Methods for purifying monoclonal antibodies

Abstract

A method for purifying a liquid feedstock containing a monoclonal antibody and impurities, comprising passing the liquid feedstock through an apparatus comprising at least two processing units, each of which produces a product stream containing the purified monoclonal antibody and, optionally, a waste stream containing at least some of the impurities, and each of which comprises specific components (i)-(v) including a multi-inlet flow controller comprising two or more variable flow inlet valves for in situ production of a bioprocessing liquid by combining at least two liquids in a desired ratio. One unit performs chromatography, and another unit performs viral inactivation. The units can be essentially identical except for the devices they contain, leading to advantages in terms of simplicity, cost and ease of operation, reduced risk of operator error, easier maintenance, and reduced spare parts inventory.

Description

[Technical field]

[0001] The present invention relates to a method for purifying monoclonal antibodies. [Background technology]

[0002] Monoclonal antibodies ("mAbs") have received much attention in recent years, especially for therapeutic applications. mAbs are generally produced by culturing recombinant host cells that have been genetically engineered to express the desired mAb. The mAb is then typically recovered from the culture medium by a process that includes several processing operations (sometimes called "unit operations").

[0003] Equipment for purifying mAbs is known in the art and typically includes several very different processing units arranged in series, each unit performing an individual processing operation. The processing units usually include at least two pumps to move liquids through the unit. Typically, the units are provided by various manufacturers that specialize in devices to perform specific processing operations (e.g., filtration, chromatography, etc.). The equipment used in commercial-scale production of mAbs is very bulky and requires large floor space and infrastructure. Furthermore, while some commonality of processing units can be achieved in some processing operations, the design of units for specific unit operations, such as viral inactivation and / or ultrafiltration, differs substantially from the design for, for example, chromatographic purification. This means that either more space is needed to accommodate two or more sets of processing units, or the interoperability of the stages and the control of the equipment is very complex. Furthermore, operators require extensive training on each of the various types of processing units they use, as the units are very different from each other. The lack of similarity between the many processing units currently used in mAb production means that a large inventory of spare parts is required. Furthermore, the lack of similarity between many units complicates routine maintenance, as engineers must learn how to repair processing units of very different construction. Devices that require different bioprocessing liquids (e.g., buffers, eluents, etc.) for different processing operations also pose a large risk of operator error and possible risk of failure of a batch of very expensive mAb if the wrong externally prepared bioprocessing liquid is provided at the wrong stage. Therefore, a simplified, widely applicable device with a low risk of operator error is desirable. Summary of the Invention [Means for solving the problem]

[0004] According to a first aspect of the present invention, there is provided a method for purifying a liquid feedstock comprising a monoclonal antibody (mAb) and impurities, comprising passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream comprising a purified monoclonal antibody and optionally a waste stream comprising at least a portion of the impurities, each unit comprising the following components (i) to (v): (i) An inlet for liquid feedstock; (ii) a multiple inlet flow controller including two or more variable flow inlet valves for producing a bioprocessing liquid from at least two other liquids that are combined in a desired ratio; (iii) means for combining a liquid feedstock and a bioprocessing liquid to generate a device feed, and / or for combining at least two other liquids to prepare a bioprocessing liquid; (iv) a device for performing processing operations on a liquid feed or device feed, the device including an inlet for the device feed and / or liquid feed and an outlet for a product stream; and (v) means for providing flows of a liquid feedstock, at least two liquids, a bioprocessing liquid, a device feed, a product stream, and an optional waste stream; where: (I) a liquid feedstock is fed through inlet (i), combined with a bioprocessing liquid by means (iii) and then fed through device (iv) or combined with the bioprocessing liquid in device (iv), in either case to provide a product stream and optionally a waste stream which exit the unit; (II) the units are arranged in series such that the liquid feed of the second and any subsequent units comprises the product stream from the preceding unit; (III) a device (iv) in at least one of the at least two processing units performs a chromatographic processing operation; and (IV) A method is provided, wherein a device (iv) in at least one of the at least two processing units performs a processing operation of virus inactivation.

[0005] The present invention provides a process that can be carried out on a manufacturing scale and offers many advantages over previous processes, 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. Component (iii) is often abbreviated to "mixer (iii)" for brevity. Component (iv) is often referred to as "device (iv)" for brevity.

[0007] In the accompanying drawings: [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows an outline of an embodiment of the method. [Diagram 2] FIG. 2 is a schematic diagram of one processing unit that can be used to perform the processing operations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] FIG. 1 shows how a feedstock from a cell culture harvest can be purified by the method according to the invention. The purified feed cell culture harvest serves as the feedstock for the first processing unit, which includes a Protein A affinity chromatography device. The liquid feedstock for the second and each subsequent unit then includes the product stream from the preceding unit. Each unit can also generate a waste stream (not shown), but this is not always the case. For example, a virus inactivation unit typically only generates a product stream that differs from the feedstock only in that the viruses present in the feedstock have been inactivated. Furthermore, a simple filtration unit can also only generate a product stream (i.e., no waste stream) if the impurities are retained on the filter of that unit. Viruses inactivated in the virus inactivation unit 2 can be removed by a later unit (e.g., a virus filtration / removal unit). The order of the units in the preferred method according to the invention is shown as steps 1-6. Finally, a product stream containing the mAb purified by units 1-6 is collected.

[0010] FIG. 2 is explained in more detail in the Examples section below. 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., drug product for pharmaceutical preparations. In some embodiments, the apparatus comprises two units (e.g., for performing two processing operations). In other embodiments, the apparatus comprises more than two units, e.g., (at least) three, four, five, six, seven, eight, nine or more units, preferably each unit having features (i)-(v) above. The method may further comprise two or more units operating in parallel, but it is preferred that all units used in the method are arranged in series. In many highly preferred embodiments, the method operation performed in each unit is different from the method operation used in all other units. Thus, the apparatus may comprise multiple units, e.g., for performing chromatography, but each such chromatography unit or the method it is used in is preferably different from the other units for performing chromatography.

[0011] In certain embodiments, each unit contains flow paths that are substantially identical to at least half, and more preferably all, of the flow paths of the other units. Preferably, each unit prepares its mixed bioprocessing liquid (in situ) during the method, thus avoiding the risk of the operator using the wrong externally prepared bioprocessing liquid intended for another method operation.

[0012] Preferably, the apparatus further comprises one or more units for performing one or more of the following method operations, in each case the units comprising components (i) to (v): - Remove all viruses from liquid materials; - Ultrafiltration of the feedstock to provide a concentrate containing the desired mAb; - diafiltration of the feedstock to buffer the target material at a pH where the desired mAb is stable; - Tangential flow filtration, including recirculation and single pass tangential flow filtration.

[0013] In a preferred embodiment, the apparatus comprises processing units containing devices for performing the following processing operations, preferably in the order listed: Protein A chromatography, viral inactivation, cation exchange chromatography, anion exchange chromatography, viral filtration (removal of inactivated viruses) and concentration of the product feed (e.g., by ultrafiltration and / or diafiltration performed in a single unit or in separate units).

[0014] The size of each unit is not particularly limited. However, for industrial-scale purification, each unit is preferably 1 to 2.2 meters high (more preferably 1.5 to 2 meters high), 0.5 to 1.2 meters wide (more preferably 0.75 to 1 meter wide) and 0.5 to 1 meter deep (more preferably 0.6 to 0.9 meters deep).

[0015] Preferably, each unit is fitted with wheels and optionally brakes to prevent unwanted movement of the unit, which allows for easy positioning, movement and replacement of the units.

[0016] Preferably, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i) to (v), and the product feed of each unit being used as a feedstock for the next unit or, if there is no next unit, the product feed being collected: a. a unit for performing a first chromatographic purification of the mAb, 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 a second chromatographic purification of the mAb, preferably by ion exchange chromatography; d. A unit for performing a tertiary chromatographic purification of the mAb, preferably by ion 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.

[0017] In some embodiments, the second chromatographic purification comprises anion exchange and the third chromatographic purification comprises cation exchange, in other embodiments, the second chromatographic purification comprises cation exchange and the third chromatographic purification comprises anion exchange.

[0018] Thus, in one preferred embodiment, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i) to (v), and the product feed of each unit being used as a feedstock for the next unit or the product feed being collected if there is no next unit: a. A unit for performing chromatographic purification of mAbs 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 performing chromatographic purification of mAbs by ion exchange chromatography.

[0019] In another preferred embodiment, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i)-(v), and the product feed of each unit being used as a feedstock for the next unit or the product feed being collected if there is no next unit: a. A unit for performing chromatographic purification of mAbs 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 mAb by cation exchange chromatography; and d. A unit for performing chromatographic purification of mAbs by anion exchange chromatography.

[0020] In another preferred embodiment, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i)-(v), and the product feed of each unit being used as a feedstock for the next unit or the product feed being collected if there is no next unit: a. A unit for performing chromatographic purification of mAbs 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 mAbs by cation exchange chromatography; d. A unit for performing chromatographic purification of the mAb by anion exchange chromatography; and e. A unit for the removal of any inactivated viruses.

[0021] In another preferred embodiment, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i)-(v), and the product feed of each unit being used as a feedstock for the next unit or the product feed being collected if there is no next unit: a. A unit for performing chromatographic purification of mAbs 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 mAbs by cation exchange chromatography; d. A unit for performing chromatographic purification of mAbs 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.

[0022] Preferably, the units are arranged in series in the order listed herein, e.g., in a device comprising units a.-f., the units are preferably arranged in series in the order a., b., c., d., e., then f.

[0023] 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).

[0024] Preferably, at least one of the units (preferably at least half of the units, more preferably all the units) further comprises a pressure sensor located downstream of component (iv).

[0025] 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).

[0026] Preferably, at least one of the units (preferably at least half of the units, more preferably all the units) further comprises a pH sensor located downstream of component (iv).

[0027] Preferably, at least one of the units (preferably at least half of the units, more preferably all the units) further comprises a conductivity sensor located downstream of component (iv).

[0028] In preferred embodiments, 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 95% of the component parts of each unit, other than component (iv), are identical to the component parts used in at least 80%, more preferably at least 90%, of the other units of the apparatus. In particularly preferred embodiments, all component parts of each unit, other than component (iv), are identical to all component parts used in all other units of 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 component parts of each unit means that less spare parts inventory is required. Furthermore, routine maintenance of the apparatus is simplified because the units are similar, and it is easier to operate the apparatus because each unit is similar to the other units of the apparatus. In contrast to the prior art, which uses widely different processing units from multiple manufacturers, technicians avoid having to learn how to service a large number of widely different processing units. Component (iv) of each unit is typically different from component (iv) of the other units (so that each unit can perform a separate processing operation), hence the term "other than component (iv)."

[0029] In a preferred embodiment, all units of the apparatus are substantially identical except for device (iv), which in this embodiment may be identical in two or more units, but more typically device (iv) differs from unit to unit such that each unit can perform a separate processing operation, for example as shown in FIG.

[0030] In a preferred embodiment, the flow paths used in at least half of the units (preferably all of the units) have substantially the same configuration. The flow passage is preferably constructed from a plastic material.

[0031] Preferably, the flow path through each unit is substantially identical to the flow paths through every other unit. In certain embodiments, one or more of the units (preferably all of the units) include a versatile flow path constructed from a material, such as stainless steel, to allow for a significant number of reuses before replacement is required.

[0032] In certain embodiments, one or more of the units (preferably all of the units) include a single-use flow path, preferably constructed from a commonly selected material that can be sterilized by gamma irradiation, e.g., a plastic material, typically designed with a limited life span, e.g., for use as a disposable consumable.

[0033] In one embodiment, the product stream from each unit except the final unit is fed directly to the next unit. In many 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 any). In this way, it is possible to test the product stream before it enters the next unit, pause the process, etc.

[0034] The feedstock of the first unit can also be supplied from a storage vessel or, if desired, directly from a cell culture device, such as a bioreactor, preferably after clarification. Examples of suitable storage vessels include tanks and bags.

[0035] The inlet (i) for the liquid feedstock preferably comprises a valve (3) and a tube fitted with a check valve to avoid contamination of the liquid feedstock with liquid flowing from the multi-inlet flow controller (ii).

[0036] mAbs that can be purified by this method include, but are not limited to, complete monoclonal antibodies, as well as biologically active fragments of monoclonal antibodies, including any of the multivalent and / or multispecific forms described above.

[0037] Naturally occurring mAbs often comprise four polypeptide chains, e.g., two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain typically contains a variable region (V H ) and the constant region (C H ), including C H The region, in its native form, consists of three domains: C H 1. C H 2 and C H 3. Each light chain typically comprises a variable region (V L ) and one domain C L The constant region comprises:

[0038] 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 typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0039] A monoclonal antibody fragment typically comprises a portion of an intact mAb, said portion possessing the desired biological activity. mAb fragments generally contain at least one antigen-binding site. Examples of mAb fragments include: (i)V L , C L , V H and C H Fab fragment with one domain; (ii) a divalent fragment can be formed by disulfide bridging between two Fab derivatives, CH 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 Fd fragment with 1 domain; (iv) C H 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 Fv fragments with domains; (vi) V L and V H single-chain antibody molecules, such as single-chain Fv (scFv) antibodies, in which the domains are covalently linked; (vii) another variable domain (V H or V L V having no constant region domain linked to a V domain polypeptide H or V L Domain polypeptides (e.g., V H -V H , V H -V L , or V L -V L ); (viii) a V, such as an isolated CDR region; H Domain, or V L Domain, and V H or V L Domain antibody fragments, such as fragments consisting of an antigen-binding fragment of any of the domains; (ix) Two antigen-binding sites in 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"; (x) So-called linear antibodies include those which contain a pair of tandem Fd segments which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0040] Impurities that can be removed by the methods typically include by-products from the process used to prepare the desired mAb, components of the media used to prepare the mAb (e.g., nutrients), cells, cellular, host cell proteins, DNA, RNA, other proteins present in or secreted from mammalian cells, endotoxins and viruses.

[0041] The multiple inlet flow controller (ii) preferably includes a variable flow, preferably intermittent flow, inlet valve that controls the flow of liquid through the flow controller. The multiple inlet flow controller includes at least two inlet valves, and often includes up to eight inlet valves (iia), such as three, four, five, six, or seven, but may include more inlet valves if desired. The inlet valves may each have the same dimensions, or one or more inlet valves may have different dimensions with respect to some or all of the other inlet valves. In certain preferred embodiments, the measured volume from each inlet valve to the outlet of the flow controller (ii) is the same for each inlet, and it is highly preferred that both the measured volume and path length from each inlet valve to the outlet of the flow controller (ii) are the same for each inlet.

[0042] The multiple inlet flow controller (ii) used in the present invention also comprises at least one outlet, and although there can be more than one outlet, it is preferred that a single outlet is used.

[0043] The valves of the multi-inlet flow controller (ii) can control the flow rate between a first relatively low flow rate that maintains liquid flow and at least a second higher flow rate. In a preferred embodiment, the valves of the multi-inlet flow controller (ii) are intermittent flow valves, which prevent flow in a first position but allow flow in at least a second position. Most preferably, all valves are intermittent flow valves. The valves may include actuators, such as pneumatic actuators, or more preferably solenoid actuators.

[0044] Preferably, the valves of the multi-inlet flow controller (ii) are most preferably controlled by a programmable control unit (not shown in the drawings) 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 (ii). This is preferably achieved by cycling through the inlet valves of the flow controller at a predetermined period or cycle rate and controlling the opening and closing of each valve according to the percentage of cycle time required to produce the desired bioprocessing liquid composition. The cycle rate can be either constant or variable. Most preferably, intermittent flow inlet valves are used and are controlled such that only one valve is open at any given time during operation. In many embodiments, the cycle rate of the multi-inlet flow controller (ii) is maintained constant and the desired relative amounts of input liquids used to make the bioprocessing liquid are maintained constant.

[0045] In many embodiments, the method includes multiple cycles of flow through the multiple inlet flow controller (ii). The number of cycles used depends on many factors, such as the duration of the method, the amount of liquid feedstock being processed, and the flow rate and maximum operating pressure of the equipment. 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.

[0046] 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, for example 0.05 to 0.5 Hz.

[0047] In a preferred embodiment, the bioprocessing liquid is provided by combining at least three liquid streams (2ab), (2cd), (2ef), where at least two (preferably all three) of the at least three liquid streams are provided by combining at least two further liquid streams each (e.g., using valves (3a) and (3b) or (3c) and (3d)). This mixing is preferably performed in a mixer (iii). The at least three liquid streams (2ab), (2cd), (2ef) may be prepared by combining liquid streams (2a) and (2b), (2c) and (2d) and (2e) and (2f), respectively.

[0048] 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, especially if the unit comprises a chromatography column and the bioprocessing liquid acts as an eluent.

[0049] Liquids (e.g., at least two other liquids) that can be used to prepare a bioprocessing liquid (e.g., as an additional liquid stream described above) 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 in the range of 2.5 to 14. 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 3M, including salts, such as sodium, calcium, potassium and ammonium salts, such as phosphates, hydrochlorides, acetates, citrates and sulfates; buffers, examples of which are well known in the art; reducing agents, such as DTT (DL-dithiothreitol) and TCEP (tris(2-carboxyethyl)phosphine); amino acids, such as histidine, arginine and glycine; surfactants, such as Tween™ 20 and Triton™-X100; water-miscible organic solvents, such as polyols, such as glycerin and polyethylene glycol; and mixtures comprising two or more of the above.

[0050] Although some mixtures may occur from the in-line combination of flows from inlet (i) and flow controller (ii) outlet and / or in the means for providing flow (v), the module further comprises a mixer (iii). Mixer (iii) provides a means for combining the liquid feedstock and liquids (2ab), (2cd), (2ef), and / or liquids from inlet (2g) used to generate device feed and, if desired, to form a bioprocessing liquid. Furthermore, mixer (iii) may also be used to combine at least two other liquids (2ab), (2cd), (2ef) and / or (2g) to prepare a bioprocessing liquid (e.g., an eluent for chromatography) that is subsequently fed to device (iv). Mixer (iii) may be, for example, an in-line mixer, or more preferably a mixing chamber. Preferred mixers (iii) include static mixers, most preferably time-delay, split-flow static mixers. In many embodiments, the means (iii) is an in-line mixer located downstream of the means for imparting flow (v) (e.g., a pump) and upstream of the device (iv) for achieving a processing operation.

[0051] In many embodiments, the mixer (iii) further comprises a means for trapping air bubbles, for example an air bubble trap. In one embodiment, the means (iii) is used to combine at least two liquids received from the multi-inlet flow controller (ii) either in the presence or absence of a liquid feedstock. Combining at least two liquids received from the multi-inlet flow controller (ii) in the absence of a liquid feedstock is useful for preparing a bioprocessing liquid to be passed through the device (iv) before or after the liquid feedstock enters the component (iv). For example, when the device (iv) includes a chromatography column, the means (iii) can be used to provide a bioprocessing liquid for pre-treating the column before the liquid feedstock is loaded onto the chromatography column, for preparing a bioprocessing liquid for washing the mAb on the chromatography column, for preparing a bioprocessing liquid for eluting the mAb from the chromatography column, and for preparing a bioprocessing liquid for removing impurities remaining in the column after the mAb has been successfully eluted from the column in purified form. Preferably, at least one of the units (preferably at least half of the units, more preferably all of the units) further comprises a device for removing air bubbles from one or more liquids passing through the unit.

[0052] The device feed inlet may also be used to receive bioprocessing liquid, for example, from components (ii) or (iii). In addition to the devices specified in (III) and (IV) for performing the method operations of chromatography and viral inactivation, the apparatus optionally further comprises one or more units for performing one or more of the following processing operations: chromatography (which may be the same as or different from the chromatography operation performed in device (III)), viral inactivation (which may be the same as or different from the viral inactivation performed in device (IV)), filtration (e.g. ultrafiltration, microfiltration, dead-end filtration and / or diafiltration), viral removal, refolding, aggregation and in-line pretreatment.

[0053] Chromatographic bioprocessing operations that can be performed using device (iv) 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, size exclusion chromatography (SEC). In many embodiments, at least one of the units performs a processing operation of Protein A affinity chromatography. The device for performing the chromatographic operation includes a suitable chromatographic device, such as a membrane, a fiber monolith, or a resin. The number and arrangement of units performing the chromatography are selected according to the properties of the mAb.

[0054] Preferably, the apparatus comprises at least two, more preferably three, units comprising components (i)-(v) for performing chromatographic purification of mAbs. In this case, the chromatographic purification performed in each unit preferably uses different conditions and / or chromatographic columns packed with different materials (e.g. different resins, membranes or monoliths) than the chromatographic columns used in all of the 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.

[0055] The device (iv) for carrying out the method of virus inactivation in the method of the present invention generally comprises a storage vessel capable of storing a liquid containing mAb under conditions that inactivate any viruses present. In certain embodiments, the outlet and inlet of the virus inactivation device (iv) can be fluidically connected to create a recirculation loop. In one such embodiment, the device is provided with a vessel or bag fluidly connected between the "device" inlet and the "device" outlet, one of the device outlets being fluidically connected to one of the inlets of the multi-inlet flow controller (ii). The vessel or bag between the "inlet" and "outlet" of the device (iv) fluidly connected to the liquid feed inlet (i) is filled by a means for providing a flow (v), typically a pump, or is pre-treated with at least one other liquid through at least one of the inlets of the other multi-inlet flow controller (ii). In certain embodiments, the vessel or bag is a mixing vessel or bag. The bioprocessing liquid is recirculated through the inlet of the multi-inlet flow controller (ii) to a container or bag and back to the inlet of the multi-inlet flow controller (ii) as the solution containing the mAb is pre-treated by at least one additional liquid fluidly connected to at least one other inlet on the multi-inlet flow controller (ii).

[0056] Viral inactivation can be carried out by several techniques using conditions known in the art. For example, using a chromatography column, a chromatography membrane, or a holding tank in which the fluid containing the liquid feedstock can be incubated at a pH below about 4.0, for example at a pH between about 3.0 and about 4.0, preferably at a pH between about 3.2 and about 3.9, particularly at a pH between about 3.4 and about 3.8, more particularly at a pH 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, for example at a period of about 30 minutes to 1.5 hours, preferably at a period of about 30 minutes to 1.25 hours, more preferably at a period of about 0.75 hours to 1.25 hours, particularly at a period of about 1 hour. In each case, the conditions selected are such that the mAb is not damaged or destroyed.

[0057] Inactivated viruses can be removed by filtration using normal flow filters (NFF) or tangential flow filtration (TFF) filters, for example as described 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 between 20 and 100 nanometers (nm). Such membranes retain inactivated viruses on the membrane surface while allowing passage of mAbs through the membrane.

[0058] 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 fluoride (PVDF), and the like, and are known as VIRESOLVE.RTM. and RETROPORE™ membranes, available from EMD Millipore, Billerica, Mass. These can be supplied either in cartridge (NFF) form, such as VIRESOLVE™ NFP virus filters, or as cassettes (for TFF), such as PELLICON™ cassettes, available from EMD Millipore, Billerica, Mass.

[0059] The filtration operations that can be performed using the device (iv) include virus filtration, depth and absolute filtration, ultrafiltration, diafiltration, and microfiltration. In many embodiments, the filtration device (iv) 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 (ii), and the feed containing the target mAb can be fluidically connected to the feed inlet (i). The processing of the liquid feed through the filtration device (iv) is achieved through a means for providing flow (v) that is fluidically connected downstream to the multi-inlet flow controller outlet (ii) and the feed inlet (i) and is upstream of the filtration device (iv). The filter is often modular and can use configurations known in the field of purifying biomolecules.

[0060] Ultrafiltration and / or diafiltration may be used to perform concentration or buffer exchange. Viral filtration, depth filtration and absolute filtration method operations are known in the art and can be carried out using commercially available filtration devices.In many embodiments, one or more filtration devices are located between the inlet and outlet of device (iv) to carry out filtration as a processing operation.In another embodiment, an additional filter device is located downstream of the device outlet, which allows the device to carry out most purification steps, such as chromatography, virus inactivation, tangential flow filtration, viral filtration or depth filtration, followed by a secondary filtration operation outside the device in certain embodiments.

[0061] Tangential flow filtration ("TFF") processing operations that can be performed using the device of the present invention include conventional recirculating TFF and single pass TFF ("SPTFF"). In certain embodiments, the outlet and inlet of the device can be fluidly connected to create a recirculating loop, as an example, recirculating tangential flow filtration.

[0062] In one embodiment, the apparatus includes a unit including a TFF device (iv), for example a TFF device including a flat sheet, hollow fiber or spiral wound membrane between the device (iv) inlet and the device (iv) outlet, and the retentate from the unit including the TFF device (iv) can be led from one of the device outlets to a fluidly connected inlet on a container or bag, including at least one inlet and one outlet. The outlet of the container or bag can be fluidly connected to the liquid feed inlet (i). The container or bag can be maintained at a constant level using an auxiliary means for feeding feed or liquid to the container or bag by being fluidly connected to a second inlet on the container or bag. In another embodiment, the apparatus is provided with a unit including a TFF device (iv) including either a flat sheet, hollow fiber or spiral wound membrane between the device inlet and the device outlet, and the retentate from the unit including the TFF device (iv) can be fluidly connected from one of the device outlets back to one of the inlets of the multi-inlet flow controller valve (ii). In certain embodiments, the recirculation loop from the device outlet to its inlet (i) includes a break vessel or bag. The solution containing the target mAb 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 unit containing the TFF device (iv), preferably through one of the multi-inlet flow controller inlets. The multi-inlet flow controller (ii) can be used to mix the retentate with at least one other liquid. The operation of recirculating the TFF is known in the art and can be controlled by setting the cross flow rate and transmembrane pressure.

[0063] In certain embodiments, the apparatus comprises a unit comprising an SPTFF device (iv). The SPTFF device preferably comprises either a flat sheet, hollow fiber or spiral wound membrane between the device (iv) inlet and the device (iv) outlet, as described, for example, in WO2017 / 118835. In some embodiments, a hybrid of SPTFF and (recirculating) TFF can be used, where the retentate resulting from the use of a variable flow valve downstream of the device (iv) is returned to the feed inlet (i).

[0064] The device (iv) preferably includes an outlet for a waste stream, optionally. However, this is not required in many embodiments, since the waste stream, if present, may leave the device using the same outlet as the product stream. For example, if the device (iv) includes a chromatography column, only one stream leaves the column, but typically this stream starts as a waste stream containing impurities until the desired mAb is eluted from the column. Then, for a period of time, the stream leaving the column contains mAb minus many impurities. Finally, the amount of mAb leaving the column decreases, and the liquid leaving the device typically contains mainly impurities.

[0065] The processing units and devices used in the method preferably include a bubble trap, a pressure sensor, a temperature sensor, a pH sensor, a flow sensor, a conductivity sensor, an air sensor and / or a UV sensor, e.g., a uv / visible multi-wavelength sensor. One or more of each of the foregoing may be present.

[0066] Means (v) for providing a flow of liquid are well known in the art and include the application of gas pressure to the liquid, in particular an inert gas such as nitrogen or helium. Preferably, means (v) for providing a flow of liquid includes one or more pumps. Pumps that can be used include peristaltic pumps, diaphragm pumps, lobe pumps, and centrifugal pumps. Both disposable and reusable pumps can be used. In many preferred embodiments, each unit includes a single pump (v) (i.e., the unit has only one pump), preferably located downstream of component (ii) and upstream of component (iv). The type and size of the pump (v) selected usually depends on the flow capacity and pressure profile appropriate to adjust and design the parameters of the device. In certain highly preferred embodiments, the pump (v) is a quaternary diaphragm pump.

[0067] In one particular embodiment of the invention, one or more units (preferably all units) comprise one or more of the following: (vii) a device for preparing each of the at least two liquids used to form the bioprocessing liquid; (viii) a device for trapping air bubbles from one or more liquids passing through the unit; (ix) a device for controlling pressure; (iv) means downstream of the outlet; (x) several sensors (27) suitable for monitoring the process operation, said sensors (27) being located upstream and / or downstream of the device (iv) inlet and the device (iv) outlet; (xi) at least one outlet in fluid communication with the feed inlet;

[0068] Preferably, one or more units (preferably all units) further comprise a means located downstream of device (iv) for applying additional pressure to the liquid flowing through device (iv). Means for applying pressure are known in the art and include pinch valves, diaphragm valves, in particular variable position diaphragm valves.

[0069] In many embodiments, the method is operated under the control of a programmable control unit, preferably a computer. In some embodiments, a single control unit controls the operation of two or more units. In other embodiments, each unit is under the control of a separate control unit. In these other embodiments, the units preferably use a common programming language, which allows simplified communication between the units.

[0070] In one embodiment, at least one (preferably half, more preferably all) of the units of the apparatus further comprises at least one switched bypass assembly (vi) which directs the flow of liquid feedstocks and / or liquids to be combined to form the bioprocessing liquid to (a) mixer (iii) for combining the liquid feedstocks and liquids (2ab), (2cd), (2ef) and / or (2g) to generate the device feed or (b) bypassing means (iii) and instead sending the liquids and / or feedstocks to device (iv) without passing through means (iii). The units of this embodiment have the advantage that they can be used for the purification of fragile mAbs, where combining the liquid feedstocks and bioprocessing liquids in mixer (iii) may damage or degrade the mAbs.

[0071] The switching bypass (vi) is particularly useful when the device (iv) comprises a chromatography column. The switching bypass (vi) can be used to load the mAb into the chromatography column (iv) without passing through the mixer (iii), and the mixer (iii) can then be used to prepare a bioprocessing liquid that acts as an eluent for the mAb already in the column (iv). Furthermore, bypassing the mixing means (iii) may be useful when the device (iv) otherwise performs method steps of ultrafiltration and / or diafiltration, where hold-up volume and product stability may be an issue.

[0072] The diverter bypass assembly (vi) preferably comprises piping and two or three valves that direct the flow of liquid feed and liquids (2ab), (2cd), (2ef) and / or (2g) to either mixer (iii) or device (iv) without passing through mixer (iii).

[0073] One embodiment of the device according to the invention is described with reference to Figure 2. The first processing unit comprises an inlet (i) for liquid raw materials, inlets (2a)-(2f) for six different buffer solutions, and an inlet (2g) for water for injection. Each inlet is fitted with a valve, such as a straight-through diaphragm valve (3) and (3a)-(3g), so that the flow can be switched on or off. In the embodiment shown, the buffer feeds from inlets (2a) and (2b), (2c) and (2d), and (2e) and (2f) are combined downstream of the valves (3a) and (3b), (3c) and (3d), and (3e) and (3f) to form three buffer feed lines (2ab), (2cd), and (2ef), respectively, which, along with the water injection inlet (2g), are fluidly connected to different inlets of a multi-inlet flow controller (ii), which includes a four valve manifold with a single outlet having a fast acting pneumatic actuator. This configuration also allows selection between buffers from 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), improving the operational flexibility of the unit. The outlet (ii) from the multi-inlet flow controller is fluidly connected to a feed of liquid containing the target mAb at a location (5) upstream of the pump (v), which provides a flow of the combined feed through a static mixer (iii) (8) equipped with a bubble trap to the inlet of the first chromatography column (iv). The line supplying the output from the pump (v) to the chromatography column (iv) 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 (v) is controlled via a programmable control unit in response to a feedback signal (29) from the flow meter (10).

[0074] In some embodiments, optionally, the multi-inlet flow controller (ii) is controlled via a programmable control unit in response to feedback signals (28) from the conductivity and temperature sensor (11). The outlet line from the chromatography column (iv) 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 sensor (16), and a variable position valve (30), which can be used to control pressure and, if desired, apply back pressure. Preferably, the operation of the pump (v) and the variable position valve (30), and thereby the control of pressure within the apparatus, are controlled via a programmable control unit in response to feedback signals (26) and (27) from the pressure sensors (7) and (13). The outlet line passes through a series of valves (17), (19), and (20) which allow the flow to be controlled between an outlet (18) for a product stream containing the purified monoclonal antibody, an outlet (21) for a waste stream containing at least some of the impurities, or an outlet (22) for example to allow collection or sampling. The apparatus is further fitted with valves (23a) and (23b) which allow the flow to be diverted to bypass column (iv) when required during operation, as well as further valves (24) and (25) which allow the flow through column (iv) to be stopped. The product stream passing through outlet (18) can then be used as a feedstock comprising the target mAb in a second unit for performing a second processing operation, configured as shown in FIG. 2, but preferably the chromatography column (iv) is replaced by a different means for performing a processing operation, such as a different type of chromatographic or non-chromatographic unit operation, where in the second unit for performing the second processing operation the feedstock supplied to inlet (i) comprises the product stream leaving the previous unit from outlet (18).

[0075] In one method of operation, valves (3a)-(3g) are closed while valve (3) is opened and a liquid feed containing the monoclonal antibody and impurities is provided by pump (v) to column (iv) to load the monoclonal antibody and impurities onto a column, for example a column containing Protein A affinity resin, such that the monoclonal antibody selectively binds to the Protein A affinity resin. Once the desired loading is completed, valve (3) is closed and one or more valves (3a)-(3g) are opened to allow one or more liquids from inlets (2a)-(2g) forming the bioprocessing liquid to be pumped through column (iv). In some embodiments, initially only valve (3a) is opened and multiple inlet valve (ii) is operated to open an inlet valve through which a buffer, which may be a wash buffer, is provided from inlet (2a) to wash the loaded column (iv) with a buffer from inlet (2a). Once the desired wash step is completed, valve (3a) may remain open or may be closed and one or more of valves (3b)-(3g) may be opened. Inlet valve (ii) of the multi-inlet flow controller is opened to pump the liquid from inlets (2b)-(2g), or a mixture containing two or more thereof, to column (iv). By opening and closing the valves of multi-inlet valve (ii) and / or controlling valves (3a)-(3g), the composition of the bioprocessing liquid fed to column (iv) can be varied and controlled as desired. For example, when valves (3b), (3c) and (3e) are open, it is possible to vary the composition of the bioprocessing liquid stepwise or gradually by varying which inlet valves are open in multi-inlet flow controller (ii) and closing others. In another example, two or more inlet valves of multi-inlet flow controller (ii) are opened and closed at a given frequency and for a selected period of time such that a given mixture of liquids is fed to column (iv). By adjusting the times and / or frequency at which the inlet valves of the multiple inlet valve (ii) are open or closed, the composition of the bioprocessing fluid can be altered.If the time and / or frequency are changed stepwise, the composition of the bioprocessing liquid is also changed stepwise. If the time and / or frequency are changed gradually over a period of time, the composition bioprocessing liquid is also changed gradually, allowing the application of a gradient to the column (iv). By any desired method, the liquid composition of the bioprocessing liquid fed to the column (iv) can be changed to a composition that elutes the mAb from the column (iv). Before elution, the liquid leaving the column (iv) (which may contain impurities) is collected via outlet (22), or sent to waste (21), and valves (17), (19), and (20) are set accordingly. When the product stream containing the purified monoclonal antibody mAb reaches valve (17), valves (19) and (20) are closed, valve (17) is opened, and the purified mAb passes through outlet (18) to the second unit and enters the second unit through its inlet (i) as the liquid feed of the second unit.

[0076] The operation of the second unit and any further units may be substantially as described above for the first unit. It will be appreciated that the mAb exiting the second unit through its outlet (18) may be recovered and used as is, or may be further purified by acting as a feedstock for further processing operations. Such further processing operations may use conventional equipment, or may use further units according to the configuration shown in Figure 2, or may be others according to the invention.

[0077] The method according to the invention further comprises the step of mixing the obtained purified monoclonal antibody with one or more pharma- ceutically acceptable carriers to form a medicament. The entire subject matter of the claims is incorporated herein by reference thereto.

[0078] The present application is illustrated, but not limited, by the following examples. EXAMPLES

[0079] An apparatus can be constructed that comprises two units in series, each having the general structure shown in Figure 2, and each unit comprising the flow paths shown in Figure 3. The first unit, device (iv), comprises a chromatography column, and the second unit, device (iv), comprises the means for carrying out viral inactivation.

[0080] Construction of the first and second processing units Each processing unit can be constructed to include the components shown in Figure 2, except that valve (23b) is replaced with a simple fluid connection. Each unit includes substantially identical flow paths.

[0081] Preparation of bioprocessing liquids The stock solutions SS1-SS7 described below can be used as streams for inlets (2a)-(2g) as indicated: Inlet (2a)- 1M Sodium Chloride (SS1); Inlet (2b)- 4M Sodium Chloride (SS2); Inlet (2c)- 250 mM dibasic sodium phosphate (SS3); Inlet (2d)- 100 mM sodium acetate (SS4); Inlet (2e)- 175 mM sodium phosphate monobasic (SS5); Inlet (2f)- 1M acetic acid (SS6); Inlet (2g) - Distilled water (SS7).

[0082] Using the multiple inlet flow controller (ii), the seven stock solutions SS1-SS7 can be combined in the ratios shown in Table 1 to obtain the five bioprocessing liquids BPL1-BPL5 shown in Table 1.

[0083] [Table 1]

[0084] 1st Processing Unit – Chromatography The processing unit includes a chromatography column as device (iv) (2.5 L mAbSelect SuRe resin column). The chromatography column (iv) is loaded with the raw material containing mAb and impurities from the inlet (i) and chased with 2 L of a ready-to-use solution of BPL1.

[0085] Bioprocessing liquids BPL1-BPL5 can be generated by proportionally selecting each of the stock solutions SS1-SS7 using a multi-inlet flow controller (ii), a downstream pump (v) and a static mixer (iii). During the establishment of the correct buffer composition as the bioprocessing liquid, column (iv) is bypassed by valve (23a), valves (24) and (25) are closed and the unwanted buffer is directed to waste (21). When the buffer is homogeneous, as indicated by a stable reading from the upstream conductivity sensor (11), the buffer is delivered to the chromatography column (iv) as the bioprocessing liquid by opening valves (24) and (25) and closing the bypass line of valve (23a). The process conditions can be monitored using conductivity, UV and pH sensors (14), (15) and (16) downstream of column (iv). During pretreatment of column (iv) prior to binding of mAb to the column, and water rinsing after use, liquid is sent to waste (21). Once pretreated with BPL1, the chromatography resin is loaded into column (iv) by the action of pump (v), bypassing static mixer (iii), with liquid feed containing monoclonal antibody and impurities drawn from feed inlet (i). The first buffer wash BPL2 is collected through outlet (22), and the second buffer wash BPL3 is collected through outlet outlet (21), while the flow-through from column (iv) is collected through outlet outlet (21). The purified mAb is recovered from column (iv) using elution buffer BPL4 as the bioprocessing liquid and collected through outlet (18). Finally, remaining impurities can be removed from column (iv) using strip buffer BPL5 as the bioprocessing liquid and collected through outlet (22).

[0086] Second Processing Unit – Viral Inactivation The processing unit contains a viral inactivation device (iv) (50L Stedim Magmix™ bag with impeller drive) with an outlet (21) connected to an inlet (2g) to provide a recirculation loop.

[0087] Preparation of bioprocessing liquids Stock solutions SS1 to SS2 described below were installed in the following order into the inlets (2a) to (2g): Inlet (2a)- 1M acetic acid (SS1); Inlet (2b) - no feed; Inlet (2c)- 1M Tris base (SS2); Inlet (2d) - no feed; Inlet (2e) - no feed; Inlet (2f) - no feed; Inlet (2g) - Device bag (iv).

[0088] The product stream from the first processing unit is used as the feedstock for the second processing unit by loading it through inlet (i) into the viral inactivation bag (iv) of the second processing unit. The feedstock thus contains mAb and virus (25 L ~ 7.5 g / L mAb in 50 mM sodium acetate pH 4.6). The feedstock is directed to device (iv) by bypassing device (iii) through pump (v) using the switched bypass assembly (vi) and by opening valve (24) and closing the bypass line of valve (23a) and valve (25). Once device (iv) is charged, the multi-flow controller valves connected to valves (25), (19), (3g) and (3g) may be opened. Valve (3) is closed and the switched bypass assembly (vi) is left in bypass mode. The impeller drive of the viral inactivation bag is turned on and set to a speed of 450 rpm. The pH of the feedstock is lowered to a target of pH 3.6 by intermittent addition of 1 M acetic acid at (2a) connected to a multi-inlet flow controller (ii) through (2ab). During the pH titration to pH 3.6, valve (3a) is opened and the multi-inlet flow controller (ii) is operated to dose approximately 100 mL of 1 M acetic acid into the virus inactivation bag (iv) every 30 seconds, each time by operation of the valve. After eight doses of 1 M acetic acid, the feedstock is recirculated around the flow path and allowed to mix in the virus inactivation bag (iv) for 15 minutes. During the 15 minutes of recirculation, acetic acid dosing is repeated as above until the pH reaches pH 3.6, as determined by sensor (15). The pH-titrated feedstock is then recirculated around the flow path and allowed to mix in the virus inactivation bag (iv) for 60 minutes.

[0089] The pH of the feedstock is raised to a target of pH 5.0 by intermittent addition of 1M Tris base at (2c) connected to a multi-inlet flow controller (ii) through (2cd). During the pH titration to pH 5.0, valve (3c) is opened and the multi-inlet flow controller (ii) is operated to dose approximately 100 mL of 1M Tris base into the virus inactivation bag (iv) every 30 seconds, each time by operation of the valve. After eight doses of 1M Tris base, the feedstock is recirculated around the flow path and allowed to mix in the bag (iv) for 15 minutes. During the 15 minutes of recirculation, the dosing of Tris base is repeated as above until the pH reaches pH 5.0, as determined by sensor (15). Finally, the product stream containing the mAb and inactivated virus is recovered by closing outlet (19), opening valve (17) and collecting the contents of the virus inactivation bag (iv) as a product stream through outlet feed (18). Aspects of the invention [Embodiment 1] A method for purifying a liquid feedstock comprising a monoclonal antibody and impurities, comprising passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream comprising a purified monoclonal antibody and, optionally, a waste stream comprising at least a portion of the impurities, each unit comprising the following components (i)-(v): (i) An inlet for liquid feedstock; (ii) a multiple inlet flow controller including two or more variable flow inlet valves for producing a bioprocessing liquid from at least two other liquids that are combined in a desired ratio; (iii) means for combining a liquid feedstock and a bioprocessing liquid to generate a device feed, and / or for combining at least two other liquids to prepare a bioprocessing liquid; (iv) a device for performing processing operations on a liquid feed or device feed, the device including an inlet for the device feed and / or liquid feed and an outlet for a product stream; and (v) means for providing flows of a liquid feedstock, at least two liquids, a bioprocessing liquid, a device feed, a product stream, and an optional waste stream; where: (I) a liquid feedstock is fed through inlet (i), combined with a bioprocessing liquid by means (iii) and then fed through device (iv) or combined with the bioprocessing liquid in device (iv), in either case to provide a product stream and optionally a waste stream which exit the unit; (II) the units are arranged in series such that the liquid feed of the second and any subsequent units comprises the product stream from the preceding unit; (III) a device (iv) in at least one of the at least two processing units performs a chromatographic processing operation; and (IV) A method, wherein a device (iv) in at least one of the at least two processing units performs a processing operation of virus inactivation. [Aspect 2] The method described in aspect 1, wherein the viral inactivation treatment is performed after the chromatography treatment. [Embodiment 3] The method of embodiment 1 or 2, wherein the apparatus comprises a further unit comprising components (i) to (v) for carrying out additional chromatographic purification of the mAb. [Embodiment 4] The method of embodiment 3, wherein the chromatographic purification carried out in each unit uses chromatographic columns packed with different conditions and / or different resins than the chromatographic columns used in all of the other units. [Embodiment 5] The method of any one of embodiments 1 to 4, wherein each chromatographic purification is independently selected from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, reverse phase chromatography, expanded bed chromatography, mixed mode chromatography, membrane chromatography, and size exclusion chromatography. [Embodiment 6] A method according to any of embodiments 1 to 5, wherein each unit prepares a mixed bioprocessing liquid in situ during the method. [Aspect 7] A method according to any one of aspects 1 to 6, wherein the apparatus further comprises a unit comprising components (i) to (v) for removing any viruses from the liquid raw material. [Embodiment 8] The method of any one of embodiments 1 to 7, wherein the apparatus further comprises a unit comprising components (i) to (v) for ultrafiltration of the liquid feedstock to provide a concentrate comprising the target material. [Embodiment 9] The method of any one of embodiments 1 to 8, wherein the apparatus further comprises a unit comprising components (i) to (v) for diafiltration of the feedstock to buffer the target material at a pH at which the target material is stable. [Embodiment 10] The method of any one of embodiments 1 to 9, wherein the means for imparting flow to each unit comprises a single pump, the single pump preferably being positioned downstream of component (ii) and upstream of component (iv). [Aspect 11] The apparatus comprises the following units arranged in series: a. A unit for performing chromatographic purification of mAbs 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 performing chromatographic purification of mAbs by ion exchange chromatography. wherein each unit comprises components (i)-(v), and the product feed of each unit is used as a feedstock for the next unit, or if there is no next unit, the product feed is collected. [Aspect 12] The apparatus comprises the following units arranged in series: a. A unit for performing chromatographic purification of mAbs 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 mAb by cation exchange chromatography; and d. A unit for performing chromatographic purification of mAbs by anion exchange chromatography. wherein each unit comprises components (i)-(v), and the product feed of each unit is used as a feedstock for the next unit, or if there is no next unit, the product feed is collected. [Aspect 13] The apparatus comprises the following units arranged in series: a. A unit for performing chromatographic purification of mAbs 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 mAbs by cation exchange chromatography; d. A unit for performing chromatographic purification of the mAb by anion exchange chromatography; and e. Unit for removal of any inactivated viruses wherein each unit comprises components (i)-(v), and the product feed of each unit is used as a feedstock for the next unit, or if there is no next unit, the product feed is collected. [Aspect 14] The apparatus comprises the following units arranged in series: a. A unit for performing chromatographic purification of mAbs 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 mAbs by cation exchange chromatography; d. A unit for performing chromatographic purification of the mAb by anion exchange chromatography; and e. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange. wherein each unit comprises components (i)-(v), and the product feed of each unit is used as a feedstock for the next unit, or if there is no next unit, the product feed is collected. [Embodiment 15] The method of any one of embodiments 11 to 14, wherein the units are arranged in series in the order listed. [Embodiment 16] A method according to any of embodiments 1 to 15, wherein the bioprocessing liquid is provided by combining at least three liquid streams, at least two of the at least three liquid streams being each provided by combining at least two additional liquid streams. [Embodiment 17] A method as described in any of embodiments 1 to 16, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a device for removing air bubbles from one or more liquids passing through the unit. [Embodiment 18] The method of any one of embodiments 1 to 17, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a pressure sensor disposed upstream of component (iv). [Embodiment 19] The method of any one of embodiments 1 to 18, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a pressure sensor disposed downstream of component (iv). [Embodiment 20] The method of any one of embodiments 1 to 19, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a UV sensor disposed downstream of component (iv). [Embodiment 21] The method of any one of embodiments 1 to 20, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a pH sensor disposed downstream of component (iv). [Embodiment 22] The method of any one of embodiments 1 to 21, wherein at least one unit (preferably at least half of the units, more preferably all of the units) further comprises a conductivity sensor disposed downstream of component (iv). [Embodiment 23] A method according to any of embodiments 1 to 22, wherein at least one unit further comprises at least one switched bypass assembly (vi) for either (a) directing the flow of liquid feedstock and bioprocessing liquid to a means (iii) for combining the liquid feedstock and bioprocessing liquid to generate a device feed, or (b) bypassing the means (iii) and instead directing the liquid feedstock and bioprocessing liquid to the device (iv) without passing through the means (iii). [Embodiment 24] The method of embodiment 23, wherein the switched bypass assembly (vi) is present in all units. [Embodiment 25] The method of any one of embodiments 1 to 24, wherein the flow paths used in at least half of the units (preferably all of the units) have substantially the same configuration. [Embodiment 26] The method of any of embodiments 1 to 25, wherein at least 75% of the component parts of each unit other than component (iv) are identical to component parts used in at least 80% of the other units of the apparatus. [Embodiment 27] The method of any one of embodiments 1 to 26, wherein all of the component parts of each unit, except for component (iv), are identical to all of the component parts used in all other units of the apparatus. [Embodiment 28] The method of any one of embodiments 1 to 27, wherein the flow path through each unit is substantially identical to the flow paths through all other units. [Embodiment 29] A method according to any of embodiments 1 to 28, wherein the composition of the bioprocessing liquid used in at least one unit is changed stepwise or gradually during the method. [Aspect 30] A method according to any one of aspects 1 to 29, wherein each unit includes only one pump. [Aspect 31] The method according to any one of aspects 1 to 30, further comprising mixing the obtained purified monoclonal antibody with one or more pharma- ceutically acceptable carriers to form a pharmaceutical agent.

Claims

1. 1. A method for purifying a liquid feedstock comprising a monoclonal antibody and impurities, comprising passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream comprising a purified monoclonal antibody and a waste stream comprising at least a portion of the impurities, each unit comprising the following components (i) to (v): (i) an inlet for liquid feedstock; (ii) a multi-inlet flow controller including two or more variable flow inlet valves for producing a bioprocessing liquid from at least two other liquids that are combined in a desired ratio; (iii) a means for combining a liquid feedstock and a bioprocessing liquid to generate a device feed, and / or for combining at least two other liquids to prepare a bioprocessing liquid; (iv) a device for performing processing operations on a liquid feed or device feed, the device comprising an inlet for the device feed and / or liquid feed and an outlet for a product stream; and (v) means for providing flows of a liquid feedstock, at least two liquids, a bioprocessing liquid, a device feed, a product stream, and a waste stream; Where: (I) a liquid feed is fed through inlet (i), combined with a bioprocessing liquid in means (iii) and then fed through device (iv) or combined with the bioprocessing liquid in device (iv), in either case to provide product and waste streams which exit the unit; (II) the units are arranged in series such that the liquid feed of the second and any subsequent units comprises the product stream from the preceding unit; (III) a device (iv) in at least one of the at least two processing units performs a chromatographic processing operation; and (IV) A method, wherein a device (iv) in at least one of the at least two processing units performs a processing operation of virus inactivation.

2. 2. The method of claim 1, wherein the viral inactivation treatment step is carried out after the chromatography treatment step.

3. The method of claim 1 or 2, wherein each unit prepares a mixed bioprocessing liquid in situ during the method.

4. 4. A method according to any one of claims 1 to 3, wherein the means for imparting flow to each unit comprise a single pump, preferably located downstream of component (ii) and upstream of component (iv).

5. The apparatus comprises the following units arranged in series: a. a unit for performing chromatographic purification of mAbs 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 performing chromatographic purification of the mAb by ion exchange chromatography. wherein each unit comprises components (i) to (v), and the product feed of each unit is used as a feed for the next unit or, if there is no next unit, the product feed is collected.

6. The apparatus comprises the following units arranged in series: a. a unit for performing chromatographic purification of mAbs 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 mAb by cation exchange chromatography; and d. A unit for performing chromatographic purification of mAbs by anion exchange chromatography. wherein each unit comprises components (i) to (v), and the product feed of each unit is used as a feed for the next unit or, if there is no next unit, the product feed is collected.

7. The apparatus comprises the following units arranged in series: a. a unit for performing chromatographic purification of mAbs 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 mAb by cation exchange chromatography; d. a unit for performing chromatographic purification of the mAb by anion exchange chromatography; and e. A unit for the removal of any inactivated viruses. wherein each unit comprises components (i) to (v), and the product feed of each unit is used as a feed for the next unit or, if there is no next unit, the product feed is collected.

8. The apparatus comprises the following units arranged in series: a. a unit for performing chromatographic purification of mAbs 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 mAb by cation exchange chromatography; d. a unit for performing chromatographic purification of the mAb by anion exchange chromatography; and e. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange. wherein each unit comprises components (i) to (v), and the product feed of each unit is used as a feed for the next unit or, if there is no next unit, the product feed is collected.

9. 9. The method of any one of claims 1 to 8, wherein the bioprocessing liquid is provided by combining at least three liquid streams, at least two of the at least three liquid streams being provided by combining at least two further liquid streams each.

10. 10. The method of claim 1, wherein at least one unit further comprises a device for removing air bubbles from one or more liquids passing through the unit.

11. A method described in any one of claims 1 to 9, wherein at least half of the units further comprise a device for removing air bubbles from one or more liquids passing through the unit.

12. A method according to any one of claims 1 to 9, wherein every unit further comprises a device for removing air bubbles from one or more liquids passing through the unit.

13. 13. The method of any one of claims 1 to 12, wherein at least one unit further comprises at least one switched bypass assembly (vi) for either (a) directing the flow of liquid feedstock and bioprocessing liquid to a means (iii) for combining the liquid feedstock and bioprocessing liquid to generate a device feed, or (b) bypassing the means (iii) and instead sending the liquid feedstock and bioprocessing liquid to the device (iv) without passing through the means (iii).

14. 14. The method of claim 13, wherein a switched bypass assembly (vi) is present in every unit.

15. 15. The method of claim 1, wherein the flow paths used in at least half of the units have the same configuration.

16. A method described in any one of claims 1 to 14, wherein the flow paths used in all units have the same configuration.

17. 17. A method according to any one of claims 1 to 16, wherein at least 75% of the component parts of each unit other than component (iv) are identical to component parts used in at least 80% of the other units of the apparatus.

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