Apparatus for purifying liquid comprising target substance

Abstract

To provide a simplified and broadly applicable apparatus for treating solutions containing target substances.SOLUTION: Provided is an apparatus for converting a liquid feedstock comprising a target substance and impurities into a product stream containing purified target substances and one or more waste stream comprising at least some of the impurities, the apparatus comprising at least two processing units arranged in series such that the feed stream of the second and any subsequent units comprises the product stream from a downstream unit, each processing unit comprising the following components (i) to (vi): (i) an inlet 1 for the liquid feedstock; (ii) a multiple inlet flow-controller 4 for providing at least two liquids in a desired ratio; (iii) mixing means 8; (iv) a device 12 for separating the impurities from the target substance; (v) means 6 for imparting the flow of liquids through the unit; and (vi) a switchable bypass assembly 31 for liquids passing through the unit.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to units, flow path assemblies and devices for processing liquids containing target substances, in particular liquids containing biomolecules, such as recombinant polypeptides. [Background technology]

[0002] Many biomolecules, especially recombinant polypeptides and nucleic acids such as plasmids (pDNA), have attracted considerable attention, particularly for therapeutic applications. Such biomolecules are generally produced by culturing recombinant host cells that have been genetically engineered to express the desired biomolecule. The biomolecule is then typically recovered from the culture medium by a process involving several unit operations.

[0003] Devices for processing solutions containing target substances are known in the art. However, equipment for use in the commercial manufacture of such compounds is typically very bulky and requires large amounts of floor space and infrastructure. Furthermore, while some commonality of equipment can be achieved across some unit operations, the design of equipment for specific unit operations, such as viral inactivation and / or ultrafiltration, is significantly different from that of, for example, chromatographic purification. This means that more space is required to accommodate two or more sets of equipment, or the interoperability and control of equipment for multiple stages is very complex. Furthermore, operators require training on the various types of equipment used. Therefore, simplified, broadly applicable equipment would be desirable. It would also be desirable to identify equipment that allows multiple processing steps to be performed using a common flow path. Summary of the Invention [Means for solving the problem]

[0004] According to a first aspect of the present invention, there is provided an apparatus for converting a liquid feedstock comprising a target substance and impurities into a product stream comprising the purified target substance, and optionally one or more waste streams comprising at least a portion of the impurities, the apparatus comprising at least two processing units arranged in series such that the feed stream of the second and any subsequent unit comprises the product stream from the downstream unit, each processing unit comprising the following components (i) to (vi): (i) Inlet for liquid raw material (1); (ii) a multiple inlet flow controller (4) including two or more variable flow inlet valves (4a) for providing at least two liquids in a desired ratio; (iii) mixing means (8); (iv) a device (12) for performing a processing operation that separates at least a portion of the impurities from the target material; (v) means for imparting a flow of liquid through the unit (6); (vi) a switchable bypass assembly (31) for directing liquid passing through the unit either into the mixing means (8) or bypassing the mixing means (8); An apparatus is provided, comprising:

[0005] The present invention provides an apparatus that can be implemented on a manufacturing scale and offers many advantages over previous apparatus, particularly with regard to simplicity, cost and ease of operation, reduced risk of operator error, easier maintenance, and reduced spare parts inventory.

[0006] In this specification, the phrase "processing unit" is often abbreviated to "unit" and The two are used interchangeably. In the accompanying drawings: [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a schematic diagram of one possible processing unit in the apparatus of the present invention. [Figure 2]FIG. 10 is a schematic diagram of a flow path assembly that may be present in a unit of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0008] FIG. 1 is described in more detail in the Examples section below. FIG. 2 schematically illustrates a flow path assembly that can be used in one or more (preferably at least half, more preferably all) of the processing units. The flow path assembly includes identified components connected by tubing, e.g., tubing made of a material that can be sterilized by gamma irradiation (e.g., a plastic material), and is preferably treated or washed and reused after each use. The flow path assembly includes six inlets (2a) through (2f) for six different liquids (e.g., buffer solution, acidic solution, alkaline solution, organic solvent, etc.) and a seventh inlet (2g) for another liquid, e.g., water. The six liquids pass through respective valves (3a, 3b), (3c, 3d), and (3e, 3f) to generate three liquid streams composed of the six liquids in desired proportions that flow into a multi-inlet flow controller (4). In this embodiment, the flow path assembly further includes a fourth tube fitted with a valve (3g) for introducing an additional liquid (e.g., water) into the multi-inlet flow controller (4) (from inlet (2g)). The inlet (1) fitted with a valve (3) has a check valve (3h) that can be used to introduce a liquid feed containing a target substance (e.g., a monoclonal antibody) and impurities into the flow path assembly. Downstream of the multi-inlet flow controller (4), the liquid stream passes through a means for providing liquid flow (6) (e.g., an impeller blade or pump head attached to a motor located outside the flow path), a block for a pressure sensor (7), and then flows into a switching bypass assembly (31). The switching bypass assembly (31) has three connections: one leading to a mixing means (8) fitted with a bubble trap, one leading to the outside of the mixing means (8), and one leading to the device feed inlet (12a). After flowing through a device (12) for performing a processing operation (device (12) not shown in Figure 2), the purified liquid feed passes through an outlet (12b) and a combination of a pressure sensor, pH sensor, and UV sensor (32).Finally, the purified liquid feed passes through an outlet line containing a series of valves (17), (19), and (20), which allow for control of flow between an exit feed outlet (18), a waste stream outlet (21), and a product stream outlet (22).

[0009] A flow path assembly comprising components (i)-(iii), (v) and (vi) as defined above in relation to a unit forms a further feature of the present invention, as does a unit comprising components (i)-(vi) and a unit comprising a flow path assembly and component (iv).

[0010] Typically, each unit performs one processing operation. The number of processing units is not particularly limited and depends on the majority of purification steps required to convert the liquid feedstock into a form suitable for the desired purpose, e.g., a drug product for a pharmaceutical formulation. In some embodiments, the apparatus includes two units (e.g., to perform two processing operations). In other embodiments, the apparatus includes more than two units, e.g., (at least) three, four, five, six, seven, eight, nine, or more units, preferably each unit having characteristics (i)-(vi) above.

[0011] The apparatus may further include two or more units operating in parallel, but all units Preferably, the units are arranged in series (e.g., connected in series, optionally with break bags between each unit, if desired). In many highly preferred embodiments, the method operations performed in each unit are different from the method operations used in all other units. Thus, an apparatus may include multiple units, for example, for performing chromatography, but each such chromatography unit (or the method in which it is used) is preferably different from the other units for performing chromatography.

[0012] In certain embodiments, each unit includes flow path assemblies that are substantially identical to at least half, and more preferably all, of the flow path assemblies of the other units. Preferably, each unit prepares its mixed bioprocessing liquid (in situ) during operation of the apparatus, thus avoiding the risk of the operator using the wrong externally prepared bioprocessing liquid intended for another process operation.

[0013] In certain embodiments, each unit includes a flow path assembly that is substantially the same as the other units. In one embodiment, the product stream from each unit is fed directly to the next unit (if there is one). In other embodiments, it is often convenient for the product stream of one or more units to be fed to a storage vessel (e.g., a "break bag") and then used as a feed for the next unit (if there is one). In this way, it is possible to test the product stream before it enters the next unit, pause the process, etc.

[0014] The raw material of the first unit may also be supplied from a storage container, or if desired, it may be supplied directly from a cell culture device, e.g., a bioreactor. Examples of suitable storage containers include tanks and bags.

[0015] The inlet (1) for the liquid feedstock typically comprises a tube fitted with a valve (3) and, optionally, a check valve (3h). The check valve (3h) is useful to avoid contamination of the liquid feedstock with the liquid flowing through the multi-inlet flow controller (4). The liquid feedstock for the second and any subsequent processing units typically comprises the product from the preceding processing unit.

[0016] The multi-inlet flow controller (4) preferably includes variable flow inlet valves (4a), more preferably intermittent flow inlet valves, that control the flow of at least two liquids (e.g., two, three, four, five, six, seven, or eight liquids) through the multi-inlet flow controller (4). The multi-inlet flow controller (4) includes at least two variable flow inlet valves (4a), and often includes up to eight, such as three, four, five, six, or seven, variable flow inlet valves (4a). The variable flow inlet valves (4a) may each have the same dimensions, or one or more of the variable flow inlet valves (4a) may have different dimensions. In certain preferred embodiments, the measured volume from each variable flow inlet valve (4 a) to the outlet of the multi-inlet flow controller (4) is the same for each variable flow inlet valve, and it is highly preferred that both the measured volume and path length from each variable flow inlet valve (4 a) to the outlet of the multi-inlet flow controller (4) are the same for each variable flow inlet valve.

[0017] The multiple inlet flow controller (4) used in the present invention also includes at least one outlet, and although there can be more than one outlet, it is preferred that a single outlet is used.

[0018] The variable flow valve (4a) can adjust the flow between a first, relatively low flow rate that maintains liquid flow and at least a second, higher flow rate. In a preferred embodiment, the variable flow inlet valve (4a) is an intermittent variable flow inlet valve, which prevents flow in a first position but allows flow in at least a second position. Most preferably, all valves are intermittent flow valves. The valves may include actuators known in the art, such as pneumatic actuators, or preferably solenoid actuators.

[0019] Preferably, the variable flow inlet valves (4a) of the multi-inlet flow controller (4) are most preferably controlled by a programmable control unit, which controls the opening and closing of the valves to achieve the required relative amounts of input liquids flowing through the multi-inlet flow controller. This is preferably achieved by cycling through the variable flow inlet valves (4a) of the multi-inlet flow controller (4) for a predetermined period or cycle rate to produce the desired composition, and controlling the opening and closing of the valves according to the required percentage of cycle time. The cycle rate can be either constant or variable. Most preferably, an intermittent flow inlet valve is used, controlled so that only one valve (4a) is open at any given time during operation. In many embodiments, the cycle rate of the multi-inlet flow controller (4) is maintained constant, and the desired relative amounts of input liquids are maintained constant.

[0020] In many embodiments, multiple cycles are used. The number of cycles used depends on many factors, such as the duration of the method, the amount of liquid to be treated, the flow rate and maximum operating pressure of the device. In certain embodiments, at least 10 cycles can be used, for example, at least 50, 100, 500, 750, 1000, 1500, 2000, 3000, 5000, 7500, 10000 or more cycles.

[0021] It will be appreciated that a range of cycle frequencies can be used. In many cases, the frequency will be less than 100 Hz, typically less than 50 Hz, usually less than 10 Hz, and preferably less than 5 Hz. In certain preferred embodiments, the frequency will be 2 Hz or less, most preferably 1 Hz or less, e.g., 0.05 to 0.5 Hz.

[0022] Mixing of at least two liquids (e.g., for preparing a bioprocessing liquid or for mixing a liquid with a liquid feedstock) can be achieved by simply combining the flow of the liquids through the outlets of the multi-inlet flow controller (4), optionally in combination with the action of the means for imparting a flow of liquids through the unit (6), while the unit includes a mixing means (8), preferably a mixing chamber, preferably including a static mixer, most preferably a time-delay, split-flow static mixer.

[0023] One of the at least two liquids is optionally a liquid ingredient. In many embodiments, the mixing means (8) is located downstream of the means for providing a flow of liquid through the unit (6) and upstream of the device for performing the processing operation (12). In some preferred embodiments, the mixing means (8) comprises a bubble trap.

[0024] The mixing means (8) is preferably suitable for combining the liquid raw material with one or more liquids to produce a device feed. The mixing means (8) is also preferably suitable for combining at least two other liquids to prepare a bioprocessing liquid.

[0025] Processing operations that can be performed by each unit include chromatography, viral inactivation, This includes filtration (e.g., viral removal), refolding, ultrafiltration, diafiltration, microfiltration, concentrating and / or performing buffer exchange, in-line pretreatment and refolding.

[0026] In some embodiments, the device includes at least two units for performing chromatographic purification of the target substance, and in many cases even at least three units for performing chromatographic purification of the target substance. The first unit for performing chromatographic purification of the target substance preferably includes an affinity chromatography column, such as a Protein A affinity column. The second unit for performing chromatographic purification of the target substance preferably includes an anion exchange chromatography column. The third unit for performing chromatographic purification of the target substance, if present, preferably includes a cation exchange chromatography column.

[0027] Thus, in one preferred embodiment, the device comprises the following units arranged in series, preferably in the order listed: a. A unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material Each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the raw material for the next unit.

[0028] In another preferred embodiment, the device comprises the following units arranged in series, preferably in the order listed: a. A unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography Each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the raw material for the next unit.

[0029] In another preferred embodiment, the device comprises the following units arranged in series, preferably in the order listed: a. A unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; d. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography. Each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the raw material for the next unit.

[0030] In another preferred embodiment, the device comprises the following units arranged in series, preferably in the order listed: a. A unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; d. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; e. Unit for removal of any inactivated viruses Each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the raw material for the next unit.

[0031] In another preferred embodiment, the device comprises the following units arranged in series, preferably in the order listed: a. A unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; d. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; e. A unit for the removal of any inactivated viruses; f. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange. Each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the raw material for the next unit.

[0032] Preferably, the units are arranged in series in the order listed herein, for example, in a device comprising units a. to g., the units are preferably arranged in series in the order a., b., c., d., e., then f.

[0033] Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a pressure sensor located upstream of component (iv).

[0034] Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a pressure sensor located downstream of component (iv).

[0035] Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a UV sensor located downstream of component (iv).

[0036] Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a pH sensor located downstream of component (iv).

[0037] Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a conductivity sensor located downstream of component (iv).

[0038] In a preferred embodiment, at least 75% of the component parts of each unit other than component (iv) are identical to the component parts used in at least 80% of the other units of the apparatus. Preferably, at least 85% of the component parts of each unit other than component (iv) are identical to the component parts used in at least 80% of the other units of the apparatus. Particularly preferably, at least 85% of the component parts of each unit other than component (iv) are identical to the component parts used in at least 80% of the other units of the apparatus. At least 95% of the components in each unit are identical to those used in at least 80%, more preferably at least 90%, of the other units in the apparatus. In particularly preferred embodiments, all components in each unit, except for component (iv), are identical to all components used in all other units in the apparatus. For the avoidance of doubt, the liquid flowing through the apparatus is not a component part of the apparatus. These embodiments are advantageous because the high degree of commonality between the components in each unit means that less spare parts inventory is required. Furthermore, because the units are similar, routine maintenance of the apparatus is simplified, and because each unit is similar to the other units in the apparatus, the apparatus is easier to operate (with less risk of destroying expensive target materials). In contrast to prior art techniques that use very different processing units from multiple manufacturers, technicians avoid having to learn how to repair a large number of very different processing units. Component (iv) in each unit is typically different from component (iv) in the other units (so that each unit can perform a distinct processing operation), hence the term "other than component (iv)."

[0039] In a preferred embodiment, all units of the apparatus are substantially identical except for device 12. In this embodiment, device 12 may be identical in two or more units, but more typically device 12 differs from unit to unit so that each unit performs a separate processing operation, as shown, for example, in FIG.

[0040] The device feed inlet can also be used to accept bioprocessing fluids, for example, from a multiple inlet flow controller (4) or from a mixing means (8). Bioprocessing fluids are useful for removing impurities from liquid feedstocks, for example, as conditioners or eluents in chromatography, as a means for inactivating viruses, as a means for washing target materials through filters, etc.

[0041] The apparatus optionally further comprises processing units for performing one or more of the following processing operations: chromatography, viral inactivation, filtration (e.g., ultrafiltration, microfiltration, dead-end filtration and / or diafiltration), viral removal, refolding, concentration and / or buffer exchange, flocculation and in-line pretreatment.

[0042] Chromatographic bioprocessing operations that can be performed using device (12) include affinity chromatography (e.g., Protein A affinity chromatography), ion exchange (either or both anion and cation exchange) chromatography, hydrophobic interaction chromatography (HIC), reversed-phase chromatography, expanded bed chromatography, mixed-mode chromatography, membrane chromatography, and size exclusion chromatography (SEC). In many embodiments, at least one of the units performs a Protein A affinity chromatography processing operation. Devices for performing chromatographic operations include suitable chromatographic devices, such as membranes, fiber monoliths, or resins. The number and arrangement of units performing chromatography are selected according to the nature of the target material.

[0043] Preferably, the apparatus comprises at least two, more preferably three, units comprising components (i) to (iv) for carrying out chromatographic purification of a target substance, where the chromatographic purification carried out in each unit is preferably carried out under different conditions and / or using chromatographic columns packed with different materials (e.g., different resins, membranes or monoliths) than those used in all other units. In a particularly preferred embodiment, at least one of the units performs affinity chromatography, at least one of the units performs cation exchange chromatography, and at least one of the units performs anion exchange chromatography.

[0044] A device (12) for performing a viral inactivation process typically includes a storage vessel capable of storing a liquid containing a target substance under conditions that inactivate any viruses present. In certain embodiments, the inlet and outlet of the viral inactivation device (12) can be fluidly connected to create a recirculation loop. In one such embodiment, the apparatus can be provided with a container or bag fluidly connected between the "device" inlet and the "device" outlet, one of which is fluidly connected to one of the inlets of the multi-inlet flow controller (4). The container or bag between the "inlet" and "outlet" of the device (12) fluidly connected to the liquid feedstock inlet (1) is filled by a means for providing flow (6), typically a pump, or pre-treated with at least one other liquid passed through at least one of the inlets of the other multi-inlet flow controller (4). In certain embodiments, the container or bag is a mixing vessel or bag. The bioprocessing liquid is recirculated through the inlet of the multi-inlet flow controller (4) to a container or bag and back to the inlet of the multi-inlet flow controller (4) as the solution containing the target substance is pre-treated by at least one additional liquid fluidly connected to at least one other inlet on the multi-inlet flow controller (4).

[0045] Virus inactivation can be carried out by several techniques using conditions known in the art. For example, a chromatography column, chromatography membrane, or holding tank can be used to incubate a fluid containing a liquid feedstock at a pH below about 4.0, e.g., between about 3.0 and about 4.0, preferably between about 3.2 and about 3.9, particularly between about 3.4 and about 3.8, and more particularly between about 3.45 and about 3.7. Preferably, the liquid feedstock is held at the aforementioned pH for a period of at least 25 minutes, e.g., between about 30 minutes and 1.5 hours, preferably between about 30 minutes and 1.25 hours, more preferably between about 0.75 and 1.25 hours, and particularly about 1 hour. In each case, the conditions selected are such that the target material is not damaged or destroyed.

[0046] Inactivated viruses can be removed by filtration using normal flow filters (NFF) or tangential flow filtration (TFF) filters, as described, for example, in U.S. Patent No. 6,365,395. In either TFF or NFF mode, filtration to remove inactivated viruses is typically performed under conditions that retain inactivated viruses, using membranes with average pore sizes of 20 to 100 nanometers (nm). Such membranes retain inactivated viruses on their surface while allowing passage of target substances through the membrane.

[0047] The unit for removing inactivated viruses can also remove viruses that survive the virus inactivation step. Representative suitable ultrafiltration membranes that can be used to remove inactivated viruses (along with any viruses that remain active) include membranes formed from regenerated cellulose, polyethersulfone, polyarylsulfone, polysulfone, polyimide, polyamide, polyvinylidene difluoride (PVDF), and the like, known as VIRESOLVE.RTM. and RETROPORE™ membranes, available from EMD Millipore, Billerica, Mass. These are available either in cartridge (NFF) form, such as the VIRESOLVE™ NFP virus filter, or as cassettes (for TFF), such as the PELLICON™ cassette, available from EMD Millipore, Billerica, Mass. It can be supplied either

[0048] Filtration operations that can be performed using device (12) include viral filtration, depth and absolute filtration, ultrafiltration, diafiltration, and microfiltration. In many embodiments, filtration device (12) includes a filter module between the device inlet and the device outlet. The filter module can be flushed and tracked using at least two liquid feeds attached to the multi-inlet flow controller inlet (4), and a liquid feed containing the target substance can be fluidly connected to the feed inlet (1). Processing of the liquid feed through filtration device (12) is accomplished through a flow-imparting means (6) fluidly connected downstream from the multi-inlet flow controller outlet (4) and the feed inlet (1), and upstream from filtration device (12). Filters are often modular and may use configurations known in the field of biomolecule purification.

[0049] Viral filtration, depth filtration, and absolute filtration process operations are known in the art and can be performed using commercially available filtration devices. In many embodiments, one or more filtration devices are located between the inlet and outlet of the device (12) to perform filtration as a processing operation. In other embodiments, additional filter devices are located downstream of the device outlet, which allows the device to perform most purification steps, such as chromatography, viral inactivation, tangential flow filtration, viral filtration, or depth filtration, followed by a secondary filtration operation outside the device, in certain embodiments.

[0050] Tangential flow filtration ("TFF") unit operations that can be performed using the device of the present invention include conventional recirculating TFF and single-pass TFF. In certain embodiments, the outlet and inlet of the device can be fluidly connected to create a recirculating loop, for example, recirculating tangential flow filtration. In one embodiment, as known in the art, the device is provided with a TFF module containing either a flat sheet, hollow fiber, or spiral-wound membrane between the device inlet and the device outlet, and the retentate from the TFF module is directed from one of the device outlets to a fluidly connected inlet on a container or bag that includes at least one inlet and one outlet. The outlet of the container or bag is fluidly connected to a liquid feed inlet. The container or bag is maintained at a constant level using an auxiliary means that is fluidly connected to a second inlet on the container or bag to supply feed or liquid to the container or bag. In another embodiment, the device is provided with a TFF module containing either a flat sheet, hollow fiber, or spiral-wound membrane between the device inlet and the device outlet, and the retentate from the TFF module is fluidly connected from one of the device outlets back to one of the inlets of the multi-inlet flow controller valve. In certain embodiments, the recirculation loop from the device outlet to its inlet includes a break vessel or bag. A solution containing the target substance or liquid is drawn into the recirculation loop through the liquid feed inlet by a means for providing flow, typically a pump. The retentate is recirculated through the TFF module, preferably through one of the multi-inlet flow controller inlets. A multi-inlet flow controller can be used to mix the retentate with at least one other liquid. The operation of recirculating TFF is known in the art and is controlled by setting the cross-flow rate and transmembrane pressure.

[0051] In certain embodiments, a single-pass TFF can be configured with a TFF module comprising either flat sheets, hollow fibers, or spiral-wound membranes between the device inlet and the device outlet, such as in the case of a single-pass TFF as described in WO2017 / 118835.

[0052] In some embodiments, a hybrid of single-pass and recirculating TFF is used. where a variable flow valve downstream of the TFF module is used to return the resulting retentate back to the feed vessel.

[0053] The device of the present invention optionally further comprises one or more of a bubble trap, a pressure sensor, a temperature sensor, a pH sensor, a flow sensor, a conductivity sensor, an air sensor, and a uv sensor such as a uv / visible multi-wavelength sensor.

[0054] Means for imparting liquid flow (6) are well known in the art and include the application of gas pressure to the liquid, particularly an inert gas such as nitrogen or helium. Preferably, the means for imparting liquid flow through the unit includes one or more pumps. Pumps that can be used include peristaltic pumps, diaphragm pumps, lobe pumps, and centrifugal pumps. Disposable and reusable pump designs can be used. In many preferred embodiments, a single pump is used for each means for performing a unit operation, located downstream of the fluid connection between the feedstock and the outlet from the flow controller (4). Most preferably, the pump is located upstream of the device (12). The type and size of the pump selected generally depends on the flow capacity and pressure profile appropriate for adjusting and designing the parameters of the apparatus. In certain highly preferred embodiments, the pump is a quaternary diaphragm pump.

[0055] The means (6) present in the flow path assembly typically includes one or more impellers, such as diaphragm impellers, which may be coupled to and driven by means for driving the impeller, such as a motor located outside the flow path assembly.

[0056] The switchable bypass assembly (31) is useful for providing the option of not allowing liquid ingredients to enter the mixing means (8) when desired, which provides the advantage that the device may also be used for the purification of fragile target materials where combining the liquid ingredients with bioprocessing liquids in the mixing means (8) could damage or degrade the target material.

[0057] The diverter bypass assembly (31) is also useful for mixing two or more fluids to prepare a bioprocessing fluid that can then be fed to the device (12) for processing operations.

[0058] The switching bypass assembly (31) is particularly useful when the device (12) includes a chromatography column. The switching bypass assembly (31) can be used to load a liquid feed containing a target substance and impurities into the chromatography column (12) without passing through the mixing means (8), and the mixing means (8) can then be used to prepare a bioprocessing liquid (the composition of which can be varied on demand using the multi-inlet flow controller (4)) that serves as an eluent for the target substance loaded into the column (12). Additionally, bypassing the mixing means (8) can be useful when the device (12) performs ultrafiltration and / or diafiltration process steps where hold-up volume and product stability may otherwise be an issue.

[0059] The diverter bypass assembly (31) preferably includes tubing and two or three valves that direct the flow of liquid raw materials and bioprocessing liquids either to the mixing means (8) or to the device (12) without passing through the mixing means (8).

[0060] In one particular embodiment of the present invention, each unit comprises the following components (i) to (vi): (i) Inlet for liquid raw material (1); (ii) a multiple inlet flow controller (4) including two or more variable flow inlet valves (4a) for providing at least two liquids in a desired ratio; (iii) mixing means (8); (iv) an outlet for supplying liquid to and an inlet for receiving liquid from the device (12) for performing a processing operation; (v) means for imparting liquid flow through the flow path assembly (6); (vi) a switchable bypass assembly (31) for directing liquid into or bypassing the mixing means (8); The fluid path assembly includes:

[0061] This flow path assembly forms a feature of the present invention. Regarding the flow path assembly: preferably, component (v) is upstream of component (iii) and downstream of component (ii); preferably, component (iii) is downstream of component (ii); preferably, the flow path assembly is constructed of a plastic material; preferably, the flow path assembly is constructed of a material that allows sterilization of the assembly by gamma irradiation, such as silicone, particularly braided silicone, polyethylene, or polypropylene; in another embodiment, the flow path assembly is constructed of stainless steel. Preferably, the flow path assembly is sterile.

[0062] Preferably, the flow path assembly further comprises one or more blocks for receiving a conductivity meter, a pH sensor and / or a pressure sensor. Preferably, at least one of the one or more blocks is located downstream of the means (6) and upstream of the mixing means (8). Furthermore, it is preferred that at least one of the one or more blocks is located downstream of the device (12). In a particularly preferred embodiment, at least one of the one or more blocks is located downstream of the device (12) and is adapted to receive a conductivity meter, a pH meter and a pressure sensor.

[0063] A unit comprising the aforementioned flow path assembly and preferably a device (12) for carrying out a processing operation to separate at least a portion of the impurities from the target material forms a further aspect of the invention.

[0064] The apparatus of the present invention preferably further comprises means for applying additional pressure (i.e., in addition to the pressure provided by the means for imparting flow (6)) to the liquid flowing through device (12), said means being located downstream of device (12). Means for applying additional pressure are known in the art and include pinch valves, with diaphragm valves and variable position diaphragm valves being particularly preferred.

[0065] In a preferred embodiment: (A) Each unit includes a flow path assembly; (B) The flow path assemblies used in at least half of the units (preferably all of the units) have substantially the same configuration.

[0066] The replaceable tubing used to make the flow path assembly is preferably constructed from a plastic material, such as silicone, especially braided silicone. Preferably, the flow path assembly through each unit is substantially identical to the flow path assembly through all of the other units.

[0067] In certain embodiments, one or more of the units (preferably all of the units) are constructed from materials such as stainless steel or other suitable materials that allow them to be reused a significant number of times before needing to be replaced. The present invention includes a multi-use flow path assembly constructed from:

[0068] In certain embodiments, one or more of the units (preferably all of the units) comprise a single-use flow path assembly, preferably constructed from a material, e.g., a plastic material, e.g., silicone, particularly braided silicone, polyethylene, or polypropylene, that has a limited lifespan and is designed to be utilized as a disposable consumable.

[0069] In many embodiments, each processing operation is performed under the control of a programmable control unit, preferably a computer. In some embodiments, a single control unit controls two or more processing operations. In other embodiments, each processing operation is under the control of a separate control unit. In these other embodiments, the control units preferably use a common programming language, thereby simplifying communication between the control units.

[0070] In a preferred embodiment, the bioprocessing liquid is provided by combining at least three liquids, with at least two (preferably all) of the at least three liquids each being provided by combining at least two additional liquids (e.g., using valves (3a) and (3b) or (3c) and (3d)). This combining is preferably performed by mixing means (8). The at least three liquids may be prepared by combining liquids (2a) and (2b), (2c) and (2d), and (2e) and (2f), respectively.

[0071] The composition of the bioprocessing liquid used in the unit may remain the same throughout the method, or the composition may change during the method. For example, the composition of the bioprocessing liquid may change gradually or stepwise during the method, particularly when the unit includes a chromatography column and the bioprocessing liquid serves as an eluent.

[0072] Liquids (e.g., at least two liquids) that can be used to prepare a bioprocessing liquid include those known in the art for carrying out suitable processing operations. Examples of such liquids include acidic, neutral, and basic solutions, e.g., those having a pH ranging from 2.5 to 14, as well as solutions of various salts at various concentrations. Examples include aqueous solutions containing one or more of the following: sodium, potassium, or ammonium hydroxide, phosphoric acid, sulfuric acid, hydrochloric acid, or acetate; aqueous solutions having salt concentrations up to about 3 M, including salts, e.g., sodium, calcium, potassium, and ammonium salts, such as phosphate, hydrochloride, acetate, citrate, and sulfate; buffers, examples of which are well known in the art; reducing agents (e.g., DTT (DL-dithiothreitol) and TCEP (tris(2-carboxyethyl)phosphine); amino acids (e.g., histidine, arginine, and glycine); surfactants (e.g., Tween™ 20 and Triton™-X100); water-miscible organic solvents, such as polyols, e.g., glycerin and polyethylene glycol; and mixtures comprising two or more of the above.

[0073] Target materials that can be processed using the units, devices and flow path assemblies of the present invention include biomolecules, e.g., pDNA; cell therapy, vaccines such as viral vaccines, gene therapy products, sugars, inclusion bodies, particularly inclusion bodies containing polypeptides; particularly recombinant polypeptides.

[0074] pDNA may be in one or more of several forms, such as supercoiled, linear, and open-circular (i.e., nicked or relaxed) isoforms. Supercoiled pDNA isoforms have a covalently closed circular conformation, while pDNA is negatively supercoiled in the host cell by the action of host enzyme systems. In the open circular isoform, one strand of the pDNA duplex is cleaved at one or more locations.

[0075] Methods for generating pDNA are well known in the art. pDNA can be natural or artificial, for example, a cloning vector with a foreign DNA insert. In many embodiments, pDNA is in the size range of 1 kilobase to 50 kilobases. For example, pDNA encoding expressed interfering RNA is typically in the size range of 3 kilobase to 4 kilobase.

[0076] Polypeptides, particularly recombinant polypeptides, include therapeutic proteins and peptides, including cytokines, growth factors, antibodies, antibody fragments, immunoglobulin-like polypeptides, enzymes, vaccines, peptide hormones, chemokines, receptors, receptor fragments, kinases, phosphatases, isomerases, hydrolases, transcription factors, and fusion polypeptides.

[0077] Antibodies include biologically active monoclonal antibodies, polyclonal antibodies, and antibody fragments, including any multivalent and / or multispecific forms of the foregoing.

[0078] A naturally occurring antibody typically comprises four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a variable region (V H ) and constant region (C H ), including C H The region, in its native form, consists of three domains: C H 1. C H 2 and CH 3. Each light chain contains a variable region (V L ) and one domain C L The constant region comprises:

[0079] V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0080] Antibody fragments that can be expressed include portions of an intact antibody, said portions possessing the desired biological activity. Antibody fragments generally contain at least one antigen-binding site. Examples of antibody fragments include: (i) V L , C L , V H and C H (ii) a Fab fragment having one domain; (iii) a C Fab fragment capable of forming a bivalent fragment by disulfide bridging between two Fab derivatives. H Fab derivatives such as Fab' fragments having one or more cysteine ​​residues at the C-terminus of one domain; (iii) V H and C H (iv) Fd fragment having one domain; H (v) Fd derivatives, such as Fd derivatives having one or more cysteine ​​residues at the C-terminus of one domain; (v) V of a single arm of an antibody L and V H (vi) Fv fragment having a V domain; L and V H (vii) single-chain antibody molecules, such as single-chain Fv (scFv) antibodies, in which the domains are covalently linked; (vii) separate variable domains (V) with or without constant region domains; H or VL V without a constant region domain linked to a V H or V L domain polypeptides, (e.g., V H -V H , V H -V L , or V L -V L (viii) isolated CDR regions, such as H Domain, or V L Domain, and V H or V L (ix) domain antibody fragments, such as fragments consisting of an antigen-binding fragment of either of the domains; (ix) two antigen-binding sites within the same polypeptide chain, e.g., a light chain variable domain (V L ) linked to a heavy chain variable domain (V H ), so-called "diabodies" including (x) Included are so-called linear antibodies, which comprise a pair of tandem Fd segments which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0081] Inclusion bodies include insoluble aggregates that form in the cytoplasm of bacterial cells, such as E. coli, and most commonly contain polypeptides, particularly recombinant polypeptides. Methods for processing target substances, such as recombinant polypeptides, in particular for purifying or isolating recombinant polypeptides, form further aspects of the present invention.

[0082] One embodiment of an apparatus according to the present invention is described with reference to FIG. 1. The first device for performing bioprocessing operations includes an inlet (1) for a liquid feed containing target biomolecules and impurities, as well as inlets (2a) through (2f) for six different buffer solutions and an inlet (2g) for water. Each inlet is fitted with a valve, such as a straight-through diaphragm valve (3) and (3a) through (3g), that can switch the flow on or off. In the embodiment shown, the buffer feeds passing through inlets (2a) and (2b), (2c) and (2d), and (2e) and (2f) are combined downstream of the valves, and (3a) through (3f) form three buffer feed lines, respectively, which are fluidly connected to different inlets of a multi-inlet flow controller (4), along with the injection feed through inlet (2g) and water for inflow. The multi-inlet flow controller (4) includes a four-valve manifold with a single outlet having a fast-acting solenoid actuator. This configuration also allows for selection between buffer solutions entering through inlets (2a) and (2b), (2c) and (2d), or (2e) and (2f) by appropriately opening and closing valves (3a) and (3b), (3c) and (3d), and (3e) and (3f), enhancing the operational flexibility of the device. The outlet from the multi-inlet flow controller (4) is fluidly connected to the liquid feed inlet (1) containing the target biomolecule and impurities at a location (5) upstream of the pump (6), which provides combined feed flow through a static mixer (8) equipped with a bubble trap to the inlet of the first chromatography column (12). The line supplying the output from the pump (6) to the chromatography column (12) is fitted with a pressure sensor (7), an air sensor (9), a flow meter (10), such as an ultrasonic flow meter, and a combined temperature and conductivity sensor (11). In some embodiments, the pump (6) is controlled via a programmable control unit in response to a feedback signal (29) from the flow meter (10).In some embodiments, the multi-inlet flow controller (4) is optionally controlled via a programmable control unit in response to feedback signals (28) from the conductivity and temperature sensor (11). The programmable control unit may also control a switching bypass assembly (31) that prepares the liquid feedstock to enter the mixing means (8) or bypass the mixing means (8), as desired. Additionally, the programmable control unit may also control the switching bypass assembly (31) for onward dispatch to the device (12) so that the bioprocessing liquid is prepared in the mixing means (8). The outlet line from the chromatography column (12) is provided with a pressure sensor (13), a combined temperature and conductivity sensor (14), a UV detector (15), such as a UV / visible multi-wavelength detector, a pH meter (16), and a variable-position valve (30), which can be used to control pressure and, if desired, apply backpressure. Preferably, operation of pump (6) and variable position valve (30), and thereby control of pressure within the apparatus, is controlled via a programmable control unit in response to feedback signals (26) and (27) from pressure sensors (7) and (13). The outlet lines pass through a series of valves (17), (19), and (20), which allow flow to be controlled between outlet feed outlet (18), waste stream outlet (21), or product stream outlet (22), for example, to allow collection or sampling. The apparatus is further fitted with valves (23a) and (23b) that allow flow to be diverted and bypass column (12) if necessary during operation, as well as further valves (24) and (25) that allow flow through the column to be stopped. The outlet feed outlet (18) can then be used as a feed line to provide a product stream containing the target material and any remaining impurities in a second apparatus for carrying out further processing operations from one unit, configured as shown in Figure 1, although preferably the chromatography column (12) is replaced with a different device (12) for carrying out further processing operations, such as a different type of chromatography column or a non-chromatographic device (12), where in the second device for carrying out further processing operations the feedstock supplied through inlet (1) contains the product stream from the previous unit through product stream outlet (18).

[0083] In one method of operation, valves 3a-3g are closed while valve 3 is opened, and a liquid containing the target substance is delivered to column 12 by pump 6, thereby loading the column with the target substance. For example, if the target substance is a monoclonal antibody, a column containing a Protein A affinity resin is preferred, as monoclonal antibodies selectively bind to the Protein A resin. The switchable bypass assembly 31 allows the liquid feed containing the target substance and impurities to be loaded onto column 12 without passing through mixing means 8. Once the desired load is complete, valve 3 is closed, and one or more valves 3a-3g are opened, allowing one or more bioprocessing liquids to enter the unit through inlets 2a-2g and be pumped through column 12. In some embodiments, multiple inlet valve (4) is operated to initially open only valve (3a), then open the inlet valve through which a buffer, which may be a wash buffer, is supplied from inlet (2a), and the loaded column is washed with the buffer from inlet (2a). Once the desired wash step is complete, valve (3a) may remain open or may be closed, and one or more of valves (3b)-(3g) may be opened. Inlet valve (4a) of multiple inlet valve (4) is opened to pump the fluid from inlets (2b)-(2g), or a mixture thereof, into column (12). By controlling the opening and closing of valve (4a) of multiple inlet valve (4) (valve (4a) is not shown in FIG. 1, but see FIG. 2), and / or valves (3a)-(3g), the composition of the bioprocessing fluid supplied to the column can be varied and controlled as desired. For example, when valves (3b), (3c), and (3e) are open, varying the open inlet valves (4a) and closing the others in the multi-inlet flow controller (4) allows for stepwise changes in the composition of the bioprocessing fluid. In another example, two or more inlet valves (4a) of the multi-inlet flow controller (4) can be opened and closed at a given frequency for a selected period of time to provide a given mixture of bioprocessing fluids to the column (12).Furthermore, the switchable bypass assembly (31) allows the liquids from the inlets (2a) through (2g) to be mixed in any combination or ratio in the mixing means (8) to generate a bioprocessing liquid / eluent gradient that can then be fed to the column (12) preloaded with the target material and impurities. The composition of the liquid fed to the column can be varied by adjusting the time and / or frequency that the inlet valve (4a) of the multiple inlet valve (4) is open or closed. If the time and / or frequency are changed in steps, the composition also changes in steps. If the time and / or frequency are changed gradually over a period of time, the composition of the resulting bioprocessing liquid also changes gradually, allowing for the application of a gradient to the column (12). By any desired method, the composition of the bioprocessing liquid fed to the column (12) can be varied to elute the target material from the column at a different rate relative to the impurities, allowing the portion of the product stream containing the target material to be collected and the waste streams on either side containing the impurities to be discarded. Prior to elution, the liquid exiting the column (12) is collected via the product stream outlet (22) or sent to waste (21) and passed through valves (17), (19). ), and (20) are set accordingly. For elution of the target substance from column (12), valves (19) and (20) are closed, valve (17) is opened, and the target substance passes through outlet (18) to the second processing unit.

[0084] The operation of the second processing unit may be substantially as described above for the first unit. It will be appreciated that the target material exiting the second processing unit through a product stream outlet (18) equivalent to the product stream outlet (18) of the first unit may be recovered and used as is, or may be subjected to one or more further processing operations, for example, in further units including components (i)-(vi). Such further processing operations may use conventional equipment, or may use further equipment according to the configuration shown in FIG. 1, or may be otherwise in accordance with the present invention.

[0085] The entire subject matter of the claims is incorporated herein by reference thereto. The present invention is illustrated, but not limited, by the following examples. [Example]

[0086] In a chromatography procedure, proteins are bound to a chromatography resin, washed with buffers of different salt concentrations, and then removed (eluted) using a high-salt buffer. As an example, recombinant lactoferrin can be bound and eluted from a 2.3 L POROS-XS cation exchange resin column using pH 7.5 sodium phosphate buffers with sodium chloride concentrations ranging from 0 to 1 M. This can be performed in a single, stand-alone unit with a fully disposable flow path assembly containing the features described in Figure 1, except that valve (23b) is replaced with a simple fluid connection. Stock solutions are attached to the inlets in the following order: 2 M sodium chloride is attached to inlet (2a); 0.1 M dibasic sodium phosphate is attached to inlet (2c); 0.01 M monobasic sodium phosphate is attached to inlet (2e); water is attached to inlet (2g); and the protein feed is attached to inlet (1). The buffer solution is generated by proportionally selecting each of the stock solutions to produce the desired buffer composition through the action of a multi-inlet flow controller (4) and downstream pump (6) and static mixer (8). During establishment of the correct buffer composition, the mixing means (8) and column (12) are bypassed using a switching bypass assembly (31) and valve (23a), valves (24) and (25) are closed, and unwanted buffer is directed to waste (21). Once the buffer is homogeneous, as indicated by a stable reading from the upstream conductivity sensor (11), the buffer is delivered to the chromatography column (12) by opening valves (24) and (25) and closing the bypass line of valve (23a). Process conditions are monitored using conductivity, UV, and pH sensors (14), (15), and (16) downstream of the column (12). During column preconditioning prior to protein binding to the column and water rinsing after use, liquid is sent to waste (21). Once pretreated, the chromatography resin is loaded onto the column (12) along with the protein drawn from the liquid feed inlet (1) by the action of a pump (6) which pushes the resin through a static mixer (8).The first low salt buffer wash is collected through the product stream outlet (18), the second medium salt buffer wash is collected through the outlet feed (22), while the flow-through from the column is collected through the outlet feed. Finally, the target protein is removed from the column using a high salt elution buffer and collected through the product stream outlet (18).

Claims

1. 1. An apparatus for converting a liquid feedstock containing a target material and impurities into a product stream containing a purified target material and one or more waste streams containing at least a portion of the impurities, the apparatus comprising at least two processing units arranged in series such that the feed stream of a second and any subsequent unit comprises the product stream from a downstream unit, each processing unit comprising the following components (i) to (vi): (i) an inlet for liquid feedstock (1); (ii) a multi-inlet flow controller (4) comprising two or more variable flow inlet valves (4a) for providing at least two liquids in a desired ratio; (iii) mixing means (8); (iv) a device (12) for carrying out a processing operation to separate at least a portion of the impurities from the target material; (v) means for providing a flow of liquid through the unit (6); and (vi) A switchable bypass assembly (31) for directing liquid passing through the unit either into the mixing means (8) or bypassing the mixing means (8).

1. An apparatus comprising:

2. 2. The apparatus of claim 1, wherein the means (6) for providing a flow of liquid through the unit is located downstream of the multi-inlet flow controller (4) and upstream of the mixing means (8).

3. 3. Apparatus according to claim 1 or 2, in which each unit comprises only one means (6) for providing a flow of liquid through that unit.

4. 4. Apparatus according to any one of claims 1 to 3, wherein the switched bypass assembly (31) comprises a pipe and two or three valves for directing the flow of the liquid feedstock and / or at least two other liquids either to the mixer means (8) or to the device (12) without passing through the mixer means (8).

5. Each unit: (a) a liquid feedstock entering the unit through inlet (1); (b) at least two liquids flowing through a multi-inlet flow controller (4); (c) the liquid feedstock and at least two liquids that are mixed in the mixer means (8) or bypass the mixer means (8) and, in either case, enter a device (12) where a processing operation is performed that separates at least a portion of the impurities from the target material to produce a product stream and, optionally, a waste stream; and (d) The route by which the product stream, and waste stream, if any, exits the unit.

5. The apparatus of claim 1, further comprising a flow path assembly comprising:

6. 6. The apparatus according to any one of claims 1 to 5, comprising at least one unit comprising components (i) to (vi) for carrying out chromatography.

7. 7. The apparatus according to any one of claims 1 to 6, comprising at least one unit comprising components (i) to (vi) for carrying out viral inactivation.

8. 8. An apparatus according to any one of claims 1 to 7, comprising at least one unit comprising components (i) to (vi) for removing any viruses from a liquid feedstock.

9. At least two components (i) to (vi) for performing chromatography.

9. The device according to claim 1, comprising a unit of:

10. 10. The apparatus of claim 9, wherein the chromatographic purification carried out in each of the at least two units for carrying out the processing operation of the chromatography unit uses a chromatography column packed with different conditions and / or different resins than those used in the other of the units.

11. 11. The apparatus according to any one of claims 1 to 10, comprising at least one unit comprising components (i) to (vi) for performing affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, reversed phase chromatography, expanded bed chromatography, mixed mode chromatography, membrane chromatography or size exclusion chromatography.

12. 12. The apparatus of any one of claims 1 to 11, wherein each unit comprises means for preparing a mixed bioprocessing liquid.

13. 13. Apparatus according to any one of claims 1 to 12, wherein each unit comprises a single pump (6) for providing a flow of liquid through that unit.

14. 14. The apparatus of any one of claims 1 to 13, comprising at least one unit comprising components (i) to (vi) for performing diafiltration of the product stream to buffer the target material at a pH at which the target material is stable.

15. The following units arranged in series, preferably in the order listed: a. a unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; and b. A unit for the inactivation of any viruses that may be present in the liquid raw material.

15. The apparatus of claim 1, wherein each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the feedstock for the next unit.

16. The following units arranged in series, preferably in the order listed: a. a unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; and c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography.

16. The apparatus of any one of claims 1 to 15, comprising: each unit comprising components (i) to (vi); and wherein the product feed of each unit except for the final unit is used as the feedstock for the next unit.

17. The following units arranged in series, preferably in the order listed: a. a unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for performing chromatographic purification of the target substance by cation exchange chromatography; and d. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography.

17. The apparatus of any one of claims 1 to 16, comprising: each unit comprising components (i) to (vi); and wherein the product feed of each unit except for the final unit is used as the feedstock for the next unit.

18. The following units arranged in series, preferably in the order listed: a. a unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; d. A unit for performing chromatographic purification of the target substance by anion exchange chromatography; and e. A unit for the removal of any inactivated viruses wherein each unit comprises components (i) to (vi), and the product feed of each unit except for the final unit is used as the feedstock for the next unit.

19. The following units arranged in series, preferably in the order listed: a. a unit for carrying out chromatographic purification of the target substance, preferably by affinity chromatography; b. A unit for the inactivation of any viruses that may be present in the liquid raw material; c. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; d. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; e. A unit for the removal of any inactivated viruses; and f. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange.

19. The apparatus of any one of claims 1 to 18, comprising: each unit comprising components (i) to (v); and the product feed of each unit except for the final unit is used as the feedstock for the next unit.

20. (A) each unit includes a flow path assembly; and (B) the flow path assemblies used in at least half of the units (preferably all of the units) have substantially the same configuration; 20. Apparatus according to any one of claims 1 to 19.

21. 21. The device of claim 20, wherein the flow path assembly is constructed from a plastic material.

22. 22. An apparatus according to any one of claims 1 to 21, wherein at least 75% of the component parts of each unit other than the device (12) are identical to the component parts used in at least 80% of the other units of the apparatus.

23. 23. Apparatus according to any one of claims 1 to 22, wherein all component parts of each unit, except for the device (12), are identical to all component parts used in all other units of the apparatus.

24. 24. The apparatus of any one of claims 1 to 23, wherein the flow path assembly through each unit is substantially identical to the flow path assembly through all of the other units.

25. The following components (i) to (vi): (i) an inlet for liquid feedstock (1); (ii) a multi-inlet flow controller (4) comprising two or more variable flow inlet valves (4a) for providing at least two liquids in a desired ratio; (iii) mixing means (8); (iv) an outlet for supplying liquid to and an inlet for receiving liquid from the device (12) for performing a processing operation; (v) means for imparting liquid flow through the flow path assembly (6); and (vi) A switchable bypass assembly (31) for directing liquid into or bypassing the mixing means (8). a flow path assembly including:

26. 26. The flow path assembly of claim 25, wherein component (v) is upstream of component (iii) and downstream of component (ii).

27. 27. A flow path assembly according to claim 25 or 26, wherein component (iii) is downstream of component (ii).

28. 28. A flow path assembly according to any one of claims 25 to 27, further comprising one or more blocks for receiving a conductivity meter, a pH meter and / or a pressure sensor.

29. 29. A flow path assembly according to claim 28, wherein at least one of the one or more blocks is located downstream of the means (6) and upstream of the mixing means (8).

30. 30. A flow path assembly according to claim 28 or 29, wherein at least one of the one or more blocks is located downstream of the device (12).

31. 31. The flow path assembly of any one of claims 28 to 30, wherein at least one of the one or more blocks is located downstream of the device (12) and is adapted to receive a conductivity meter, a pH meter and a pressure sensor.

32. 32. A flow path assembly according to any one of claims 25 to 31 constructed from a plastic material.

33. 33. The flow path assembly of any one of claims 25 to 32, constructed of materials that tolerate sterilization by gamma irradiation.

34. 32. The flow path assembly of any one of claims 25 to 31 constructed of stainless steel.

35. 35. The flow path assembly of any one of claims 25 to 34, which is sterile.

36. 36. Apparatus according to any one of claims 1 to 24, comprising a flow path assembly according to any one of claims 25 to 35.

37. 1. A unit for converting a liquid feedstock containing a target material and impurities into a product stream containing a purified target material and one or more waste streams containing at least a portion of the impurities, the unit comprising the following components (i) to (vi): (i) an inlet for liquid feedstock (1); (ii) two or more variable flow injections to provide at least two liquids in a desired ratio; a multi-inlet flow controller (4) including a port valve (4a); (iii) mixing means (8); (iv) a device (12) for carrying out a processing operation to separate at least a portion of the impurities from the target material; (v) means for providing a flow of liquid through the unit (6); and (vi) A switchable bypass assembly (31) for directing liquid passing through the unit either into the mixing means (8) or bypassing the mixing means (8). Including, units.

38. 38. The unit of claim 37, wherein component (v) is upstream of component (iii) and downstream of component (ii).

39. 39. A unit according to claim 37 or 38, wherein component (iii) is downstream of component (ii).

40. 40. A unit according to any one of claims 37 to 39, comprising only one means (6) for providing a flow of liquid through the unit.

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