Separation system for isolating and purifying a target component

Abstract

The present invention relates to a separation system for separating and purifying a target component, a method for separating and purifying a target component, and the use of a single-pass cross-flow diafiltration unit to connect together first and second chromatography devices.

Description

[Technical field]

[0001] The present invention relates to a separation system for separating and purifying a target component, a method for separating and purifying a target component, and the use of a single-pass cross-flow diafiltration unit to connect together first and second chromatography devices. [Background technology]

[0002] In recent years, major improvements have been made in the upstream processing of target component producing cell lines, which has led to a situation where the production bottleneck has shifted to the operation of subsequent clarification, capture and purification units in downstream processing (DSP).

[0003] In downstream processing of biopharmaceuticals, purification is generally performed by chromatography unit operations in bind-and-elute mode. In this case, the product binds to the ligands of the chromatography medium, while impurities such as aggregates, host cell proteins (HCPs) and desoxyribonucleic acid (DNA) do not bind and therefore flow through the chromatography device. The product is then recovered by an elution step in specific buffer conditions, thereby separating it from the impurities.

[0004] However, this process is time-consuming and expensive since different steps such as loading, washing, elution, cleaning and equilibration must be performed using several different buffer systems. Further disadvantages of this mode of operation are the increased footprint of the different chromatography systems, the increased complexity of operation and the challenge of integration into a continuous production process.

[0005] The flow-through mode of operation offers several advantages. By choosing the appropriate buffer conditions, the product flows through the chromatographic medium while retaining the impurities. This reduces the complexity of the operation and allows for easy integration into a continuous process. However, between different flow-through chromatography unit operations, the buffer conditions need to be changed to allow efficient separation of the impurities from the product. This is commonly achieved by additional unit operations such as diafiltration / tangential flow filtration (TFF), which leads to drawbacks as it makes the whole process more complicated, time-consuming and expensive. Summary of the Invention [Problem to be solved by the invention]

[0006] The technical problem underlying the present invention is therefore to provide a means for downstream processing of target components, which should be significantly cheaper and have lower complexity when compared to conventional means for DSP, while still providing high purity and yield. [Means for solving the problem]

[0007] The solution to the above technical problem is achieved by the embodiments characterized in the claims.

[0008] In particular, the present invention provides a separation system configured to process a first fluid comprising a plurality of components, at least one of the plurality of components of the first fluid being a target component, the separation system comprising: a first and second chromatography device and a single-pass cross-flow diafiltration unit integrally integrated between the first and second chromatography devices; a first chromatography device configured to receive and process the first fluid and provide a second fluid containing the target component; a single-pass cross-flow diafiltration unit configured to continuously diafilter a second fluid with a diafiltration medium to obtain a permeate and a third fluid containing a target component as a retentate; The present invention relates to a separation system in which a second chromatography device is configured to receive and process a third fluid and provide a fourth fluid containing the target component.

[0009] In the separation system of the invention, it is advantageously possible to directly combine two (different) chromatographic devices in flow-through mode with buffer exchange integrated between them by single-pass tangential flow filtration (SPTFF) (see FIG. 1). This approach allows three unit operations to be combined into one. This preferably reduces the footprint and investment costs considerably. Furthermore, preferably, a high yield of the target component can be advantageously achieved in a continuous flow-through mode of operation. A first fluid containing the target component is purified by a first chromatographic device to obtain a second fluid, which is immediately subjected to diafiltration in a cross-flow diafiltration unit. The obtained third fluid is then subjected to purification by a second chromatographic device to obtain a fourth fluid. The operations can advantageously be performed continuously.

[0010] According to the present invention, the separation system comprises a first and a second chromatography device and a single-pass cross-flow diafiltration unit integrally integrated between the first and second chromatography devices. The term "integrally integrated" in this specification defines that the first and second chromatography devices form one (closed) unit with the cross-flow diafiltration unit. Therefore, no further units or unit operations such as in-line dilution, dilution in an intermediate holding tank, (additional) buffer exchange, titration, desalting steps, etc. are included between the first chromatography device and the cross-flow diafiltration unit and between the cross-flow diafiltration unit and the second chromatography device.

[0011] The first and second chromatography devices may each independently be a plurality of chromatography devices. One or more of the plurality of chromatography devices, preferably one, are operated at a time. By applying a switching means, it is possible to switch between the chromatography devices in each of the plurality of chromatography devices, for example when one of the devices reaches its capacity limit. Thus, the separation system may further comprise a switching means for (alternately) connecting or disconnecting the individual chromatography devices of the plurality of chromatography devices to a separation line (comprising a source of the first fluid, one of the plurality of first chromatography devices, the single-pass cross-flow diafiltration unit, and one of the plurality of second first chromatography devices). Suitable switching means are, for example, valves. A chromatography device of the plurality of chromatography devices that is not in operation (i.e. not currently fluidly connected to the single-pass cross-flow diafiltration unit) may be regenerated. For example, if the plurality of chromatography devices consists of two chromatography devices (in parallel), one of the plurality of chromatography devices may be operated and the other of the plurality of chromatography devices may be regenerated. Furthermore, the separation system may further comprise one or more regeneration units for regenerating one or more of the plurality of chromatography devices (disconnected from the separation line). Such a regeneration unit may comprise containers for the regeneration liquid and the waste liquid, and optionally pumps and / or valves for regulating the flow of the (each of) the liquids. Examples of regeneration liquids are equilibration buffer, sodium chloride solution, and sodium hydroxide solution. The waste liquid may be collected in at least one waste container. Furthermore, the separation system may further comprise sensors, such as UV sensors, for providing information, for example, for controlling the switching of valves so that the target component is not transferred to waste and / or so that the regeneration liquid without the target component is not transferred to the separation line, and / or for monitoring the purification performance of the chromatographic device during operation. Alternative or supplementary sensors (e.g. pH sensors) may also be provided, for example, for detecting changes in the medium.The use of suitable sensors can allow for the change of membrane absorber or automation of the purification process. The switching between operation and regeneration can be performed independently in each of the multiple chromatography devices of the first and second chromatography devices and can be selected according to the performance of the membrane absorber currently being operated. Preferably, each of the first and second chromatography devices consists of two chromatography devices (in parallel).

[0012] Non-limiting examples of separation systems with multiple chromatographic devices for each of the first and second chromatographic devices are shown in Figures 4 and 5, which show a separation system consisting of two parallel membrane absorbers as the first chromatographic device, a single-pass cross-flow diafiltration unit, and two parallel membrane absorbers as the second chromatographic device. The membrane absorbers are fluidly connected such that only one membrane absorber per chromatographic device at a time actually performs purification before and after the single-pass cross-flow diafiltration unit (path 1 shown; separation line), while other membrane absorbers for the chromatographic devices not used can be regenerated (paths 2 and 3 shown). Provide corresponding containers containing the necessary regenerating liquids such as equilibration buffer, NaOH and NaCl. Each of these liquids can be fed to the regenerating membrane absorber via at least one pump (CIP pump 1, CIP pump 2) controlled by valves (HV-101, HV-102, HV-201, HV-202, HV-203) as required. Further, waste containers (waste 1, waste 2) are provided. The change from one membrane absorber to a membrane absorber in parallel can be achieved via eight valves (for a membrane absorber before single-pass cross-flow diafiltration, these are, for example, HV-111, HV-112, HV-113 and HV-114, which are placed before the membrane absorber, and HV-121, HV-122, HV 123 and HV-124, which are placed after the membrane absorber). When switching from one membrane absorber to the other, for example, by switching the group of valves after the membrane absorber (for example, HV-121, HV-122, HV 123 and HV-124) after the group of valves before the membrane absorber (for example, HV-111, HV-112, HV-113 and HV-114), it is possible to avoid the transfer of target compounds to waste or the transfer of regeneration buffer to the separation line. In this way, the fluid in the line between the two groups of valves can still be directed to the originally desired path. The corresponding control can be performed by a UV sensor (not shown in Figures 4 and 5).The UV signal allows a determination of when changes in the media should also reach the second set of valves and switch the valves accordingly.

[0013] Any form and combination of chromatographic methods and respective ligands can be used for the first and second chromatographic devices. For example, the first and second chromatographic methods of the first and second chromatographic devices can be independently selected from the group consisting of anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed mode chromatography (MMC), and size exclusion chromatography (SEC). Preferably, the first and second chromatographic methods are selected from a combination of AEX and CEX, a combination of AEX and HIC, a combination of CEX and HIC, and vice versa. More preferably, the first and second chromatographic methods are selected from AEX and CEX, and CEX and AEX. The combination of AEX and CEX, and vice versa, can advantageously remove impurities with different isoelectric points. The combination of AEX and HIC and CEX and HIC can advantageously remove impurities / aggregates based on hydrophobicity. Examples of ligands for the above-mentioned methods are known in the art. For example, quaternary ammonium can be used as a ligand for strong AEX, and sulfonic acid can be used as a ligand for strong CEX.

[0014] The chromatographic media of the first and second chromatographic devices are not particularly limited. For example, the first and second chromatographic devices can each independently comprise a chromatographic medium selected from the group consisting of a membrane adsorber, a chromatographic resin, and a monolith. Preferably, the first and second chromatographic devices apply the same chromatographic media. More preferably, the chromatographic media of each of the first and second chromatographic devices is a membrane adsorber. The membrane adsorber preferably allows for significantly higher flow rates and higher productivity. The first and second chromatographic devices are preferably independently selected from the group consisting of an AEX membrane absorber, a CEX membrane absorber, a HIC membrane absorber, an affinity chromatography membrane absorber, an MMC membrane absorber, and an SEC membrane absorber, more preferably independently selected from an AEX membrane absorber, a CEX membrane absorber, and a HIC membrane absorber, and even more preferably independently selected from an AEX membrane absorber and a CEX membrane absorber.

[0015] Preferably, the first chromatographic device is configured to retain other components (impurity components) and not substantially retain the target component, providing a second fluid containing the target component in a higher relative amount than the first fluid. Here, "relative amount" refers to the mass of the target component relative to the total mass of the other components (impurity components) in the respective fluids. Thus, the term "higher relative amount of the target component in the second fluid than in the first fluid" means that the mass of the target component relative to the total mass of the other components (impurity components) in the second fluid is greater than the mass of the target component relative to the total mass of the other components (impurity components) in the first fluid. This can be achieved, for example, by reducing the amount of the other components (impurity components) from the first fluid to the second fluid to a greater extent than the amount of the target component (if the amount of the target component is reduced at all).

[0016] In a preferred embodiment, the first chromatographic device retains at least 20% by weight, more preferably at least 40% by weight, and most preferably at least 60% by weight of other components (impurity components) present in the first fluid, and furthermore, the first chromatographic device retains up to 10% by weight, more preferably up to 5.0% by weight, more preferably up to 2.0% by weight, more preferably up to 1.0% by weight, and most preferably up to 0.10% by weight of the target component present in the first fluid.

[0017] Preferably, the second chromatographic device is configured to retain other components (impurity components) and not substantially retain the target component, providing a fourth fluid containing the target component in a higher relative amount than the third fluid. The definitions given above with respect to the first and second fluids apply analogously.

[0018] In a preferred embodiment, the second chromatographic device retains at least 20% by weight, more preferably at least 40% by weight, and most preferably at least 60% by weight of other components (impurity components) present in the third fluid, and furthermore, the second chromatographic device retains up to 10% by weight, more preferably up to 5.0% by weight, more preferably up to 2.0% by weight, more preferably up to 1.0% by weight, and most preferably up to 0.10% by weight of the target component present in the third fluid.

[0019] The targeting component is not particularly limited. For example, the targeting component can be any biomolecule of interest, such as a protein, an antibody, a hormone, a vaccine, a nucleic acid, an exosome, and a virus, as well as a virus-like particle. In a preferred embodiment, the targeting component is an antibody, more preferably a monoclonal antibody (mAb), or a fragment or derivative thereof, or a nanobody. Examples of monoclonal antibodies are adalimumab, cetuximab, rituximab, infliximab, omalizumab, and denosumab. The targeting component can be obtained, for example, from cell cultures, such as "Chinese Hamster Ovary Cells" (CHO cells) or other mammalian cell lines.

[0020] The source of the first fluid is not particularly limited. For example, the first fluid can be obtained by applying any biological, biochemical, chemical or pharmaceutical method. This allows other purification methods to be performed beforehand, applying different purification means, to obtain the first fluid. For example, the target component can be produced by a suitable cell line, for example a CHO cell line, for example by perfusion culture. As a cell retention device, for example an alternating flow filtration membrane can be applied. Furthermore, as a potential first capture step, Protein A affinity chromatography and virus inactivation by acidification (for example pH value < 3.5) can be performed. To obtain any required (buffer) conditions in the first fluid, for example any diafiltration or titration can be performed.

[0021] The first fluid contains a plurality of components, and is not particularly limited as long as at least one of the plurality of components of the first fluid is a target component. The additional (impurity) components (apart from the components of the medium) are not particularly limited and may depend on the preparation conditions of the target component. Examples of additional components are aggregates, host cell proteins, deoxyribonucleic acid, and their fragments and charge variants.

[0022] The medium of the first fluid is not particularly limited. In principle, any fluid is suitable, with water and an aqueous salt solution being preferred. As the medium of the first fluid, for example, an aqueous buffer solution can be used. Preferably, the medium of the first fluid is selected from the group consisting of KPI buffer, sodium phosphate buffer, sodium acetate buffer, PBS, glycine, citrate buffer, Tris buffer, BIS-Tris buffer, HEPES buffer and water. The concentration of the buffer in the aqueous solution is not particularly limited, and may be, for example, 1.0 mM to 5.0 M, preferably 5.0 mM to 1.0 M, more preferably 10 mM to 200 mM, and most preferably 25 to 100 mM. The medium of the first fluid and its conditions can be appropriately selected depending on the components of the first fluid to be separated (impurities and target components) and the first chromatography device to be applied. For this purpose, for example, a statistical experimental design (DoE) approach can be applied.

[0023] Design of Experiments (DoE) is an efficient method to determine the relevant influencing factors of a process or product from a large number of parameters. With the help of experimental design, these factors are varied largely independently of each other to derive their influence on the target variable, thus resulting in a causal-effect model. During the evaluation, it is estimated whether all the desired goals are achieved or if, for example, some goals are conflicting. Examples of goals can be high purity, high yield, high quality (e.g., if applicable: activity, critical quality attributes, glycosylation (e.g., for mAbs), charge variants). Conflicting goals can be yield and purity. The aim of DoE is to achieve the best trade-off between the goals. In general, DoE consists of the following steps: screening of significant variables, analysis, optimization and validation of the process model.

[0024] The conditions of the first fluid can be appropriately set (e.g., by selecting an appropriate buffer, which can be evaluated by DoE) to optimize the yield and / or purity achieved by the first chromatographic device.

[0025] The pH of the first fluid is not subject to any particular limitation. For example, the pH of the first fluid may be 3.0 to 14.0, preferably 5.0 to 12.0, more preferably 6.0 to 10.0, and most preferably 6.0 to 9.0. The pH of the second fluid may correspond to the pH of the first fluid, i.e., the pH may not be substantially changed by passing through the first chromatography device.

[0026] The conductivity of the first fluid is not particularly limited and may be, for example, 0 mS / cm to 500 mS / cm, preferably 0 mS / cm to 150 mS / cm.

[0027] The cross-flow diafiltration unit is a single-pass cross-flow diafiltration unit configured to continuously diafilter a second fluid using a diafiltration medium to obtain a permeate and a third fluid containing the target component as a retentate, such that the second fluid flows over the surface of the filter material and the third fluid is provided by the cross-flow diafiltration unit after only one pass, i.e. without reuse.

[0028] As the single-pass cross-flow diafiltration unit, the single-pass cross-flow diafiltration unit generally known in the art can be applied.For example, as described in WO 2017 / 174192 A1, a single-pass method can be applied, or as described in US Patent No. 7 384 549 B2, a cross-flow diafiltration unit can be applied.

[0029] In a preferred embodiment, the single-pass cross-flow diafiltration unit comprises at least a diafiltration channel, a first filter material (preferably flat), a retentate channel, a second filter material (preferably flat), and a permeate collection channel, the first filter material being arranged to mutually define the diafiltration channel and the retentate channel, and the second filter material being arranged to mutually define the retentate channel and the permeate collection channel; The diafiltration channel is connected in fluid communication with at least one inlet for the diafiltration medium, the retentate channel is connected in fluid communication with at least one inlet for a second fluid and at least one outlet for a third fluid as retentate, and the permeate collection channel is connected in fluid communication with at least one outlet for the permeate. An example of a respective cross-flow diafiltration unit is, for example, shown diagrammatically in FIG. 2. A suitable cross-flow diafiltration unit is described, for example, in DE 10 2016 004 115 A1 (Cross-flow diafiltration unit for continuous diafiltration).

[0030] The shape (spatial form) of the first filter material and the second filter material is not particularly limited. Preferably, the first filter material and the second filter material are flat filter materials. The term "flat" indicates that each filter material is substantially in a single plane. Preferably, all filter materials are more or less in planes that are substantially parallel to each other.

[0031] Additionally, the cross-flow diafiltration unit can be a wound module. To this end, initially flat or planar arrangements of first and second filter materials are wound around a core such that the first and second filter materials (as well as the diafiltration, retentate and permeate channels) each have a helical cross section.

[0032] Additionally, the cross-flow diafiltration unit may be a hollow fiber module, where the first and second filter materials have a hollow fiber shape (cylinders without disk-shaped ends), and the inner diameters of the first and second hollow fiber filter materials are different to allow the first and second filter materials to be inserted into the second and first filter materials, respectively, to form the diafiltration channel, the retentate channel, and the permeate channel.

[0033] The first filter material preferably has a molecular weight cutoff (MWCO) in the range of 1 kDa to 1,500 kDa. The second filter material preferably has a molecular weight cutoff in the range of 1 kDa to 1,500 kDa. The molecular weight cutoff can be determined in accordance with US standard ASTM E1343-90 ("Standard test method for molecular weight cutoff evaluation of flat sheet ultrafiltration membranes"). The MWCO of the membrane is preferably selected such that the target component is retained in the retentate channel, while smaller buffer components permeate the membrane and are removed at the permeate outlet.

[0034] The first filter material has a pore size of preferably 0.001 to 50 μm, preferably 0.01 to 0.5 μm.The second filter material has a pore size of preferably 0.001 to 50 μm, preferably 0.01 to 0.5 μm.

[0035] For microfiltration membranes with a pore size of at least 0.1 μm, i.e. an average pore size between 0.1 and 10 μm, capillary flux porometry is used to determine the pore size. This is a gas-liquid porosimetry, in which the differential pressure and the flow rate of the gas through the membrane sample are measured first in the wet state and then in the dry state. Before the measurement, the membrane sample is brought into contact with a wetting liquid so that all the pores are filled with this liquid. After filling the pores and introducing the sample, the measuring cell must be closed and the measurement started. After starting the measurement, the gas pressure is automatically gradually increased and the pore size corresponding to the applied pressure is emptied by the gas pressure. This continues until the relevant pore range is covered, i.e. until even the smallest pores present in the measuring range have been cleared of liquid. The pressure is then reduced again and the measurement is automatically repeated on the now dry sample. The pore size distribution is calculated from the difference between the two pressure-flow curves using the Young-Laplace equation (see also A. Shrestha, “Characterization of porous membranes via porometry”, 2012, Mechanical Engineering Graduate Theses & Dissertations, Paper 38, University of Colorado at Boulder).

[0036] For the determination of pore sizes from >10 μm to 1 mm, a method based on image analysis described in Journal of Membrane Science 372 (2011), pages 66 to 74, can be used.

[0037] For pore sizes below 0.1 μm, a liquid-liquid displacement method is used that has similarities to capillary flow porometry, however, instead of the gas flow rate, the flow rate of the displaced liquid is measured as a function of the differential pressure increase (see R. Davila, “Characterization of ultra- and nanofiltration commercial filters by liquid-liquid displacement porosimetry”, 2013).

[0038] According to a preferred embodiment, the first filter material is a first filtration membrane or the second filter material is a second filtration membrane. It is particularly preferred that the first filter material is a first filtration membrane and the second filter material is a second filtration membrane.

[0039] In particular, porous membranes in the ultrafiltration and microfiltration range are suitable as the first and second filter materials.

[0040] In a preferred embodiment, the second filter material has a pore size smaller than the target component and larger than at least one of the other components of the second fluid. Preferably, the first filter material and the second filter material are identical.

[0041] Ultrafiltration membranes are characterized by a pore size of less than 0.01 μm or a molecular weight cut-off in the molecular weight range of approximately 1 to 1,500 kDa, while microfiltration membranes have a pore size in the range of 0.01 to 50 μm, preferably 0.01 to 0.5 μm, or a molecular weight cut-off of 30 to 1,500 kDa. Filtration membranes may consist, for example, of polyvinylidene fluoride, cellulose and its derivatives, polyethersulfone or polysulfone, with crosslinked cellulose hydrate being particularly preferred.

[0042] The inlet for the feed fluid (here, the second fluid) is preferably attached to a first edge region of the cross-flow diafiltration unit, and the outlet for the retentate (here, the third fluid) is attached to a second edge region of the cross-flow diafiltration unit facing the first edge region. This arrangement makes it possible to define a substantially uniform direction of retentate flow from the inlet for the feed fluid as the starting point to the outlet for the retentate as the end point. As a result, the direction of retentate flow runs substantially parallel to the flow path along the (flat) filter material, i.e. essentially without deflection, so that a stable and reliable flow of retentate can be ensured by the cross-flow diafiltration unit. In addition, due to the substantially straight flow path without deflections or loops, etc., it is possible to minimize the pressure drop in the filtration unit as well as the undesirable effects of non-linear flow on the target substances contained in the feed fluid. For the reasons mentioned above, it is also preferred that the inlet for the diafiltration medium is attached to the first edge region of the cross-flow diafiltration unit. However, it is also possible to mount the inlet for the diafiltration medium in the second edge region or in the third edge region and / or in the fourth edge region.

[0043] According to a preferred embodiment, the outlet for the permeate is attached to the second edge region of the cross-flow diafiltration unit. In each case, it is particularly preferred that at least one outlet for the permeate is attached to both the first and the second edge regions of the cross-flow diafiltration unit. In another embodiment of the invention, the outlet for the permeate is alternatively or additionally attached to the third and / or fourth edge regions of the cross-flow diafiltration unit. The third edge region is located on the left side in the flow direction when the cross-flow diafiltration unit is viewed in plan from the diafiltration channel side. Correspondingly, the fourth edge region is located on the right side and thus opposite the third edge region. The above-mentioned arrangement of the outlet(s) makes it possible to achieve not only particularly high permeation performances but also design advantages.

[0044] The first edge region preferably comprises the outer third of the length of the filtration unit opposite the direction of flow. Correspondingly, the second edge region comprises the outer third of the length of the filtration unit along the direction of flow. The same is true for the third and fourth edge regions. It is advantageous to make the first to fourth edge regions as small as possible. It is therefore particularly preferred that the edge regions comprise the respective outer 20%, even more preferably the respective outer 10%, most preferably the respective outer 3%.

[0045] In principle, there are no particular constraints on the mounting of the inlets and outlets. For example, the inlets and outlets may be mounted such that the feed fluid already enters the retentate channel in the direction of flow and leaves the retentate channel in the direction of flow. Correspondingly, the outlet for the permeate can be mounted such that the permeate leaves the permeate collection channel in the direction of flow and / or the inlet for the diafiltration medium can be mounted such that it enters the diafiltration channel in the direction of flow. However, preferably, the inlets and outlets are mounted such that the diafiltration medium enters the diafiltration channel perpendicular to the direction of flow, and then the feed fluid enters the retentate channel perpendicular to the direction of flow and leaves it as retentate perpendicular to the direction of flow. Such mounting of the inlets and outlets facilitates the arrangement of a plurality of the inventive filtration units to form a filter cassette. A respective arrangement is for example shown diagrammatically in FIG. 2. As shown in FIG. 2, the second fluid is fed via the feed inlet. Due to the selected MWCO of the membrane, the target components are retained in the retentate channel, while the smaller components of the second fluid permeate the membrane and are removed at the permeate outlet. This has the advantage of increasing the purity of the target component. At the same time, diafiltration buffer is actively supplied by an additional channel to permeate the membrane and wash other components of the second fluid (including buffer components) from the retentate into the permeate (buffer exchange).

[0046] Preferably, the cross-flow diafiltration unit comprises multiple inlets for a feed fluid (here, the second fluid), multiple outlets for a retentate (here, the third fluid), multiple inlets for a diafiltration medium, and multiple outlets for a permeate, in each of the retentate, diafiltration, and permeate channels, respectively. Each of the multiple inlets is supplied with fluid / medium by one respective inlet channel that branches into each of the multiple inlets. Each of the multiple outlets couples to one respective outlet channel and supplies a respective fluid thereto.

[0047] In continuous diafiltration, both the feed fluid (here, the second fluid) and the diafiltration medium are added continuously so that there is no need to interrupt the process. As a result, cross-flow diafiltration units make it possible to carry out the process in an efficient and economical manner.

[0048] The diafiltration medium is not particularly limited and is selected according to the second chromatographic device. In principle, any fluid is suitable, with water and aqueous salt solutions being preferred. As the diafiltration medium, for example, an aqueous buffer solution can be used. Preferably, the diafiltration medium is selected from the group consisting of KPI buffer, sodium phosphate buffer, sodium acetate buffer, PBS, glycine, citrate buffer, Tris buffer, BIS-Tris buffer, HEPES buffer and water. The concentration of the buffer in the aqueous solution is not particularly limited and may be, for example, 1.0 mM to 5.0 M, preferably 5.0 mM to 1.0 M, more preferably 10 mM to 200 mM, and most preferably 25 to 100 mM. The diafiltration medium and its conditions can be appropriately selected depending on the components of the third fluid to be separated (impurities and target components) and the second chromatographic device to be applied. For this purpose, for example, a statistical experimental design (DoE) approach as described above can be applied.

[0049] To optimize the yield and / or purity achieved by the second chromatographic device, the conditions of the diafiltration medium and the third fluid can be appropriately set (e.g., by selecting appropriate buffers, which can be evaluated by DoE).

[0050] The pH of the diafiltration medium and the third fluid is not particularly limited. For example, the pH of the diafiltration medium may be 3.0 to 14.0, preferably 5.0 to 12.0, more preferably 6.0 to 10.0, and most preferably 6.0 to 9.0. The pH of the third fluid may be 3.0 to 14.0, preferably 5.0 to 12.0, more preferably 6.0 to 10.0, and most preferably 6.0 to 9.0.

[0051] The conductivity of the diafiltration medium and the third fluid is not particularly limited and may be, for example, 0 mS / cm to 500 mS / cm, preferably 0 mS / cm to 150 mS / cm.

[0052] Preferably, the second and third fluids differ in at least one property selected from the group consisting of pH, conductivity, salt concentration (eg, ammonium sulfate), and buffer composition.

[0053] For example, if the first and second chromatographic devices are a combination of AEX and CEX or CEX and AEX, the pH can be changed, in particular, from the second fluid to the third fluid. This advantageously gives the opportunity to remove impurities, preferably with different isoelectric points. If the first and second chromatographic devices are a combination of AEX and CEX or CEX and AEX, the conductivity can be changed, in particular, from the second fluid to the third fluid. This advantageously gives the opportunity to remove impurities based on affinity for ligands (ionic interactions). If the first and second chromatographic devices are a combination of AEX and HIC or CEX and HIC, the ammonium sulfate concentration can be changed, in particular, from the second fluid to the third fluid. This advantageously gives the opportunity to remove impurities / aggregates based on hydrophobicity. If the first and second chromatographic devices are a combination of AEX and HIC or CEX and HIC, the conductivity can be changed, in particular, from the second fluid to the third fluid. This advantageously gives the opportunity to remove impurities / aggregates based on hydrophobicity. If the first and second chromatographic devices are a combination of HIC and AEX or a combination of HIC and CEX, in particular ammonium sulfate can be removed or the conductivity can be reduced in particular from the second fluid to the third fluid, which advantageously gives the opportunity to bind impurities, preferably on the AEX / CEX ligands (impurity removal).

[0054] In a preferred embodiment, the cross-flow diafiltration unit is configured such that the volumetric flow rate of the diafiltration medium supplied is 0.5 to 20 times the volumetric flow rate of the second fluid supplied (i.e., the feed fluid supplied). The volumetric flow rate of the diafiltration medium supplied is preferably 1.0 to 15 times, more preferably 2.0 to 10 times, more preferably 4.0 to 9.0 times, and most preferably 5.0 to 7.0 times the volumetric flow rate of the second fluid supplied.

[0055] The volumetric flow rate is not limited to any particular value and may depend on the exploit size of the chromatography device and the diafiltration unit. For example, the volumetric flow rate of the diafiltration medium supplied may be 1.0 mL / min to 2.0 L / min, preferably 2.0 mL / min to 1.0 L / min, more preferably 4.0 mL / min to 500 mL / min, and most preferably 10 mL / min to 175 mL / min. For example, the volumetric flow rate of the second fluid supplied may be 0.5 mL / min to 100 mL / min, preferably 1.0 mL / min to 50 mL / min, and more preferably 2.0 mL / min to 25 mL / min. For example, the volumetric flow rate of the retentate / third fluid at the outlet may be 0.5 mL / min to 100 mL / min, preferably 1.0 mL / min to 50 mL / min, and more preferably 2.0 mL / min to 25 mL / min.

[0056] In a preferred embodiment, the diafiltration medium is supplied at a pressure of 0.1 to 4 bar. More preferably, the diafiltration medium is supplied at a pressure greater than the retentate / third fluid outlet pressure.

[0057] Preferably, diafiltration is carried out continuously, ie under constant / continuous addition of diafiltration medium and feed fluid, so as to provide a particularly efficient and economical filtration method.

[0058] After cross-flow diafiltration, (the majority of) the target component is preferably contained in the retentate (third fluid). Thus, the mass ratio of the mass of the target component contained in the retentate (third fluid) to the total mass of the target components subjected to diafiltration (i.e. in the second fluid) is preferably more than 50% by mass, more preferably at least 80% by mass, more preferably at least 90% by mass, and most preferably at least 95% by mass.

[0059] The means for providing the fluids and media are not particularly limited. For example, pressure tanks, pumps, and valves can be used as means for providing (including passing) the fluids and media. Valves can be used, for example, instead of retentate pumps, and pressure can be controlled. In a preferred embodiment, the system includes at least three means, more preferably three means, for providing the fluids and media. In particular, valves or pumps can be used to provide the third fluid to the second chromatography medium. Preferably, pumps are used to provide the fluids and media. In a preferred embodiment, the system includes at least three pumps, more preferably three pumps. For example, pumps are provided for each of the first fluid, the diafiltration medium, and the third fluid. Examples of each are shown, for example, in FIG. 1. The system can operate with any type of pump, as well as with stand-alone pumps, or with a more complex system with at least three pumps. By changing the flow rates of the three pumps, the desired buffer exchange rate and the concentration or dilution of the product can be adjusted. Each location of the means for providing fluids and media may be provided with means for monitoring the condition of the respective fluid / media, such as pH meters, conductivity sensors, pressure sensors, spectrometers (e.g., Raman, UV / VIS, NIR), and flow meters.

[0060] The fourth fluid can be subjected to further purification steps, such as affinity and / or hydrophobic interaction, and / or cation and / or anion exchange chromatography, and / or sterile filtration and / or virus filtration, and / or virus inactivation, and / or precipitation and / or crystallization and / or extraction (aqueous two-phase extraction) and / or lyophilization, in any suitable order. Thus, the separation system of the present invention can further comprise respective further purification units, such as affinity and / or hydrophobic interaction, and / or cation and / or anion exchange chromatography units, and / or sterile filtration and / or virus filtration units, and / or virus inactivation units and / or precipitation units and / or crystallization units and / or extraction units (aqueous two-phase extraction units) and / or lyophilization units, in any suitable order. Between each unit, a further single-pass cross-flow diafiltration unit can be included.

[0061] Apart from the first and second chromatographic devices and the single-pass cross-flow diafiltration unit, the separation system can include further such chromatographic devices and single-pass cross-flow diafiltration units. For example, the fourth fluid obtained from the second chromatographic device can be provided to a further single-pass cross-flow diafiltration unit, and the fourth fluid is continuously diafiltered to obtain a second permeate and a fifth fluid containing the target component. The fifth fluid can then be provided to a third chromatographic device, where it is processed to provide a sixth fluid containing the target component. This can also be further extended to the above method.

[0062] In a further aspect, the present invention relates to a method for purifying a target component contained in a first fluid, the method comprising the steps of: (a) providing a first fluid to a first chromatography device; (b) processing the first fluid in a first chromatography device to obtain a second fluid containing the target component; (c) providing a second fluid to a single-pass cross-flow diafiltration unit; (d) treating the second fluid with a diafiltration medium in a single-pass cross-flow diafiltration unit to obtain a third fluid containing the target component; (e) providing a third fluid to a second chromatography device; (f) processing the third fluid in a second chromatography device to obtain a fourth fluid containing the target component; The single-pass cross-flow diafiltration unit is integrally integrated between the first and second chromatography devices. The above statements and definitions apply analogously to this aspect of the invention. Preferably, the method according to the invention is carried out using a separation system according to the invention.

[0063] The method of the present invention may further comprise the step of performing DoE on the target component. For example, these steps may include the following steps: screening of significant variables, analysis, optimization and validation of a process model. As a result, it is possible to obtain first and second chromatography devices, other components of the fluid, and / or diafiltration media that are preferably optimized (e.g., with respect to yield and / or purity) for a particular target component.

[0064] In a further aspect, the present invention relates to the use of a single-pass cross-flow diafiltration unit for connecting together a first chromatography device and a second chromatography device suitable for purifying a target component contained in a first fluid, the first chromatography device being configured to receive and process the first fluid and provide a second fluid containing the target component, The single-pass cross-flow diafiltration unit is configured to continuously diafilter a second fluid with a diafiltration medium to obtain a permeate and a third fluid containing a target component as a retentate; The second chromatography device is configured to receive and process the third fluid to provide a fourth fluid containing the target component. The above statements and definitions apply equally to this aspect of the invention. Preferably, the single-pass cross-flow diafiltration unit is a single-pass cross-flow diafiltration unit of a separation system according to the invention. Preferably, the first chromatography device and the second chromatography device are the first and second chromatography devices of a separation system according to the invention. [Brief description of the drawings]

[0065] [Figure 1] Schematic diagram of the separation system of the present invention: Direct combination of two different chromatographic media in flow-through mode with buffer exchange integrated between them by single-pass tangential flow filtration. [Diagram 2] Possible construction of a single-pass cross-flow diafiltration unit (8) of the present invention having a flat second filter material (23) and a flat first filter material (24), with the flow of the diafiltration medium (2), second fluid (21), retentate / third fluid (22) and permeate (13) indicated by arrows. The diafiltration, retentate and permeate collection channels are kept open by spacers (25) for the respective media. [Diagram 3] 1 shows the breakthrough curves of mAb and DNA as products and HCP as impurities after the second chromatography step of an embodiment of the present invention. The breakthrough was determined by the ratio of the concentration from the flow-through of the second chromatography device to the concentration of the feed solution. [Figure 4] FIG. 1 is a schematic diagram of a separation system of the present invention having two parallel membrane absorbers as a first chromatographic device, a single-pass cross-flow diafiltration unit, and two parallel membrane absorbers as a second chromatographic device, as well as a means for switching between the operation mode of the parallel membrane absorbers (path 1 shown at the top; separation line) and the regeneration mode (paths 2 and 3 shown at the bottom). [Diagram 5] Schematic diagram of the separation system of FIG. 4 after switching the membrane absorber in the operating mode in both the first and second chromatography devices to the regeneration mode (FIG. 4) (paths 2 and 3 are shown here) and after switching the membrane absorber in the regeneration mode in both the first and second chromatography devices (FIG. 4) to the operating mode (path 1 is shown here; separation line).

[0066] The present invention is further illustrated in the following non-limiting examples. EXAMPLES

[0067] A CHO cell line was used to produce mAb in perfusion culture. The cells were cultured at 36.8°C and pH 6.95 using a commercially available serum-free medium. An alternating flow filtration membrane was used as a cell retention device to collect the perfusion permeate containing the mAb as the product. As a first capture step, Protein A affinity chromatography was performed, followed by viral inactivation by acidification (pH 3.4). Diafiltration was then performed to obtain the mAb in the appropriate buffer conditions of the first chromatography medium. This resulted in a first fluid.

[0068] As the first chromatographic medium, an AEX membrane adsorber (Sartobind Q; strong anion exchanger, ligand: quaternary ammonium, membrane material: stabilized reinforced cellulose, pore size: 3-5 μm, ligand density: 2-5 μeq / cm) was used. 2 ), and as the second chromatographic medium, a CEX membrane adsorber (Sartobind S; strong cation exchanger, ligand: sulfonic acid, membrane material: stabilized reinforced cellulose, pore size: 3–5 μm, ligand density 2–5 μeq / cm 2 ) was used. Suitable buffer conditions were obtained by a DoE-based approach. In this regard, a conductivity of 4.2 mS / cm at pH 8.0 was evaluated for the first chromatographic medium and a conductivity of 4.0 mS / cm at pH 8.5 was evaluated for the second chromatographic medium.

[0069] The process setup is shown in Figure 1. Both the chromatography steps and the buffer exchange in between were performed in only one unit operation instead of three (first chromatography step, diafiltration for buffer exchange and second chromatography step) using a system with three pumps. A high yield of mAb was obtained in the flow-through of the second chromatography device throughout the complete process. This was shown by the high breakthrough of mAb shown in Figure 3 (variations and values ​​slightly above 100% were due to small variations in the pump flow rate). At the same time, a high removal of HCPs (88%, corresponding to an average amount of 29 ppm in the second fluid over all fractions) and DNA (96%, corresponding to an average amount of 2 ppm in the fourth fluid over all fractions) was obtained throughout the process. [Explanation of symbols]

[0070] Figures 1 and 2 1 First Fluid (Buffer Solution for First Chromatography Device) 2 Diafiltration medium (second buffer solution) 3 Diafiltration media pump 4. First fluid pump 5. A first chromatography device having a first chromatography medium 6 Retentate Channel 7 Diafiltration Channel 8 Single Pass Tangential Flow Filtration Cassettes 9 Permeate Channel 10 Pump for the third fluid (retentate) 11 A second chromatography device having a second chromatography medium 12 Fourth fluid (flow-through of the second chromatography device (containing the purified target component)) 13 Permeate 21 Second Fluid 22 Retentate / Third Fluid 23 Second filter material 24 First filter material 25 Spacer

Claims

1. A separation system configured to process a first fluid (1) containing multiple components, wherein at least one of the multiple components of the first fluid (1) is a target component, and the separation system The system comprises a first chromatography apparatus (5) and a second chromatography apparatus (11), and a single-pass cross-flow dialysis filtration unit (8) integrally incorporated between the first chromatography apparatus (5) and the second chromatography apparatus (11). The first chromatography apparatus (5) is configured to receive and process the first fluid (1) and provide a second fluid (21) containing the target compound. The single-pass cross-flow dialysis filtration unit (8) is configured to continuously dialysis-filter the second fluid (21) using a dialysis filtration medium (2) for obtaining a permeate (13) and a third fluid (22) containing the target component as a retaining fluid. A separation system in which the second chromatography apparatus (11) is configured to receive and process the third fluid (22) to provide a fourth fluid (12) containing the target compound.

2. A method for purifying a target component contained in a first fluid (1), wherein the method is (a) A step of providing the first fluid (1) to the first chromatography apparatus (5), (b) A step of processing the first fluid (1) in the first chromatography apparatus (5) to obtain a second fluid (21) containing the target component, (c) A step of providing the second fluid (21) to a single-pass cross-flow dialysis filtration unit (8), (d) A step of processing the second fluid (21) with a dialysis filter medium (2) in the single-pass cross-flow dialysis filtration unit (8) to obtain a third fluid (22) containing the target component, (e) A step of providing the third fluid (22) to the second chromatography apparatus (11), (f) A step of processing the third fluid (22) in the second chromatography apparatus (11) to obtain a fourth fluid (12) containing the target component, A method comprising the single-pass cross-flow dialysis filtration unit (8) being integrally incorporated between the first chromatography apparatus (5) and the second chromatography apparatus (11).

3. The use of a single-pass cross-flow dialysis filtration unit (8) for integrally connecting a first chromatography apparatus (5) and a second chromatography apparatus (11) suitable for purifying a target component contained in a first fluid (1), wherein the first chromatography apparatus (5) is configured to receive and process the first fluid (1) and provide a second fluid (21) containing the target component. The single-pass cross-flow dialysis filtration unit (8) is configured to continuously dialysis-filter the second fluid (21) using a dialysis filtration medium (2) for obtaining a permeate (13) and a third fluid (22) containing the target component as a retaining fluid. The second chromatography apparatus (11) is configured to receive and process the third fluid (22) to provide a fourth fluid (12) containing the target component.

4. The single-pass cross-flow dialysis filtration unit comprises at least a dialysis filtration channel (7), a first filter material (24), a retaining fluid channel (6), a second filter material (23), and a permeate collection channel (9), wherein the first filter material (24) is arranged to define the dialysis filtration channel (7) and the retaining fluid channel (6) from each other, and the second filter material (23) is arranged to define the retaining fluid channel (6) and the permeate collection channel (9) from each other. The separation system according to claim 1, wherein the dialysis filtration channel (7) is connected in a fluid conduction manner to at least one inlet for the dialysis filtration medium (2), the retaining fluid channel (6) is connected in a fluid conduction manner to at least one inlet for the second fluid (21) and at least one outlet for the third fluid (22), and the permeate collection channel (9) is connected in a fluid conduction manner to at least one outlet for the permeate (13).

5. The single-pass cross-flow dialysis filtration unit comprises at least a dialysis filtration channel (7), a first filter material (24), a retaining fluid channel (6), a second filter material (23), and a permeate collection channel (9), wherein the first filter material (24) is arranged to define the dialysis filtration channel (7) and the retaining fluid channel (6) from each other, and the second filter material (23) is arranged to define the retaining fluid channel (6) and the permeate collection channel (9) from each other. The method according to claim 2, wherein the dialysis filtration channel (7) is connected in a fluid conduction manner to at least one inlet for the dialysis filtration medium (2), the retaining fluid channel (6) is connected in a fluid conduction manner to at least one inlet for the second fluid (21) and at least one outlet for the third fluid (22), and the permeate collection channel (9) is connected in a fluid conduction manner to at least one outlet for the permeate (13).

6. The single-pass cross-flow dialysis filtration unit comprises at least a dialysis filtration channel (7), a first filter material (24), a retaining fluid channel (6), a second filter material (23), and a permeate collection channel (9), wherein the first filter material (24) is arranged to define the dialysis filtration channel (7) and the retaining fluid channel (6) from each other, and the second filter material (23) is arranged to define the retaining fluid channel (6) and the permeate collection channel (9) from each other. The use according to claim 3, wherein the dialysis filtration channel (7) is connected in a fluid conduction manner to at least one inlet for the dialysis filtration medium (2), the retaining fluid channel (6) is connected in a fluid conduction manner to at least one inlet for the second fluid (21) and at least one outlet for the third fluid (22), and the permeate collection channel (9) is connected in a fluid conduction manner to at least one outlet for the permeate (13).

7. The separation system according to claim 4, wherein the second filter material (23) has a pore diameter smaller than the target component and larger than at least one of the other components of the second fluid (21).

8. The method according to claim 5, wherein the second filter material (23) has a pore diameter smaller than the target component and larger than at least one of the other components of the second fluid (21).

9. The use according to claim 6, wherein the second filter material (23) has a pore diameter smaller than the target component and larger than at least one of the other components of the second fluid (21).

10. The separation system according to any one of claims 1 to 4, wherein the second fluid (21) and the third fluid (22) differ in at least one characteristic selected from the group consisting of pH, conductivity, salt concentration, and buffer composition.

11. The method according to any one of claims 2 and 5, wherein the second fluid (21) and the third fluid (22) differ in at least one property selected from the group consisting of pH, conductivity, salt concentration, and buffer composition.

12. The use according to any one of claims 3 and 6, wherein the second fluid (21) and the third fluid (22) differ in at least one property selected from the group consisting of pH, conductivity, salt concentration, and buffer composition.

13. The separation system according to any one of claims 1 and 4, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) are each independently a plurality of chromatography apparatuses.

14. The method according to either claim 2 or 5, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) are each independently a plurality of chromatography apparatuses.

15. The use according to any one of claims 3 and 6, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) are each independently a plurality of chromatography apparatuses.

16. The separation system according to any one of claims 1 and 4, wherein in each of the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is independently selected from the group consisting of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed-mode chromatography, and size exclusion chromatography.

17. The method according to either claim 2 or 5, wherein in each of the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is independently selected from the group consisting of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed-mode chromatography, and size exclusion chromatography.

18. The use according to any one of claims 3 and 6, wherein in each of the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is independently selected from the group consisting of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed-mode chromatography, and size exclusion chromatography.

19. The separation system according to claim 16, wherein in the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is selected from AEX and CEX, and CEX and AEX.

20. The method according to claim 17, wherein in the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is selected from AEX and CEX, and CEX and AEX.

21. The use according to claim 18, wherein in the first chromatography apparatus (5) and the second chromatography apparatus (11), the chromatography method is selected from AEX and CEX, and CEX and AEX.

22. The separation system according to any one of claims 1 and 4, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) each independently include a chromatography medium selected from the group consisting of a membrane adsorbent, a chromatography resin, and a monolith.

23. The method according to either claim 2 or 5, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) each independently include a chromatography medium selected from the group consisting of a membrane adsorbent, a chromatography resin, and a monolith.

24. The use according to any one of claims 3 and 6, wherein the first chromatography apparatus (5) and the second chromatography apparatus (11) each independently include a chromatography medium selected from the group consisting of a membrane adsorbent, a chromatography resin, and a monolith.

25. The separation system according to claim 22, wherein the first chromatography apparatus and the second chromatography apparatus are independently selected from an AEX membrane absorber and a CEX membrane absorber.

26. The method according to claim 23, wherein the first chromatography apparatus and the second chromatography apparatus are independently selected from an AEX membrane absorber and a CEX membrane absorber.

27. ​​The use according to claim 24, wherein the first chromatography apparatus and the second chromatography apparatus are independently selected from the AEX membrane absorber and the CEX membrane absorber.

28. The separation system according to any one of claims 1 and 4, wherein the system comprises at least three means for providing a fluid and medium selected from the group consisting of a pressure tank, a pump, and a valve.

29. The method according to any one of claims 2 and 5, wherein the system comprises at least three means for providing a fluid and medium selected from the group consisting of a pressure tank, a pump, and a valve.

30. The use according to any one of claims 3 and 6, wherein the system comprises at least three means for providing a fluid and medium selected from the group consisting of a pressure tank, a pump, and a valve.

31. The separation system according to claim 28, wherein three pumps (3, 4, 10) are used as means for providing the fluid and medium.

32. The method according to claim 29, wherein three pumps (3, 4, 10) are used as means for providing the fluid and medium.

33. The use according to claim 30, wherein three pumps (3, 4, 10) are used as means for providing the fluid and medium.

34. The separation system according to any one of claims 1 and 4, wherein the single-pass cross-flow dialysis filtration unit is configured such that the volumetric flow rate of the supplied dialysis filtration medium is 0.5 to 20 times the volumetric flow rate of the supplied second fluid.

35. The method according to any one of claims 2 and 5, wherein the single-pass cross-flow dialysis filtration unit is configured such that the volumetric flow rate of the supplied dialysis filtration medium is 0.5 to 20 times the volumetric flow rate of the supplied second fluid.

36. The use according to any one of claims 3 and 6, wherein the single-pass cross-flow dialysis filtration unit is configured such that the volumetric flow rate of the supplied dialysis filtration medium is 0.5 to 20 times the volumetric flow rate of the supplied second fluid.

37. The separation system according to any one of claims 1 and 4, wherein the target component is selected from proteins, antibodies, hormones, vaccines, nucleic acids, exosomes, viruses, and virus-like particles.

38. The method according to any one of claims 2 and 5, wherein the target component is selected from proteins, antibodies, hormones, vaccines, nucleic acids, exosomes, viruses, and virus-like particles.

39. The use according to any one of claims 3 and 6, wherein the target component is selected from proteins, antibodies, hormones, vaccines, nucleic acids, exosomes, viruses, and virus-like particles.

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