EP4694993A1

EP4694993A1 - A chromatography system, use thereof, and a method for separating enveloped or membranous biological particles

Info

Publication number: EP4694993A1
Application number: EP24716812.3A
Authority: EP
Inventors: Jean-Luc Maloisel; Åsa HAGNER MCWHIRTER; Ola Lind; Brigitta NÉMETH
Original assignee: Cytiva Bioprocess R&D AB
Current assignee: Cytiva Bioprocess R&D AB
Priority date: 2023-04-12
Filing date: 2024-04-08
Publication date: 2026-02-18
Also published as: WO2024213496A1; CN120936418A; KR20250172638A; JP2026512897A

Abstract

The present disclosure is directed to a chromatography system comprising a buffer valve arrangement configured to allow independent control of a first buffer feed and a second buffer feed; a pump arrangement configured to supply a first buffer feed, a second buffer feed, and a feed comprising a biological target compound and one or more impurities; a selection valve arrangement, comprising a first chromatography device selection valve; a first chromatography device, comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for a biological target compound; a second chromatography device, comprising a second chromatography material, which comprises a conditioning chromatography material; wherein the selection valve arrangement is configured to enable separation of a biological target compound from impurities by allowing a feed, comprising a biological target compound and one or more impurities, to continuously pass through the first chromatography device and the second chromatography device, wherein the first chromatography device and the second chromatography device are configured to be connected in series. The disclosure also provides use of the chromatography system and a method for separation of enveloped or membranous biological particles from one or more impurities.

Description

A CHROMATOGRAPHY SYSTEM, USE THEREOF, AND A METHOD FOR SEPARATING ENVELOPED OR MEMBRANOUS BIOLOGICAL PARTICLES

FIELD OF DISCLOSURE

The present disclosure relates to the field of separation of biological target compounds, such as enveloped virus particles. The disclosure is directed to a chromatography system and use thereof for separating enveloped or membranous biological particles from impurities, as well as a method for separating enveloped or membranous biological particles from impurities.

BACKGROUND OF THE DISCLOSURE

Liquid chromatography is a separation technique that is used to separate and analyse complex liquid mixtures of compounds. The process involves a stationary phase and a liquid mobile phase, where the liquid mixture to be separated is introduced to the mobile phase and passes through the stationary phase. The different components of the mixture will interact with the stationary phase to varying degrees, causing them to separate and be collected at different times. There are various types of liquid chromatography techniques that differ based on the stationary and mobile phases used, for example ion exchange chromatography, size-exclusion chromatography, and affinity chromatography. Different types of chromatography require different ways of sample preparation, different process conditions and buffers.

Various biological target compounds, such as antibodies and viral vectors, are produced by fermentation of host cells. The biological target compounds must then be separated from host cell material and other impurities contained in the fermentation broth before they can be used for example in medical and analytical applications. A combination of several steps of different types of liquid chromatography is often applied with the object of achieving a high recovery of the biological target molecule at a high level of purity. However, processes for separation of biological target compounds often require manually switching between different chromatography devices and sample preparation between chromatography steps to condition the sample for the next step, which altogether makes the processes time-consuming.

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

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

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

However, current downstream purification processes of lentivirus are often synonymous with low recovery (typically 10-20 %) of infectious viruses, since lentiviruses are unstable and sensitive to shear forces, buffer components such as salt, and degrade quickly in room temperature. Timeconsuming multistep processes are also not regarded as beneficial. Lentiviruses have been reported to be stable in a very narrow pH range, 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 NaCI (Process development of lentiviral vector expression, purification and formulation for gene therapy applications, Doctoral thesis, Sara Nilsson, UCL, 2016), of processing solutions, which makes the downstream purification process challenging.

Hence, there is a continuous need in the art for novel chromatography systems and purification processes providing higher capacities and faster processing, leading to a generally better process economy.

SUMMARY OF THE INVENTION

One object of the present disclosure is to provide a chromatography system enabling a faster and simplified separation of biological target compounds from impurities, with similar performance as previously known chromatography systems. The chromatography system is constructed such that a feed, comprising biological target compounds and impurities, can be subjected to several purification steps uninterruptedly thanks to being continuously passed through the system. Several chromatography devices are connected in-line, within the chromatography system. Thereby, a significantly reduced time is achieved compared to when using conventional chromatography systems. Traditionally, purification process flows are typically interrupted and prolonged for example due to manually changing of buffers and/or chromatography devices within the system, and since some steps are performed in separate containers or devices, outside of the chromatography system, e.g., conditioning of chromatography media and/or performing of certain filtration/purification steps. With the presently disclosed chromatography system, less human intervention is required compared to when operating previously known chromatography systems.

More particularly, the present disclosure is directed to a chromatography system comprising: a buffer valve arrangement configured to allow independent control of a first buffer feed and a second buffer feed; a pump arrangement configured to supply a first buffer feed, a second buffer feed, and a feed comprising a biological target compound and one or more impurities; a selection valve arrangement, comprising a first chromatography device selection valve; a first chromatography device, comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for a biological target compound; a second chromatography device, comprising a second chromatography material, which comprises a conditioning chromatography material; wherein the selection valve arrangement is configured to enable separation of a biological target compound from impurities by allowing a feed, comprising a biological target compound and one or more impurities, to continuously pass through the first chromatography device and the second chromatography device, wherein the first chromatography device and the second chromatography device are configured to be connected in series.

The present disclosure also provides use of the herein disclosed chromatography system for separation of a biological target compound from one or more impurities, wherein the biological target compound is selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles, optionally wherein the extracellular vesicles are exosomes.

Additionally, the present disclosure is directed to a method for separating enveloped or membranous biological particles from one or more impurities, said method comprising: a. Adding a feed comprising enveloped or membranous biological particles, and one or more impurities, to a first chromatography device, comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for said enveloped or membranous biological particles; b. Eluting the enveloped or membranous biological particles from the first chromatography device in at least one eluate fraction; c. Adding the at least one eluate fraction comprising enveloped or membranous biological particles, obtained in step b, to a second chromatography device, comprising a second chromatography material comprising a conditioning chromatography material; d. Obtaining the enveloped or membranous biological particles, in at least one flow-through fraction from the second chromatography device; wherein the feed is continuously passed through the first chromatography device and the second chromatography device to enable separation, wherein the first chromatography device and the second chromatography device are connected in series.

Preferred aspects of the present disclosure are described below in the detailed description and in the dependent claims. It is noted that the present disclosure relates to all possible combinations of features recited in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 1 A-B are schematic outlines of a chromatography system according to embodiments of the present disclosure.

Fig. 2 is a schematic outline of a chromatography system according to a further embodiment.

Fig. 3 is a flow chart outlining the steps of a method for separating lentivirus particles from impurities according to the present disclosure.

Fig. 4 shows chromatograms for separation of lentivirus particles on different chromatography system set-ups, as described in Example 1 herein.

As illustrated in the figures, some features may be exaggerated for illustrative purposes and, thus, are provided to illustrate the general structures of embodiments of the present disclosure. DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure solves or at least mitigates the problems associated with existing chromatography systems and processes for separation of biological target compounds from impurities by providing, as illustrated in Fig. 1A, a chromatography system 10 comprising: a buffer valve arrangement 20 configured to allow independent control of a first buffer feed and a second buffer feed; a pump arrangement 40 configured to supply a first buffer feed, a second buffer feed, and a feed comprising a biological target compound and one or more impurities; a selection valve arrangement 60, comprising a first chromatography device selection valve 62; a first chromatography device 70, comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for a biological target compound; a second chromatography device 72, comprising a second chromatography material, which comprises a conditioning chromatography material; wherein the selection valve arrangement is configured to enable separation of a biological target compound from impurities by allowing a feed, comprising a biological target compound and one or more impurities, to continuously pass through the first chromatography device and the second chromatography device, wherein the first chromatography device and the second chromatography device are configured to be connected in series.

The buffer valve arrangement 20 has at least one inlet (indicated by an arrow in Fig. 1) and a corresponding outlet for buffer feed, normally two or more inlets and corresponding outlets, for independent control of two or more buffer feeds.

The pump arrangement 40 includes at least one pump, optionally two or more pumps.

Fig. IB illustrates an embodiment, in which the chromatography system 10 in addition to the components shown in Fig. 1A further comprises a third chromatography device 74 comprising a third chromatography material 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 wherein the pore size of the shell prevents particles having a size of >20 nm from contacting the core, wherein the third chromatography device 74 is configured to be connected in series with said first chromatography device 70 and said second chromatography device 72, and wherein the selection valve arrangement 60 is configured to enable separation by allowing the feed to continuously pass through said first, second and third chromatography devices. The selection valve arrangement 60 makes it possible to connect two or three chromatography devices in series and thereby to perform a two-step or three-step chromatography purification process in-line in the chromatography system. Interruptions and delays, traditionally encountered due to manual handling of various steps during the process, can hereby be avoided, which saves time and work efforts by users operating the equipment. The chromatography system may be completely set up and programmed before starting the purification process, after which the process may be started by one push of a button and allowed to continue to the end without further involvement by the user. The present disclosure may thus be said to provide a so-called "plug and play" chromatography system, meaning a system intended to work perfectly when first used or connected, without reconfiguration or adjustment by the user.

More particularly, the presently disclosed chromatography system 10 provides the following general advantages compared to a traditional chromatography system:

- Connected desalting and pH adjustment step after affinity capture. This potentially saves time since no dilution is needed to reduce the conductivity and adjust the pH of the sample before polishing, which leads to lower volume of sample feed, and thereby a shorter loading time. Further, it gives full control over the sample conductivity to allow high performance separation of virus particles such as lentivirus;

- No hold times;

- No freeze-thaw cycles;

- No sample conditioning (pH and conductivity) or sample handling between chromatography steps.

For purification of lentivirus vectors, an additional advantage is that the lentivirus vectors, which are sensitive to high salt concentration, are exposed to high salt conditions for shorter time (minutes vs hours). Direct buffer exchange into a suitable pH with a stabilizer (such as sucrose) is advantageous for improving the infectious yield. However, it is still surprising that the lentivirus particles can withstand such an in-line connected two-step or three-step purification process, considering that lentivirus is unstable and sensitive to shear forces, buffer components such as salt, and can degrade easily in room temperature.

The chromatography system 10 may be applied with various formats of chromatography devices and chromatography materials as described in more detailed elsewhere herein. Non-limiting examples of chromatography materials suitable for in-line connected purification of lentivirus vectors are combinations of membrane adsorbers, convection-based membranous structures comprising nanofibres, monoliths, and resins (beads). The herein disclosed chromatography system provides a significant time reduction (up to 70%) and simplified lentivirus purification process while achieving similar performance as a conventional purification set-up in terms of recovery of lentivirus. It is surprising that a full capture and polishing process using such an in-line connected two-step or three-step purification process can be successfully implemented considering the combined chromatography challenge to keep the lentivirus stable with optimal conditions without manual intervention and maintaining high chromatographic performance.

Fig. 2 illustrates a currently preferred, non-limiting embodiment of the chromatography system 10, in which the buffer valve arrangement consists of a first buffer selection valve 22, and a second buffer selection valve 24.

The first buffer selection valve 22 may be configured to control a first buffer feed, herein alternatively called buffer A, which is used when equilibrating a chromatography device before loading a sample feed comprising biological target compounds onto the chromatography device, and the second buffer selection valve 24 may be configured to control a second buffer feed, herein alternatively called buffer B, which is used when eluting the biological target compounds from the chromatography system 10.

More particularly, the first buffer selection valve 22 may be configured to control a feed of a first buffer 30 for equilibration of the first chromatography device 70, herein alternatively called buffer Al, and the first buffer selection valve 22 may further be configured to control a feed of a first buffer 32 for equilibration of the second chromatography device 72, herein alternatively called buffer A2. Buffer 32 may also be used for equilibration of the third chromatography device 74. In cases where the biological target compounds are not binding to the second chromatography device but instead exiting the second chromatography device in flow-through, buffer 32 may also be used for passing the biological target compounds from the second chromatography device to the third chromatography device. This is for example applicable where the biological target compounds are lentivirus particles.

Further, the second buffer selection valve 24 may be configured to control a feed of a second buffer 34 for elution of biological target compounds from the first chromatography device 70, herein alternatively called buffer Bl, and to control a feed of a second buffer 36 for elution of a biological target compound from the second chromatography device 72, herein alternatively called buffer B2. Buffer B2 may optionally also be used for elution of a biological target compound from the third chromatography device 74. Alternatively, a separate buffer feed B3 may be used for elution of biological target compounds from the third chromatography device 74 (not shown). The system 10 according to Fig. 2 further includes a pump arrangement consisting of a system pump 42 and a sample pump 44. The system pump 42 is configured to supply the feeds of buffer 30, buffer 32, buffer 34, and buffer 36. The sample pump 44 is configured to supply a feed of sample 46, comprising a biological target compound and one or more impurities.

The system 10 according to Fig. 2 also includes an injection valve 50. It is to be understood that any injection valve conventionally used in the art can be used.

According to the embodiment shown in Fig. 2, the selection valve arrangement consists of a first chromatography device selection valve 62, a second chromatography device selection valve 64 and a third chromatography device selection valve 66. The chromatography device selection valve 62 connects the first chromatography device 70, the second chromatography device selection valve 64 connects the second chromatography device 72, and the third chromatography device selection valve 66 connects the third chromatography device 74, wherein the three chromatography devices are connected in series in-line within the system 10.

However, it is contemplated that the selection valve arrangement 60 may consist of a single chromatography device selection valve 62, which connects the first, second and third chromatography devices 70, 72 and 74 in series (as shown in Fig. 1). Alternatively, the selection valve arrangement 60 may consist of a combination of a first chromatography device selection valve 62 and a second chromatography device selection valve 64 (not shown), which combination connects the first, second and third chromatography devices 70, 72 and 74 in series. It is to be understood that in embodiments where the number of chromatography device selection valves is lower than the number of chromatography devices, at least one of the selection valves can handle different flow paths, which makes it possible to perform a multi-step chromatography purification on a chromatography system including fewer selection valves than the number of chromatography devices.

A non-limiting example of a chromatography system which may be used is an AKTA pure chromatography system (Cytiva, Sweden). The AKTA pure system in its standard set-up has only one column valve (corresponding to a chromatography device selection valve of system 10). The AKTA pure system used in the experimental section herein has been modified by addition of two additional chromatography device selection valves, to obtain a system according to the embodiment shown in Fig. 2. Another non-limiting example of a chromatography system which may be used is an NGC Chromatography System (BioRad, USA). It may be modified by addition of versatile valves, to obtain a system according to the embodiment shown in Fig. 2. Non-limiting examples of chromatography device selection valves that may be part of the presently disclosed system 10 are so-called versatile valves, such as for example versatile valve V9-V (Cytiva, Sweden).

The chromatography system 10 according to Fig. 2 further includes a UV detector 80, a conductivity detector 82, an outlet valve 84 and a fraction collector 86, all of which are standard components of chromatography systems.

The herein disclosed chromatography system 10 is primarily intended for use in preparatory applications, for feed material of volumes ranging from a few mL to several hundreds of litres. The system may also be used for analytical applications. The distinguishing features of the system 10 provide more significant advantages when applied in large-scale processes than in small-scale processes.

The term "chromatography material" is used herein to denote a type of separation matrix.

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

The chromatography materials referred to herein may comprise a linker connecting the ligand to the support, i.e., the coupling of the ligand to the support is provided by introducing a linker between the support and ligand. The coupling may be carried out following any conventional covalent coupling methodology such as by use of epichlorohydrin; epibromohydrin; allyl-glycidylether; bisepoxides such as butanedioldiglycidylether; halogen-substituted aliphatic substances such as di- chloro- propanol; and divinyl sulfone. Non-limiting examples of suitable linkers comprise vinyl sulfone, vinyl sulfone in combination with glycidol, polyethylene glycol (PEG) having 2-6 carbon atoms, carbohydrates having 3-6 carbon atoms, or polyalcohols having 3-6 carbon atoms.

Alternatively, the ligand may be coupled to the support via a longer linker molecule, also known as a "surface extender", or simply "extender". Extenders are well known in this field, and commonly used to sterically increase the distance between ligand and support. Extenders are sometimes denoted tentacles or flexible arms. For a more detailed description of possible chemical structures, see for example US 6,428,707, which is hereby included herein by reference. In brief, the extender may be in the form of a polymer such as a homo- or a copolymer. Hydrophilic polymeric extenders may be of synthetic origin, i.e., with a synthetic skeleton, or of biological origin, i.e., a biopolymer with a naturally occurring skeleton. Typical synthetic polymers are polyvinyl alcohols, polyacrylamides and polymethacrylamides, polyvinyl ethers etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.

In this context, "ligand" is a molecule that has a known or unknown affinity for a given analyte and includes any functional group, or capturing agent, immobilized on its surface, whereas "analyte" includes any specific binding partner to the ligand. The term "ligand" may herein be used interchangeably with the terms "specific binding molecule", "specific binding partner", "capturing molecule" and "capturing agent".

Herein, the molecules in a liquid sample which interact with a ligand are referred to as a "biological target compound" or an "analyte". The term "biological target compound" encompasses various types of biological molecules and compounds. Non-limiting examples include plasmids, exosomes, mRNA, virus particles, and proteins, such as monoclonal antibodies.

Analytes of particular interest according to the present disclosure are enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles (e.g., exosomes), and virus-like particles. Particularly interesting examples of enveloped virus particles are lentivirus particles.

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

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

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

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

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

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

A non-limiting example of a membranous structure is a Mustang® membrane (Pall Corporation, USA).

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

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

A non-limiting example of porous particles is Capto beads (Cytiva, Sweden), which are substantially spherical particles having a diameter of approx. 90 pm.

A non-limiting example of expanded bed media is STREAMLINE resins (Cytiva, Sweden).

As mentioned further above, the ligand of the first chromatography material may comprise an anion exchange group or an affinity group having a binding affinity for said enveloped or membranous biological particles.

Where the ligand of the first chromatography material comprises an anion exchange group, it may be a strong or partially strong anion exchange group, more particularly a quaternized amine group. A quaternary amine group is a strong anion exchange group, which is always positively charged irrespective of to which pH it is subjected. For DEAE-based types of chromatography materials, the degree of quaternization of the amine group may vary among the amine groups included in a chromatography material. A degree of quaternization of the amine group of from about 12% to about 100% globally in a chromatography material is generally considered to result in a chromatography material which behaves like a strong, or at least partially strong, anion exchange chromatography material since these at least 12% of all amine groups are always charged.

More particularly, where the ligand of the first chromatography material comprises a strong or partially strong anion exchange group, the ligand may be defined by Formula I: wherein Ri is selected from H and C1-C3 alkyl, and R? and R3 are independently selected from H, Cl- C3 alkyl, CH2OH, and CH2CHOHCH3, preferably wherein each of Rl, R2, and R3 is CH3.

The wavy moiety represents the support material, including the linker. The ligand may be attached to a carbon atom of the linker.

There are currently available chromatography materials comprising a ligand defined by Formula I, wherein each of Rl, R2, and R3 is CH3; e.g., a chromatography material made available under the name Capto Q. (Cytiva, Sweden). Capto Q. further comprises dextran as surface extender. Capto Q. is a non-limiting example of a strong anion exchange chromatography material having about 100% quaternized amine groups.

Another non-limiting example of a chromatography material comprising a quaternary amine ligand is the monolithic CIMmultus® QA (Sartorius, Germany).

Where the ligand of the first chromatography material comprises a strong or partially strong anion exchange group, it may alternatively be defined by Formula II: wherein: m is an integer of from 1 to 3;

Ri and R? are independently selected from a C1-C3 alkyl; Rs, and R₄ are independently selected from C1-C3 alkyl and CH2CHOHCH3; and R₅ is selected from hydrogen, a C1-C3 alkyl and CH2CHOHCH3; provided that if m is 1, the ligand is defined by the following Formula III: wherein n is an integer of from 0 to 3; provided that if n is 0, R3 and R₄ are independently selected from C1-C3 alkyl, and R₅ is hydrogen or CH2CHOHCH3.

As a non-limiting example, the ligand is defined by Formula III and comprises a combination of two or more of the following structures (i)-(iv):

(i) n is 0; R3 and R₄ are ethyl; and R₅ is hydrogen or CH2CHOHCH3;

(ii) n is 1; Ri, R?, R3, R₄ are ethyl; and R₅ is hydrogen or CH2CHOHCH3;

(iii) n is 2; each Ri and R? is ethyl; R3 and R₄ is ethyl; and R₅ is hydrogen or CH2CHOHCH3;

(iv) n is 3; each Ri and R? is ethyl; R3 and R₄ is ethyl; and R₅ is hydrogen or CH2CHOHCH3.

One currently available chromatography material comprising a ligand defined by Formula III and comprising a combination of the above-mentioned structures (i)-(iv) is the chromatography resin called Capto DEAE (Cytiva, Sweden). Capto DEAE further comprises dextran as surface extender. Capto DEAE is a non-limiting example of a strong, or partially strong, anion exchange chromatography material having a degree of quaternization of the amine groups of about 15%.

Where the ligand of the first chromatography material comprises an anion exchange group, it may alternatively comprise a weak anion exchange group. In contrast to the above-defined quaternized amine groups, almost all other ionic exchange groups are weak, i.e., their charge varies from fully charged to not charged within a reasonable range of pH used (such as pH 2-11) and having a neutral charge (same amount of + and - charges) at pl.

More particularly, where the ligand of the first chromatography material comprises a weak anion exchange group, the ligand may be defined by Formula IV: wherein X, for each occurrence independently, is selected from H, OH and a C1-3 group, and Ri, R?, R3 and R₄ are independently selected from H and a C1-3 group, wherein a C3 group is straight or branched, wherein a C1-3 group comprises groups independently selected from OH, O-C1-2, S-C1-2, NH, NHR, and NR2, wherein R is selected from H and a C1-3 group.

Non-limiting examples of ligands defined by Formula IV are N,N,N'-triethylethylenediamine, diethylenetriamine, N,N'-dimethylethylenediamine, N-methylethylenediamine, 1,3-diaminopropane, l,3-diamino-2-hydroxypropane, 2-methyl-l,3-propanediamine and N,N-diethylethylenediamine. A currently preferred ligand comprising a weak anion exchange group is N,N-diethylethylenediamine.

Where the ligand of the first chromatography material comprises an affinity group, it may have a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles (e.g., exosomes), and virus-like particles. For example, the ligand may have a binding affinity for lentivirus particles.

The second chromatography material is a conditioning chromatography material, wherein the term "conditioning chromatography material" is intended to mean that it conditions or prepares a sample feed or solution, which is loaded onto and being passed through the chromatography material, for subsequent purification steps and/or final formulation of a product. The conditioning may for example include or consist of desalting, i.e., decreasing of the salt concentration, of the sample feed or solution. Alternatively, conditioning may include or consist of increasing the salt concentration of the sample feed or solution. Alternatively, or additionally, conditioning may include or consist of changing the pH of the sample feed or solution, i.e., decreasing or increasing the pH. Further, a conditioning chromatography material may additionally achieve removal of any low molecular weight impurities.

When the conditioning consists of desalting, the conditioning chromatography material may suitably comprise a size exclusion chromatography material. Salt is retarded by the size exclusion material while the biological target compounds, which are much bigger than salt molecules, pass through the size exclusion material without retardation and thus are obtained in flow-through fractions from the second chromatography material. The third chromatography material comprises porous beads having an inner porous core and an outer porous shell. The porosity of the core and the shell may be the same or different. However, at least the porosity of the shell prevents particles having a size of >20 nm, such as enveloped or membranous biological particles, from permeating through the shell to contact the core.

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

The shell is typically hydrophilic. Thus, the surface of the porous bead that is accessible to large entities, such as enveloped or membranous biological particles, 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, for instance comprising oxygen and/or nitrogen atoms. Examples of such polar groups are hydroxyl, amino, carboxy, sulphonate (S and SP ligands) ester, ether of lower alkyls (such as (-CH2CH2O-)nH where n is an integer 2, 3, 4 and higher).

The core strongly binds biomolecules such as proteins and DNA through hydrophobic interactions. In the present context, impurities such as remaining host cell proteins and DNA (preferably fragmented DNA), can enter the porous bead and bind, whereas the target enveloped or membranous biological particles cannot enter, but are obtained in the flow-through fraction.

The core may be hydrophobic. Preferably, the hydrophobic core is hydrophilic per se 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. However, alternatively, the core may be hydrophobic per se, based on a hydrophobic polymer. For example, styrene/ethylstyrene/DVB, vinylethers and acrylates containing hydrophobic substituents as well as fluoroalkane-containing polymers are contemplated.

Hydrophobic interaction ligands may comprise aliphatic hydrocarbons, such as C1-C30 alkyl, preferably C4-C16 alkyl, and/or aromatic hydrocarbons, such as phenyl, antracene, naphtalene.

A hydrophilic polymer on which the shell and optionally the core may be based is a polysaccharide, such as agarose. For example, the core and the shell may both comprise cross-linked agarose. The porous core-shell beads may be produced as described in WO2009131526. In particular, the core and the shell may be made of agarose, and the core may be functionalized with hydrocarbon interaction ligands comprising 4-16 carbons, preferably octyl ligands. Useful chromatography media are available under the trade names Capto™ Core 400 and Capto™ Core 700, respectively, available from Cytiva, Sweden. The beads of Capto™ Core 700 have a shell with 700 kDa size exclusion cut-off and a core with multimodal octylamine ligand.

The present disclosure further provides use of the above-described chromatography system 10, including the above-described first and second chromatography materials, and optionally the abovedescribed third chromatography material, for separation of a biological target compound from one or more impurities, wherein the biological target compound is selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles (e.g., lentivirus), extracellular vesicles (e.g., exosomes), and virus-like particles.

Currently preferred, non-limiting examples of the first chromatography material include: i. a support material in the form of porous particles functionalised with a diethylethanolamine ligand; optionally wherein the ligand is connected to the support material by a polymer, such as dextran; ii. a support material in the form of a convection-based membranous structure comprising a nonwoven web of polymer nanofibres functionalised with a diethylethanolamine ligand; ill. a support material in the form of a convection-based membranous structure comprising a nonwoven web of polymer nanofibres functionalised with a N,N-diethylethylenediamine ligand; iv. a support material in the form of a convection-based membranous structure comprising a nonwoven web of polymer nanofibres functionalised with a ligand comprising an affinity group which has a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles, optionally wherein the extracellular vesicles are exosomes; and v. a support material in the form of porous particles functionalised with a ligand comprising an affinity group which has a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles, optionally wherein the extracellular vesicles are exosomes.

The present disclosure additionally provides, as illustrated in Fig. 3, a method 100 for separating enveloped or membranous biological particles from one or more impurities, the method comprising: a. Adding (110) a feed comprising enveloped or membranous biological particles and one or more impurities, to a first chromatography device (70), comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for said enveloped or membranous biological particles; b. Eluting (120) the enveloped or membranous biological particles from the first chromatography device in at least one eluate fraction; c. Adding (130) the at least one eluate fraction comprising enveloped or membranous biological particles, obtained in step b, to a second chromatography device (72), comprising a second chromatography material comprising a conditioning chromatography material; d. Obtaining (140) the enveloped or membranous biological particles in at least one flow-through fraction from the second chromatography device; wherein the feed is continuously passed through the first chromatography device and the second chromatography device to enable separation, wherein the first chromatography device and the second chromatography device are connected in series.

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

Preferably, the eluate volume from step (b) is <15% of the total volume of the second chromatography material.

As indicated in Fig. 3, the method 100 may further comprise the following steps: e. adding (150) the at least one flow-through fraction comprising enveloped or membranous biological particles, obtained in step d, to a third chromatography device (74), comprising a third chromatography material 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 wherein the pore size of the shell prevents particles having a size of >20 nm from contacting the core; f. obtaining (160) the enveloped or membranous biological particles in at least one flow-through fraction from the third chromatography device; wherein the at least one flow-through fraction obtained in step d is continuously passed on to and through the third chromatography device, which is connected in series with the first and the second chromatography devices.

It is to be understood that the first, the second, and the third chromatography materials, and the corresponding chromatography devices 70, 72 and 74, referred to in the method 100 are as defined and exemplified in detail further above in connection with the description of the chromatography system 10. In the method 100, the chromatography material referred to in steps (a) and (b) may be referred to as a capture chromatography material, meaning that the chromatography material is applied in a capture step, which in the context of liquid chromatography refers to the initial step(s) of a separation procedure. Herein, the capture step performed in steps (a) and (b) of the method 100 achieves a significant purification of the biological target compound from soluble impurities. Additional steps such as clarification and filtration (e.g., tangential flow filtration) may be performed prior to steps (a) and (b).

Further, the object of steps (c) and (d) of the method 100 is to condition or prepare the biological target compound for the conditions required to perform the subsequent purification steps (e) and (f). More particularly, for separation of lentivirus vectors, it is crucial to stabilise the virus quickly by removing salt after an anion exchange capture step and possibly also to adjust the pH. This in-line connected desalting performed in steps (c) and (d) is an important part of the present disclosure, helping to achieve good separation results while reducing the process time. Conventionally, a desalting step would be performed in a separate device, not connected in-line to the other chromatography devices.

In the method 100, the chromatography material referred to in steps (e) and (f) may be referred to as a polishing chromatography material, meaning that the chromatography material is applied in a polishing step.

The term "polishing step" refers in the context of liquid chromatography to a final purification step, wherein trace impurities are removed to leave an active, safe product. Impurities removed during the polishing step are often conformers of the target molecule, i.e., forms of the target molecule having particular molecular conformations, or suspected leakage products. A polishing step may alternatively be called "secondary purification step".

In the method 100 illustrated in Fig. 3, the flow rate used is dependent on the type of chromatography material used and the dimensions of the chromatography material or the chromatography device containing said chromatography material), and the residence time chosen. For example, a much higher flow rate can be applied to convection-based membranous support material, such as Fibro™, than to porous particles, such as Capto beads.

Further, as appreciated by persons skilled in the art, different buffers used in the method 100 are selected based on which chromatography materials are applied and which biological target compounds are to be purified. In steps (a) and (b) of the method, the pH of the buffer used may vary depending on which type of ligand is used. For affinity ligands, the buffer may for example have a pH around 7-8 for both binding in step (a) and for elution in step (b). To achieve elution of the bound biological target compounds, the buffer in step (b) comprises additional components compared to the buffer used in step (a). Non-limiting examples of such additional components are amino acids, such as arginine or proline. For anion exchange ligands, the buffer pH may be around 6.5-8.5 for both binding in step (a) and elution in step (b). To achieve elution of the bound biological target compounds, the buffer in step (b) comprises a salt concentration not applied in the buffer of step (a). Said buffer is suitably selected from buffers generally recommended for affinity chromatography or anion exchange chromatography, respectively, and which are suitable for the above-mentioned pH ranges. Nonlimiting examples include tris(hydroxymethyl)amino-methane (i.e., Tris), 1,3- bis(tris(hydroxymethyl)methylamino) propane (i.e., bis-Tris propane), triethanolamine, N- methyldiethanolamine, diethanolamine, 1,3-diaminopropane, ethanolamine, phosphate buffer, bisTris, Imidazole, MOPS, and HEPES.

Steps (e) and (f) of method 100 may comprise applying a buffer having a pH of from about 6.5 to about 7.5.

As described above, the object of steps (c) and (d) is to condition or prepare the biological target compounds for the conditions required to further purify and/or formulate the biological target compounds obtained in step (d) into a pharmaceutically acceptable composition. Any further purification may be performed by carrying out the subsequent steps (e) and (f) of method 100, in which case the buffer used in steps (c) and (d) will normally be the same as the buffer used in step (e). In general, the buffer 34 used in step (b) of the method, for elution of the enveloped or membranous biological particles from the first chromatography material, is not suitable for use in steps (e) and (f). Hence, buffer 32, used in step (c) and step (e), for equilibration and loading of a feed onto the second chromatography material and onto the third chromatography material, will be a different buffer than buffer 34.

A person skilled in the art is able to choose a suitable concentration for any one of the above-listed buffers.

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

Al: 50 mM Tris-HCI pH 7.4

A2: 50 mM Tris-HCI pH 7.4, 130 mM NaCI, 4% sucrose Bl: 50 mM Tris-HCI pH 7.4, 1.3 M NaCI B2: 1 M NaOH

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

Al: 50 mM sodium phosphate pH 7.0

A2: 50 mM sodium phosphate pH 7.0, 130 mM NaCI, 4% sucrose

Bl: 50 mM sodium phosphate pH 7.0, 1.3 M NaCI

B2: 1 M NaOH

The method 100 suitably comprises applying a selection valve arrangement 60 configured to enable the separation of enveloped or membranous biological particles from impurities, by allowing the feed to continuously pass through the first and the second chromatography devices 70, 72, and optionally the third chromatography device 74. The selection valve arrangement comprises a first chromatography device selection valve 62, and may optionally further comprise a second chromatography device selection valve 64, and optionally also a third chromatography device selection valve 66, as described in more detail elsewhere herein.

As seen in Fig. 3, the method 100 may further comprise an optional step of equilibrating 125 the second chromatography material before step c to conditions required for the enveloped or membranous biological particles to be obtained in step d, and to be obtained in step f.

Similarly, the method 100 may further comprise an optional step of equilibrating 145 the third chromatography material before step e to conditions required for the enveloped or membranous biological particles to be obtained in step f.

A currently preferred, non-limiting embodiment of the method 100 comprises separating lentivirus particles from impurities.

The herein disclosed method 100 may be a preparative method (preferably) or an analytical method.

The distinguishing features of the presently disclosed chromatography system 10 makes it possible to equilibrate the second chromatography material and/or the third chromatography material in-line while the method 100 is running, as opposed to equilibrating them separately, as isolated steps, thereby interrupting the continuous flow of feed in-line, within the system 10. This is advantageous since it enables quick neutralisation of pH and reduction of conductivity of the viral vector sample and contributes to reducing the overall time required to perform the process.

Additional reduction of time can be achieved since loading of the different chromatography devices within the system 10 may be done partly simultaneously. For example, it is not required to wait until the sample feed has passed through the second or third device before more sample is loaded into the first device, etc.

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

EXPERIMENTAL SECTION

Example 1: Separation of lentivirus particles from impurities by in-line connected capture, desalting, and polishing chromatography

A three-step chromatographic process was performed in one run by use of an AKTA pure™ chromatography system (Cytiva, Sweden), which has been modified by addition of two versatile valves. An in-line connected set-up for capture and polishing of lentivirus particles using resin columns was evaluated. A hands-free walk away set-up with one desalting column in between an anion exchange capture column and a polishing column was used to condition the sample and ensure low conductivity with a low salt buffer containing sucrose for stabilization of the lentivirus particles to maximise the infectious recovery.

The performance of said three-step in-line connected set-up (Table 1) was compared to results obtained by applying a two-step in-line connected set-up including an anion exchange capture column and a desalting column for buffer exchange into dilution buffer (Table 2), and a one-step setup including an anion exchange capture column with direct dilution (Table 3), respectively.

Each of the separation processes was carried out at pH 7.0 and at pH 7.4, respectively.

Three-step set-up

The modified AKTA pure™ chromatography system comprised: a 1^st chromatography device selection valve, herein called column valve 1 (CV1), a 1^st column comprising anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden), a 2^nd chromatography device selection valve, herein called versatile valve 1 (VV1) which has been added compared to a standard AKTA instrument, a 2^nd column comprising a desalting material, 50 mL HiPrep 26/10 Desalting (Cytiva, Sweden), a 3^rd chromatography device selection valve, herein called versatile valve 2 (VV2), which has been added compared to a standard AKTA instrument, and a 3^rd column comprising polishing bead material, 1 mL Capto™ Core 700 HiTrap (Cytiva, Sweden).

The three in-line connected columns were equilibrated separately before sample loading. Clarified Lentivirus-GFP feed material with a total particle titer of 3 x IO¹⁰ VP/mL was loaded onto the first column and the protocol of Table 1 was followed.

Table 1. Protocol (Unicorn™ software) for three-step set-up.

Two-step set-up

The modified AKTA pure™ chromatography system comprised: a 1^st chromatography device selection valve, column valve 1 (CV1), a 1^st column comprising anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden), a 2^nd chromatography device selection valve, versatile valve 1 (VV1), added compared to a standard AKTA instrument, a 2^nd column comprising a desalting material, 50 mL HiPrep 26/10 Desalting (Cytiva, Sweden), and a 3^rd chromatography device selection valve, versatile valve 2 (VV2), added compared to a standard AKTA instrument (however not needed in this set-up; switched off during the entire protocol).

The two in-line connected columns were equilibrated separately before sample loading. Clarified Lentivirus-GFP feed material with a total particle titer of 3 x IO¹⁰ VP/mL was loaded onto the first column and the protocol of Table 2 was followed.

Table 2. Protocol (Unicorn™ software) for two-step set-up. pH 7.4 buffer system for both three-step and two-step set-up:

Al: 50 mM Tris-HCI pH 7.4

A2: 50 mM Tris-HCI pH 7.4, 130 mM NaCI, 4% sucrose

Bl: 50 mM Tris-HCI pH 7.4, 1.3 M NaCI

B2: 1 M NaOH pH 7.0 buffer system for both three-step and two-step set-up:

Al: 50 mM sodium phosphate pH 7.0

A2: 50 mM sodium phosphate pH 7.0, 130 mM NaCI, 4% sucrose

Bl: 50 mM sodium phosphate pH 7.0, 1.3 M NaCI

B2: 1 M NaOH One-step set-up

The modified AKTA pure™ chromatography system comprised: a 1^st chromatography device selection valve, herein called column valve 1 (CV1), a 1^st column comprising anion exchange capture bead material, 5 mL Capto DEAE HiTrap (Cytiva, Sweden), a 2^nd chromatography device selection valve, herein called versatile valve 1 (VV1) which has been added compared to a standard AKTA instrument (however not needed in this set-up; switched off during the entire protocol), and a 3^rd chromatography device selection valve, herein called versatile valve 2 (VV2), which has been added compared to a standard AKTA instrument (however not needed in this set-up; switched off during the entire protocol).

Clarified Lentivirus-GFP feed material with a total particle titer of 3 x IO¹⁰ VP/mL was loaded onto the only column with direct dilution in 50 mM Tris-HCI pH 7.4 and 50 mM NaPi pH 7.0, 130 mM NaCI, 5% sucrose. The protocol of Table 3 was used.

Table 3. Protocol (Unicorn™ software) for one-step set-up. pH 7.4 buffer system for one-step set-up:

Al: 50 mM Tris-HCI pH 7.4

Bl: 50 mM Tris-HCI pH 7.4, 1.3 M NaCI

B2: 1 M NaOH pH 7.0 buffer system for one-step set-up:

Al: 50 mM sodium phosphate pH 7.0 Bl: 50 mM sodium phosphate pH 7.0, 1.3 M NaCI

B2: 1 M NaOH

Results

Fig. 4 shows the results of separation of lentivirus particles from impurities by use of three different processes (numbered 1-3) at pH 7.4 and at pH 7.0, respectively: 1: Capto DEAE capture column followed by direct dilution,

2: In-line connected Capto DEAE capture column and desalting column,

3: In-line connected Capto DEAE capture column, desalting column, and polishing column.

The results show that separation at pH 7 gave higher infectious recovery compared to separation at pH 7.4 and that the connected columns did not affect the total or infectious particle recovery negatively. More particularly, the 1-step chromatography consisting of a capture step with Capto DEAE followed by direct dilution resulted in 60-70% physical and infectious recovery (data not shown). The 2-step chromatography with a Capto DEAE capture step with in-line connected desalting step resulted in close to 100% physical recovery and 70-90% infectious recovery for both pH 7.4 and pH 7.0 (Fig. 4). The 3-step chromatography set-up with in-line connected Capto DEAE capture step, desalting step and polishing Capto Core step, resulted in 60% physical particle recovery and only 30% infectious particle recovery at pH 7.4 for the second and third step. Using pH 7, which is more optimal for the Capto Core step, resulted in about 90% physical particle recovery and close to 100% infectious recovery. The third, polishing step is needed to remove the DNA impurities that are coeluting together with the lentivirus particles in the Capto DEAE capture step (data not shown).

A p24 ELISA was used to determine the total particle titer and a cell-based transduction assay (counting GFP expressing cells by flow cytometry) was used to determine infectious titer.

Impurity levels in the final samples after Capto Core 700 were below detection limit of Micro BCA total protein assay and Picogreen total DNA assay (data not shown). Highly purified lentivirus was obtained.

Example 2

Experimental designs for separation of lentivirus particles from impurities are performed with equipment and samples as in Example 1 above, with the following variations:

In terms of the first chromatography material (used in the capture step): Different support material, for example a membrane or monolith;

Different Capto™ Core molecular weight cut-off, e.g., CC400 (Cytiva, Sweden); Additional capture step(s), for example by applying an affinity ligand and an anion exchange ligand or semi-affinity ligand.

In terms of the biological target compound:

Lentivirus pseudo types; - Different insert (gene of interest) than GFP;

Exosome instead of lentivirus;

Other enveloped virus instead of lentivirus.

REFERENCES

F. Higashikawa et al., Virology 280, 124-131 (2001), Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors

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

WO2009131526

Claims

1. A method (100) for separating enveloped or membranous biological particles from one or more impurities, the method comprising: a. Adding (110) a feed comprising enveloped or membranous biological particles and one or more impurities, to a first chromatography device (70), comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for said enveloped or membranous biological particles; b. Eluting (120) the enveloped or membranous biological particles from the first chromatography device in at least one eluate fraction; c. Adding (130) the at least one eluate fraction comprising enveloped or membranous biological particles, obtained in step b, to a second chromatography device (72), comprising a second chromatography material comprising a conditioning chromatography material; d. Obtaining (140) the enveloped or membranous biological particles in at least one flow- through fraction from the second chromatography device; wherein the feed is continuously passed through the first chromatography device and the second chromatography device to enable separation, wherein the first chromatography device and the second chromatography device are connected in series.

2. The method according to claim 1, wherein the method comprises applying a selection valve arrangement (60) configured to enable the separation, by allowing the feed to continuously pass through the first chromatography device and the second chromatography device; wherein the selection valve arrangement comprises a first chromatography device selection valve (62).

3. The method according to claim 2, wherein the selection valve arrangement further comprises a second chromatography device selection valve (64).

4. The method according to any one of claims 1-3, further comprising: e. Adding (150) the at least one flow-through fraction comprising enveloped or membranous biological particles, obtained in step d, to a third chromatography device (74), comprising a third chromatography material 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 wherein the pore size of the shell prevents particles having a size of >20 nm from contacting the core; f. Obtaining (160) the enveloped or membranous biological particles in at least one flow- through fraction from the third chromatography device; wherein the at least one flow-through fraction obtained in step d is continuously passed on to and through the third chromatography device, which is connected in series with the first and the second chromatography devices.

5. The method according to claim 4, wherein the selection valve arrangement further comprises a third chromatography device selection valve (66).

6. The method according to any one of claims 1-5, further comprising equilibrating (125) the second chromatography material before step c to conditions required for the enveloped or membranous biological particles to be obtained in step d, and optionally to be obtained in step f.

7. The method according to any one of claims 1-6, further comprising equilibrating (145) the third chromatography material before step e to conditions required for the enveloped or membranous biological particles to be obtained in step f.

8. The method according to any one of claims 1-7, wherein the enveloped or membranous particles are selected from enveloped virus particles, extracellular vesicles, and virus-like particles.

9. The method according to claim 8, wherein the enveloped virus particles are lentivirus particles.

10. The method according to claim 8, wherein the extracellular vesicles are exosomes.

11. The method according to any one of claims 1-10, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula I: wherein Ri is selected from H and C1-C3 alkyl, and R? and R3 are independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably wherein each of Ri, R?, and R3 is CH3.

12. The method according to any one of claims 1-10, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula II: wherein: m is an integer of from 1 to 3;

Ri and R2 are independently selected from a C1-C3 alkyl; Rs, and R₄ are independently selected from C1-C3 alkyl and CH2CHOHCH3; and R₅ is selected from hydrogen, a C1-C3 alkyl and CH2CHOHCH3; provided that if m is 1, the ligand of the strong, or partially strong, anion exchange chromatography material is defined by the following Formula III: wherein n is an integer of from 0 to 3; provided that if n is 0, R3 and R₄ are independently selected from C1-C3 alkyl, and R₅ is hydrogen or CH2CHOHCH3.

13. The method according to any one of claims 1-10, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula IV: wherein X, for each occurrence independently, is selected from H, OH or a C1-3 group, and

Ri, R2, R3 and R₄ are independently selected from H, and a C1-3 group, wherein a C3 group is straight or branched, wherein a C1-3 group comprises groups independently selected from OH, O-C1-2, S-C1-2, NH, NHR, and NR₂, wherein R is selected from H and a C1-3 group.

14. The method according to any one of claims 1-10, wherein the ligand of the first chromatography material comprises an affinity group which has a binding affinity for said enveloped or membranous biological particles.

15. The system according to any one of claims 9-14, wherein the support material of the first chromatography material comprises a membranous structure, nanofibres, a monolith, porous particles, non-porous particles, or expanded bed media.

16. The system according to any one of claims 9-15, wherein the second chromatography material comprises a size exclusion chromatography material.

17. A chromatography system (10), the system comprising: a buffer valve arrangement (20) configured to allow independent control of a first buffer feed and a second buffer feed; a pump arrangement (40) configured to supply a first buffer feed, a second buffer feed, and a feed comprising a biological target compound and one or more impurities; a selection valve arrangement (60), comprising a first chromatography device selection valve (62); a first chromatography device (70), comprising a first chromatography material comprising a support material functionalised with a ligand, wherein the ligand comprises an anion exchange group or an affinity group having a binding affinity for a biological target compound; a second chromatography device (72), comprising a second chromatography material, which comprises a conditioning chromatography material; wherein the selection valve arrangement is configured to enable separation of a biological target compound from impurities by allowing a feed, comprising a biological target compound and one or more impurities, to continuously pass through the first chromatography device and the second chromatography device, wherein the first chromatography device and the second chromatography device are configured to be connected in series.

18. The system according to claim 17, wherein the selection valve arrangement further comprises a second chromatography device selection valve (64).

19. The system according to claim 17 or 18, further comprising a third chromatography device (74), comprising a third chromatography material 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 wherein the pore size of the shell prevents particles having a size of >20 nm from contacting the core, wherein the third chromatography device is configured to be connected in series with said first and second chromatography devices, and wherein the selection valve arrangement is configured to enable separation by allowing the feed to continuously pass through said first, second and third chromatography devices.

20. The system according to claim 19, wherein the selection valve arrangement further comprises a third chromatography device selection valve (66).

21. The system according to any one of claims 17-20, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula I: wherein Ri is selected from H and C1-C3 alkyl, and R? and R3 are independently selected from H, C1-C3 alkyl, CH2OH, and CH2CHOHCH3, preferably wherein each of Ri, R?, and R3 is CH3.

22. The system according to any one of claims 17-20, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula II: wherein: m is an integer of from 1 to 3;

Ri and R? are independently selected from a C1-C3 alkyl; R3, and R₄ are independently selected from C1-C3 alkyl and CH2CHOHCH3; and R₅ is selected from hydrogen, a C1-C3 alkyl and CH2CHOHCH3; provided that if m is 1, the ligand of the strong, or partially strong, anion exchange chromatography material is defined by the following Formula III: wherein n is an integer of from 0 to 3; provided that if n is 0, R3 and R₄ are independently selected from C1-C3 alkyl, and R₅ is hydrogen or CH2CHOHCH3.

23. The system according to any one of claims 17-20, wherein the ligand of the first chromatography material comprises an anion exchange group defined by Formula IV: wherein X, for each occurrence independently, is selected from H, OH or a C1-3 group, and Ri, R2, R3 and R₄ are independently selected from H, and a C1-3 group, wherein a C3 group is straight or branched, wherein a C1-3 group comprises groups independently selected from OH, O-C1-2, S-C1-2, NH, NHR, and NR₂, wherein R is selected from H and a C1-3 group.

24. The system according to any one of claims 17-20, wherein the ligand of the first chromatography material comprises an affinity group which has a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles; optionally wherein the extracellular vesicles are exosomes.

25. The system according to any one of claims 17-24, wherein the support material of the first chromatography material comprises a membranous structure, nanofibres, a monolith, porous particles, non-porous particles, or expanded bed media.

26. The system according to any one of claims 17-25, wherein the second chromatography material comprises a size exclusion chromatography material.

27. Use of a system according to any one of claims 17-26 for separation of a biological target compound from one or more impurities, wherein the biological target compound is selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles; optionally wherein the extracellular vesicles are exosomes.

28. The use according to claim 27 , wherein the first chromatography material is selected from a group consisting of: i. a support material in the form of porous particles functionalised with a diethylethanolamine ligand; optionally wherein the ligand is connected to the support material by a polymer, such as dextran; ii. a support material in the form of a convection-based membranous structure comprising a non-woven web of polymer nanofibres functionalised with a diethylethanolamine ligand; ill. a support material in the form of a convection-based membranous structure comprising a non-woven web of polymer nanofibres functionalised with a N,N- diethylethylenediamine ligand; iv. a support material in the form of a convection-based membranous structure comprising a non-woven web of polymer nanofibres functionalised with a ligand comprising an affinity group which has a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles, optionally wherein the extracellular vesicles are exosomes; and v. a support material in the form of porous particles functionalised with a ligand comprising an affinity group which has a binding affinity for a biological target compound selected from a group consisting of enveloped or membranous biological particles, such as enveloped virus particles, extracellular vesicles, and virus-like particles; optionally wherein the enveloped virus particles are lentivirus particles; optionally wherein the extracellular vesicles are exosomes.