formulation solutions used in the method.

[0098] While the present invention is described herein with reference to exemplary embodiments, those skilled in the art will recognize that the invention is not limited thereto. In the claims, any reference signs placed in parentheses shall not be construed as limiting the scope of the claims.

[0099] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. 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. [Example]

[0100] Determination of ionic capacity The dynamic ion binding capacity (DBC) of chromatographic materials functionalized with AIEX ligands such as diethylethanolamine (DEAE), quaternary amines (Q), or N,N-diethylethylenediamine (DAX) is described below with reference to a nanofibrous chromatographic material (Fibro™ membrane absorber, Cytiva, Sweden) and should be generally applicable to any AIEX ligand. The method uses a conductometric titration in which added HCl protonates or neutralizes deprotonated weak AIEX ligands and displaces OH- bound to strong AIEX ligands. In contrast to the protein DBC method, the conductivity signal of the permeate, rather than the UV signal, is monitored in the AKTA system.

[0101] A 25 mm diameter membrane disk was attached to a membrane holder device suitable to allow chromatography of the membrane and run on an AKTA Explorer 10 system (Cytiva, Sweden) equipped with a sample pump. The method consisted of the following steps: 1. Rinse the AKTA system and membrane (loaded into the PEEK device) 2. Loading the membrane with excess NaOH 3. Rinse the membrane with MQ water 4. Rinse the HCl solution in the bypass 5. Loading the membrane with HCl and monitoring conductivity breakthrough 6. Reload the membrane with excess NaOH (optional, prepare for normalization by mass) 7. Rinse the membrane with MQ water

[0102] Each membrane batch is analyzed on triplicate discs, normalized by disc volume and, optionally, by disc dry mass.

[0103] Determination of mean flow pore size The mean flow pore size of porous materials can be measured by capillary flow analysis using commercially available equipment. In this example, the equipment used was a POROLUX™ 100 Porometer (IB-FT GmbH, Berlin, Germany), following the manufacturer's manual and the methodology shown in Table 1.

[0104] [Table 1]

[0105] Experimental Example Lentiviral production and clarification Lentiviruses carrying green fluorescent protein (GFP) inserts were produced in HEK293T suspension cell culture media following transfection with four plasmids by standard methods. At harvest, the feed was treated with 10 U / mL nuclease (denarase) containing 2 mM MgCl2 and cells were precipitated before clarification using 10 μm, 5 μm, 0.5 μm, and 0.2 μm filters. The clarified feed lentivirus titers were ≥ E9 VP / mL and ≥ E6 TU / mL.

[0106] Capture of lentiviruses using weak AIEX chromatography The clarified feed was applied to a 5 ml HiTrap™ Capto™ DEAE chromatography column (Cytiva, Sweden) connected to an AKTA™ Pure 25 (Cytiva, Sweden) equilibrated in 50 mM Tris-HCl, pH 8.0 (Buffer A). The protocol for eluting lentivirus using a short, linear NaCl gradient ranging from 130 to 650 mM can be found in Table 2. "CV" indicates column volume. Immediately after elution, the eluate was diluted 1:5 with 5% sucrose to stabilize the virus and preserve infectivity.

[0107] The flow rate was 5 ml / min (1 min residence time) for all steps. All runs were performed in duplicate.

[0108] [Table 2]

[0109] Impurity removal using layered bead resin with HIC ligands at different pH and flow rates The eluate from the weak AIEX capture step was applied to a 1 mL HiTrap™ Capto™ Core 700 column (Cytiva, Sweden) connected to an AKTA™ Pure 25 (Cytiva, Sweden) equilibrated in 50 mM Tris-HCl at pH 8.0, 7.4, or 7.0, respectively. The lentivirus load was approximately E11 VP in 50 mM Tris-HCl, 4% sucrose, and 130 mM NaCl at pH 7.0, 7.4, or 8.0. For pH 7.0, the estimated flow rates were 0.3 mL / min, 0.9 mL / min, 1.4 mL / min, and 1.9 mL / min (corresponding to residence times of 3.3 min, 1.1 min, 0.7 min, and 0.5 min). All runs were performed in duplicate.

[0110] 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 (microBCA kit, μg / mL), and total DNA (picoGreen assay, ng / mL).

[0111] result A capture chromatogram using weak anion exchange with a short NaCl gradient elution is shown in Figure 4. Capture with Capto™ DEAE resulted in >60% infectious lentivirus recovery in the pooled gradient (Figure 5). The gradient was fractionated into five fractions, with the majority of lentivirus eluting in elution fractions 2 and 3. The majority of host cell proteins (HCPs) (98% of total protein) were obtained in the flow-through and wash, while approximately 50% of DNA co-eluted with the lentivirus (Figure 5).

[0112] The Capto™ DEAE eluate was diluted with buffers of pH 7.0, 7.4, or 8 and applied to a Capto™ Core 700 column as described above. Flow-through fractions were collected and analyzed. It was found that the buffer at pH 7.0 resulted in an 80% infection recovery, a highly significant improvement over the approximately 35% recovery obtained with buffers of pH 8.0 or 7.4 (Figure 6). All pH buffers resulted in similar p24 recovery levels. Impurity levels were below the detection limit for both HCP and DNA (BCA, picogreen) for all pHs (data not shown).

[0113] Finally, evaluation of different flow rates as described above showed that flow rates up to 1.4 mL / min and residence times of ≥ 0.7 min could be used with excellent lentiviral infection recoveries (nearly 100%). Also, 1.9 mL / min or a residence time of 0.5 min could be used at pH 7.0 with infection recoveries that were significantly improved over prior art methods (70%). Impurity levels were below the detection limit for both HCP and DNA (BCA, picogreen) at all flow rates.

[0114] (References) [Explanation of symbols]

[0115] 1 Feed 2. Impurities 3 Biological particles with envelopes or membranes 4. First Chromatography Media 5 beads 51 Shell 52 cores 100 ways 101 Feed addition process 102 Eluting the enveloped or membrane-bearing biological particles from the first chromatographic medium 103 adjusting the pH of the eluted fraction of step 102 to a pH less than 7.4 104 applying the eluted fraction to a second chromatography medium 105. Obtaining at least one flow-through fraction containing purified enveloped or membrane-bearing biological particles.

Claims

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

2. 2. The method of claim 1, wherein the enveloped or membrane-bearing biological particle is selected from an enveloped viral particle and an exosome.

3. 3. The method of claim 1, wherein the ligand for capturing the enveloped or membrane-bearing biological particle is selected from affinity ligands and anion exchange ligands.

4. 3. The method of claim 2, wherein the support material of the first chromatographic medium (4) is functionalized with an anion exchange ligand containing a diamine functional group that yields at least one weak anion exchange group to an ionic capacity of 10 to 500 μmol / mL.

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

6. The ligand is of formula (I): 【Chemistry 1】 wherein X is, independently at each occurrence, H, OH, or C １～３ is selected from the group R １ , R ２ , R ３ , and R ４ is H and C １～３ are independently selected from the group C ３ The group is linear or branched, C １～３ The groups are OH, O—C １～２ , S.-C. １～２ , NH, NHR, NR ２ and R is H and C １～３ 6. The method of claim 4 or 5, wherein the compound is selected from the group

7. 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-propanediamine, and N,N-diethylethylenediamine, preferably N,N-diethylethylenediamine.

8. 8. The method of any one of claims 1 to 7, 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. 9. The method according to any one of claims 1 to 8, wherein the support material of the first chromatographic medium (4) is a non-woven fibrous material having an average flow pore size of 0.1 to 2.0 μm.

10. 10. The method of claim 1, wherein the ligand for capturing the enveloped or membrane-bearing biological particle is connected to the support material through an extender group selected from polysaccharide and polymer structures.

11. 11. The method of any one of claims 1 to 10, wherein the eluting in step b) is effected with increasing salt concentrations.

12. 12. The method of any one of claims 1 to 11, wherein the eluting in step b) is effected by contacting the first chromatographic medium with an elution buffer having a salt concentration of at most 0.65 M.

13. 13. The method of any one of claims 1 to 12, wherein the pore size of the shell (51) prevents molecules larger than 700 kDa from accessing the core.

14. 14. The method of claim 13, wherein the pore size of the shell (51) prevents molecules larger than 400 kDa from accessing the core.

15. 15. The method according to any one of claims 1 to 14, wherein the pore size of the shell (51) prevents particles having a size of 20 nm or more, such as 30 nm or more, for example 60 nm or more, for example 100 nm or more, from contacting the core.

16. 16. The method according to any one of claims 1 to 15, wherein the porous beads (5) comprise a hydrophilic polymer, for example a polysaccharide such as agarose.

17. 17. The method according to any one of claims 1 to 16, wherein the porous core (52) is functionalized with hydrophobic interaction ligands, preferably the hydrophobic interaction ligands comprise aliphatic or aromatic C4 to C16 hydrocarbons, for example aliphatic C4 to C16 hydrocarbons.

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

19. 19. The method according to any one of claims 1 to 18, wherein the elution fraction is applied to the chromatography medium in step d) at a pH of from 6.5 to 7.2, such as from 6.5 to 7.1 or from 6.8 to 7.

1.

20. 20. The method of any one of claims 1 to 19, wherein the residence time of the eluted fraction on the chromatography medium in step d) is between 0.5 and 10 minutes.

21. 21. The method of any one of claims 1 to 20, wherein the eluted fraction is applied to the chromatography medium in step d) at a flow rate of from 0.1 mL / min to 3 mL / min.

22. 22. The method according to any one of claims 1 to 21, wherein the feed (1) has been subjected to a nuclease treatment prior to step a).

23. 23. The method of any one of claims 1 to 22, wherein at least one flow-through fraction containing purified enveloped or membrane-bearing biological particles obtained in step e) does not contain detectable DNA.

24. 1. Use of a chromatography medium comprising porous beads (5) having an inner porous core (52) and an outer porous shell (51) for the chromatographic separation of enveloped or membrane-bearing biological particles from impurities such as contaminating DNA at a pH below 7.4, wherein the core is capable of binding molecules via hydrophobic interactions and the pore size of the shell prevents particles having a size of 20 nm or larger from penetrating into the beads and interacting with the core.

25. 25. Use according to claim 24, wherein the pore size of the shell (51) is such that molecules with a molecular weight of 400 kDa or more are prevented from penetrating into the beads and interacting with the ligands of the core.

26. 26. Use according to claim 24 or 25, wherein the pore size of the shell (51) prevents particles having a size of 20 nm or more, such as 30 nm or more, for example 60 nm or more, for example 100 nm or more, from penetrating into the bead and interacting with the ligands of the core.

27. 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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