Process for purifying target substances

Abstract

To provide a method for purifying a liquid containing a target substance, in particular a biomolecule such as a recombinant polypeptide.SOLUTION: There is provided a method for purifying a composition comprising water, a target substance, impurities, and optionally cells. The method comprises the following the steps (A) and (B): (A) preparing a liquid feedstock by performing the step (Ai) and / or (Aii) on the composition: (Ai) removing at least some of the cells from the composition; (Aii) concentrating the composition by removing water from the composition; and (B) passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream containing purified target substance and optionally a waste stream comprising at least some of the impurities, the units comprising specified components.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for purifying a liquid containing a target substance ("TS"), in particular a liquid containing a biomolecule, such as a recombinant polypeptide. [Background technology]

[0002] Target substances such as biomolecules, particularly recombinant polypeptides and nucleic acids, have attracted considerable attention in recent years for their potential use in therapeutic applications. Before such substances can be used in therapeutic applications, they typically require purification to remove host cells, unwanted proteins, viruses, and the like. Biomolecules are often produced by culturing recombinant host cells that have been genetically engineered to express the desired biomolecule. The biomolecule is then recovered from the culture medium and typically purified by a method involving several processing operations (sometimes referred to as "unit operations").

[0003] Equipment for purifying biomolecules is known in the art and typically includes several very different processing units, each performing a distinct processing operation. Typically, units are provided by various manufacturers that specialize in devices for performing one specific processing operation (e.g., filtration, chromatography, etc.). Equipment currently used for commercial-scale biomolecule production is typically very bulky, requiring extensive floor space and infrastructure. Furthermore, while some commonality of processing units can be achieved across multiple processing operations, the design of units for specific unit operations, such as viral inactivation and / or ultrafiltration, is significantly different from that of, for example, chromatographic purification. This means that either more space is required to accommodate two or more sets of processing units, or the interoperability of stages and control of the equipment is significantly more complex. Furthermore, operators require extensive training on each of the various types of processing units they use because the units are so different from one another. The lack of similarity among the many processing units currently used to purify biomolecules means that large inventories of spare parts are required. Furthermore, the lack of similarity among the many units complicates routine maintenance, as engineers must learn how to repair processing units with very different designs. Devices that require different processing solutions (e.g., buffers, eluents, etc.) for different processing operations also pose a significant risk of operator error, which can risk failure of a very expensive batch of biomolecules if the wrong, externally prepared processing solution is provided at the wrong stage. Simplified, more broadly applicable devices with a lower risk of operator error are 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 composition comprising water, a target substance, impurities, and optionally cells, comprising the following steps (A) and (B): (A) Preparing a liquid feedstock by performing steps (Ai) and / or (Aii) on the composition: (Ai) removing at least some cells from the composition; (Aii) concentrating the composition by removing water from the composition; and (B) passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream comprising a purified target substance and, optionally, a waste stream comprising at least some impurities, each unit comprising the following components (i) through (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 fluid from at least two other fluids combined in a desired ratio; (iii) means for combining a liquid feedstock and a bioprocessing liquid to produce 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, including an inlet for the device feed and / or liquid feed and an outlet for a product stream; (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 a bioprocessing liquid in device (iv), in either case providing a product stream and optionally a waste stream that exits the unit; (II) step (Ai) is performed only if the composition comprises cells; (III) at least one of the units is a unit for performing chromatographic purification of a target substance; (IV) A method is provided in which 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.

[0005] 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.

[0006] Preferably, device (iv) in at least one of the at least two processing units performs a processing operation of viral inactivation. The present invention provides a method that can be carried out on a manufacturing scale and offers many advantages over previous methods, particularly with regard to simplicity, cost and ease of operation, reduced risk of operator error, easier maintenance, and reduced spare parts inventory.

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

[0008] [Figure 1] FIG. 1 shows a schematic embodiment of the method. [Figure 2] FIG. 1 shows a schematic embodiment of the method. [Figure 3] FIG. 2 is a schematic diagram of one processing unit that can be used to perform processing operations. DETAILED DESCRIPTION OF THE INVENTION

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

[0010] FIG. 2 is identical to FIG. 1 except that step (A) is performed within one or more units of the apparatus. FIG. 3 is described in more detail in the Examples section below.

[0011] Advantageously, step (A) provides the apparatus with a liquid feed having a concentration within a desired concentration range. In this way, significant fluctuations in the concentration of TS entering the apparatus can be avoided, which can increase the efficiency of the processing operations, particularly when one or more of the processing operations involves affinity or ion exchange chromatography.

[0012] Of course, if the composition does not include cells, for example, if the composition is a cell harvest from which the cells have already been removed, it may not be necessary (or possible) to remove the cells from the composition. If the composition includes cells, it may be desirable to remove some or all of the cells from the composition so that the cells do not block or interfere with the flow of the liquid feedstock through the device.

[0013] If step (Ai) is carried out, it preferably removes at least 80%, more preferably at least 90%, especially at least 98%, more especially substantially all cells from the composition.

[0014] In step (Ai), at least some cells, if any, can be removed from the composition, for example, by centrifugation or filtration, such as depth filtration or microfiltration, particularly by recirculating tangential flow filtration ("TFF"). In the TFF method, one or more retentates and permeates are typically recirculated through a TFF device. The permeate, which typically contains fewer cells than the composition, is then concentrated by removing water from the composition in step (Aii), and the resulting concentrate is then used as a liquid feedstock for the processing described in step (B). The retentate may be recycled or reused through the TFF device, for example, to recover cells and create more TS. Preferably, TFF is used to remove cells, if any, from the composition, although other methods for removing cells, such as other filtration methods and / or centrifugation, may be used if desired.

[0015] TFF devices for performing cell removal in step (A) are commercially available from EMD Millipore Corporation (Billerica, Mass.), and include, for example, the Labscale™ TFF system, the Cogent™ M1 TFF system, the Cogent™ microscale TFF system, the assembly for the Flexware™ TFF system, the ProFlux™ TFF system, and the Prostak™ TFF system.

[0016] Although not particularly limited, in step (Aii), the concentration of the composition by removing water is preferably 5 to 20 g of target material / cm 3 , especially 5-10g TS / cm 3 The removal of water also removes impurities, particularly low molecular weight impurities, e.g. It will be appreciated that impurities having a molecular weight below a particular cutoff when a medium having a given molecular weight cutoff is used for concentration, such as impurities containing residual medium or feed components, may be simultaneously removed. In some cases, the removal of water in step (Aii) may also alter the concentration of impurities. For example, the concentration of low molecular weight impurities may decrease (if removed with water), and the concentration of high molecular weight impurities may increase with the concentration of the target material.

[0017] In step (Aii), various methods can be used to concentrate the composition by removing water, including, but not limited to, single-pass tangential flow filtration (SPTFF), ultrafiltration (e.g., ultrafiltration using a centrifugal filter and / or a stirred cell), lyophilization, evaporation, precipitation, crystallization, aqueous two-phase separation, dialysis, or a combination of two or more of the above.

[0018] In a preferred embodiment, the composition is concentrated in step (Aii) by a method comprising SPTFF. When step (Aii) is carried out by a process involving SPTFF, the retentate is typically used as the liquid feed for step (B) and the permeate is sent to waste without being reused.

[0019] In one embodiment, step (Ai) comprises removing cells from the composition using TFF, and step (Aii) comprises concentrating the composition by removing water from the composition using SPTFF. In this manner, the liquid raw material feed may be prepared for step (B) of the method continuously, semi-continuously, or batchwise.

[0020] In some embodiments, TFF and / or SPTFF involves passing a feed liquid through a tangential flow filtration device under pressure, where the pressure applied to the feed liquid is cycled between at least a higher pressure and a lower pressure. Preferably, this implementation is performed inline. The pressure can be applied by a pump, and the pressure can be varied by operating a variable flow valve, e.g., an intermittent flow valve. The higher pressure is preferably used for up to 99.9% of the time the liquid feed is pressurized, preferably 85-99%. Although not particularly limited, typically, at least 10 cycles are used, typically at a cycle frequency of less than 100 Hz, preferably less than 5 Hz, e.g., 0.05-0.5 Hz. The higher pressure is preferably at least 1.05 times greater than the lower pressure, more preferably at least 1.1-2.0 times greater than the lower pressure.

[0021] In one embodiment, in step (Aii), concentrating the composition by removing water from the composition is carried out using an ultrafiltration cassette to provide a liquid feed having a concentration within the desired concentration range in a single pass.

[0022] In certain embodiments, the apparatus comprises a unit comprising an SPTFF device (iv) comprising either a flat sheet, hollow fiber, or spiral wound membrane between a device (iv) inlet and a device (iv) outlet, e.g., as described in WO2017 / 118835.

[0023] The method according to the present invention can be carried out in many different ways, including but not limited to: - the composition comprises cells, and at least some of the cells are removed in step (Ai) by a method comprising centrifugation or filtration; - the composition comprises cells, and at least some of the cells are removed in step (Ai) by a method comprising recirculating tangential flow filtration; - the composition is substantially cell-free and step (Ai) is omitted; - step (Aii) is carried out by a method comprising single-pass tangential flow filtration, ultrafiltration, freeze-drying, evaporation, precipitation, crystallization, aqueous two-phase separation, dialysis, or a combination of two or more of the foregoing, but preferably by a method comprising single-pass tangential flow filtration. - the composition comprises cells, and at least some of the cells are removed in step (Ai) by a method comprising recirculation tangential flow filtration, and step (Aii) is carried out by a method comprising single-pass tangential flow filtration; - step (Ai) is performed outside the device; and Step (Aii) is performed external to the device.

[0024] Step (Ai) typically removes cells, but may also remove cell debris and some host cell impurities from the composition. In one embodiment, step (Ai) includes one or more centrifugation steps. Centrifugation can be performed as known in the art. For example, centrifugation of the composition may be performed at a concentration of about 1×10 -8It can be performed using a normalized load of 1000 m / s and a gravitational force of approximately 5000 x g to approximately 15000 x g.

[0025] In another embodiment, step (Ai) comprises removing at least some of the cells from the composition using a microfiltration and / or ultrafiltration membrane. In some embodiments, the microfiltration or ultrafiltration membrane may be in a tangential flow filtration (TFF) mode. In one embodiment, step (Ai) comprises removing at least some of the cells from the composition by TFF, e.g., using a 0.1 μm or 750 kD molecular weight cutoff membrane (e.g., a polysulfone membrane), preferably using a pressure of 5 to 40 psig, and optionally at a temperature ranging from about 4°C to about 60°C.

[0026] In yet another embodiment, step (Ai) involves removing at least some cells from the composition using one or more depth filtration steps. Depth filtration refers to a method of removing particles from a solution using a series of filters arranged in order of decreasing pore size. The three-dimensional matrix of the depth filter forms a maze-like path through which the sample passes. The principal retention mechanism of a depth filter relies on random adsorption and mechanical capture throughout the depth of the matrix. In various embodiments, the filter membrane or sheet may be made of rolled cotton, polypropylene, rayon cellulose, glass fiber, sintered metal, porcelain, diatomaceous earth, or other known materials. In certain embodiments, the depth filter membrane may be chemically treated to impart a positive electrical charge, i.e., a cationic charge, allowing the filter to capture negatively charged particles, such as DNA, host cell proteins, or aggregates. In this embodiment, any depth filtration system available to those skilled in the art can be used. In certain embodiments, the depth filtration step can be achieved using the Millistak™ Pod Depth Filter System, XOHC media, available from Millipore Corporation. In another embodiment, the depth filtration step can be accomplished using a Zeta Plus™ depth filter available from 3M Purification Inc.

[0027] In some embodiments, the depth filter has an average pore size of about 0.1 μm to about 8 μm. In other embodiments, the depth filter has pores with an average diameter of about 2 μm to about 5 μm, particularly 0.01 μm to about 1 μm. In yet other embodiments, the depth filter has pores with an average diameter of about 1 μm.

[0028] In some embodiments, step (Ai) comprises two or more depth films arranged in series. In this embodiment, for example, a Millistak™ Mini DOHC and XOHC filter can be placed in series.

[0029] Concentrating the composition in step (Aii) may also increase the concentration of impurities (e.g., unwanted host cell proteins ("HCPs")) and viruses in addition to increasing the concentration of TS. When HCP impurities are primarily negatively charged and TS are positively charged (as is the case for many monoclonal antibodies), the apparatus preferably includes a processing unit for performing anion exchange chromatography. Generally, unwanted HCP levels in liquid feedstock are low (e.g., several hundred ppm), and much higher processing efficiencies can be obtained by concentrating the liquid feedstock before processing the feedstock in a processing unit including a chromatography unit.

[0030] 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 pharmaceutical preparations. 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) through (v) above. While the method can further include two or more units operating in parallel, it is preferred that all units used in the method be arranged in series. In many highly preferred embodiments, the method operations performed in each unit are different from the method operations used in all other units. Thus, the apparatus may include multiple units, e.g., for performing chromatography, but each such chromatography unit or the method it is used in preferably differs from the other units performing chromatography.

[0031] 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 an operator using the wrong externally prepared bioprocessing liquid intended for another method operation.

[0032] Preferably, the apparatus further comprises one or more units for performing one or more of the following method operations, in each case the unit comprising components (i) to (v): - Remove all viruses from the liquid material; - Ultrafiltration of the feedstock to provide a concentrate containing the desired target substance; - diafiltration of the feedstock to buffer the target material at a pH at which the desired target substance is stable; and - For example, tangential flow filtration, including recirculation and single pass tangential flow filtration.

[0033] In a preferred embodiment, the apparatus comprises a processing unit comprising devices for carrying out the following processing operations, preferably in the order listed: Performing affinity chromatography (especially Protein A affinity chromatography), viral inactivation, cation exchange chromatography, anion exchange chromatography, viral filtration (removal of inactivated virus), and concentration and / or buffer exchange of the product feed (e.g., by ultrafiltration and / or diafiltration performed in a single unit or in separate units).

[0034] 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).

[0035] 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 unit.

[0036] Preferably, the apparatus comprises the following units arranged in series, preferably in the order listed, each unit comprising components (i) to (v), the product feed of each unit being used as the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. a unit for carrying out a first chromatographic purification of the target substance, preferably by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out a second chromatographic purification of the target substance, preferably by ion exchange chromatography; e. A unit for carrying out a third chromatographic purification of the target substance, preferably by ion exchange chromatography; f. A unit for the removal of any inactivated viruses; and g. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange.

[0037] In some embodiments, the second chromatographic purification comprises anion exchange and the third chromatographic purification comprises cation exchange, while in other embodiments, the second chromatographic purification comprises cation exchange and the third chromatographic purification comprises anion exchange.

[0038] 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally, a unit for performing step (Ai) and / or a unit for performing step (Aii); and b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography.

[0039] In another 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. a unit for carrying out chromatographic purification of the target substance by affinity chromatography; and c. A unit for the inactivation of any viruses that may be present in the liquid raw material.

[0040] In another 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; and d. A unit for carrying out chromatographic purification of the target substance by ion exchange chromatography.

[0041] In another 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; and e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography.

[0042] In another 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; and f. A unit for the removal of any inactivated viruses.

[0043] In another 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 the feedstock for the next unit, or the product feed being collected if there is no next unit: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; f. A unit for the removal of any inactivated viruses; and g. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange.

[0044] 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., f., then g.

[0045] 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).

[0046] 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).

[0047] 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).

[0048] 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).

[0049] 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).

[0050] 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, the similarity of the units simplifies routine maintenance of the apparatus, and the similarity of each unit to the other units of the apparatus makes it easier to operate. In contrast to the prior art, which uses vastly different processing units from multiple manufacturers, technicians avoid having to learn how to service a large number of vastly different processing units. Because the components (iv) of each unit are typically different from the components (iv) of the other units (so that each unit can perform a distinct processing operation), The phrase "other than component (iv)" is used.

[0051] 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 so that each unit can perform a separate processing operation, for example as shown in Figure 1.

[0052] 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 channel is preferably constructed from a plastic material.

[0053] Preferably, the flow path through each unit is substantially identical to the flow path through every other unit. In certain embodiments, one or more of the units (preferably all of the units) include a multi-use flow path constructed from a material, e.g., stainless steel, that allows for reuse a significant number of times before replacement is required.

[0054] In certain embodiments, one or more of the units (preferably all of the units) preferably include a single-use flow path constructed from a commonly selected material that can be sterilized by gamma irradiation, e.g., a plastic material, typically designed with a limited lifespan, e.g., for use as a disposable consumable.

[0055] In one embodiment, the product stream from each unit other than 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.

[0056] 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.

[0057] The inlet (i) for the liquid feedstock preferably typically includes a tube fitted with a valve (3) and a check valve to avoid contamination of the liquid feedstock with the liquid flowing from the multi-inlet flow controller (ii).

[0058] Target substances that can be purified by this method 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.

[0059] pDNA may be in one or more of several forms, including supercoiled, linear, and open circular (i.e., nicked or relaxed) isoforms. Supercoiled pDNA isoforms have a covalently closed circular conformation, and pDNA becomes negatively supercoiled in host cells by the action of host enzyme systems. In open circular isoforms, one strand of the pDNA duplex is broken at one or more locations.

[0060] Methods for generating pDNA are well known in the art. pDNA can be natural or artificial, for example, a cloning vector carrying a foreign DNA insert. In many embodiments, the pDNA is in the size range of 1 kilobase to 50 kilobases. For example, pDNA encoding an expressed interfering RNA is typically in the size range of 3 kilobase to 4 kilobase.

[0061] 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.

[0062] Antibodies include biologically active monoclonal antibodies (mAbs), polyclonal antibodies, and antibody fragments, including multivalent and / or multispecific forms of any of the foregoing.

[0063] 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 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 contains a variable region (V L ) and one domain C L The constant region comprises:

[0064] V H and V L The region 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 in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0065] Monoclonal antibody fragments typically contain a portion of an intact mAb, which portion possesses 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) C, which can form a bivalent fragment by disulfide bridging between two Fab derivatives HFab derivatives, such as Fab' fragments with 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 with one or more cysteine ​​residues at the C-terminus of one domain; (v) V of a single arm of the 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) with or without a constant region domain H or V L 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) 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 antigen-binding fragments of any 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 ), including so-called "diabodies"; (x) So-called linear antibodies include antibodies that contain a pair of tandem Fd segments that, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0066] Impurities that can be removed by the methods typically include by-products from the method 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.

[0067] The multi-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 multi-inlet flow controller includes at least two inlet valves, and often includes up to eight inlet valves, such as three, four, five, six, or seven, although more inlet valves can be included if desired. The inlet valves may each have the same dimensions, or one or more inlet valves may have different dimensions relative 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.

[0068] The multiple inlet flow controller (ii) used in the present invention also comprises at least one outlet, and although there may be more than one outlet, it is preferred that a single outlet is used.

[0069] 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 that 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.

[0070] 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 for a predetermined period of time, or cycle rate, to produce the desired bioprocessing liquid composition, and controlling the opening and closing of each valve according to the required percentage of cycle time. The cycle rate can be either constant or variable. Most preferably, intermittent flow inlet valves are used, controlled so 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.

[0071] In many embodiments, the method includes multiple cycles of flow through the multi-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, e.g., at least 50, 100, 500, 750, 1000, 1500, 2000, 3000, 5000, 7500, 10000, or more cycles.

[0072] It will be appreciated that a range of cycle frequencies can be used. In most 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 is 2 Hz or less, most preferably 1 Hz or less, for example, 0.05 to 0.5 Hz.

[0073] 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 each provided by combining at least two additional liquid streams (e.g., using valves (3a) and (3b) or (3c) and (3d)). This mixing is preferably carried out in 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.

[0074] The composition of the bioprocessing fluid 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 fluid may change gradually or stepwise during the method, particularly if the unit includes a chromatography column and the bioprocessing fluid acts as an eluent.

[0075] 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 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.

[0076] Although some mixing 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 includes a mixer (iii). Mixer (iii) provides a means for combining the liquid feedstock and liquids (2ab), (2cd), (2ef), and / or liquid from inlet (2g) used to generate device feed and, if desired, form a bioprocessing liquid. Additionally, mixer (iii) can 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) can 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, 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 the processing operation.

[0077] In many embodiments, the mixer (iii) further comprises a means for trapping air bubbles, for example, an air bubble trap. In one embodiment, 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 device (iv) before or after the liquid feedstock enters component (iv). For example, if device (iv) includes a chromatography column, means (iii) can be used to provide a bioprocessing liquid for pretreating the column before the liquid feedstock is loaded onto the chromatography column, for preparing a bioprocessing liquid for washing the target substance on the chromatography column, for preparing a bioprocessing liquid for eluting the target substance from the chromatography column, and for preparing a bioprocessing liquid for removing impurities remaining in the column after the target substance 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 includes a device for removing air bubbles from one or more liquids passing through the unit.

[0078] The device feed inlet may also be used to receive bioprocessing fluid, for example, from component (ii) or (iii). In addition to the device designated in (III) 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 operations performed in device (III)), viral inactivation, filtration (e.g., ultrafiltration, microfiltration, dead-end filtration and / or diafiltration), viral removal, refolding, flocculation, sterile filtration, and in-line pretreatment.

[0079] Chromatographic bioprocessing operations that can be performed using device (iv) include affinity chromatography (particularly 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. 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 target substance.

[0080] Preferably, the apparatus includes at least two, more preferably three, units comprising components (i)-(v) for performing chromatographic purification of the target substance. In this case, the chromatographic purification performed in each unit preferably uses different conditions and / or a chromatographic column packed with a different material (e.g., a different resin, membrane, or monolith) 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.

[0081] The device (iv) for carrying out the virus inactivation method in the method of the present invention generally comprises a storage vessel capable of storing a liquid containing the target substance under conditions that inactivate any viruses present. In certain embodiments, the outlet and inlet of the virus inactivation device (iv) may be fluidically connected to create a recirculation loop. In such an embodiment, The apparatus is provided with a container or bag fluidly connected between the "device" inlet and the "device" outlet, one of the device outlets being fluidly connected to one of the inlets of the multi-inlet flow controller (ii). The container or bag between the "inlet" and "outlet" of the device (iv), which is fluidly connected to the liquid feedstock inlet (i), is filled by a means for providing flow (v), typically a pump, or is pretreated with at least one other liquid through at least one of the inlets of the multi-inlet flow controller (ii). In certain embodiments, the container or bag is a mixing container or bag. The bioprocessing liquid is recirculated back to the container or bag through the inlet of the multi-inlet flow controller (ii) and back to the inlet of the multi-inlet flow controller (ii), as the solution containing the target substance is pretreated with at least one additional liquid fluidly connected to at least one other inlet on the multi-inlet flow controller (ii).

[0082] 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., a pH 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 hours 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.

[0083] Inactivated viruses can be removed by filtration using normal flow filters (NFF) or tangential flow filtration (TFF) filters, such as those 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 the inactivated viruses using membranes with average pore sizes of 20 to 100 nanometers (nm). Such membranes retain the inactivated viruses on their surface while allowing the passage of target substances through the membrane.

[0084] 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), etc., 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 the VIRESOLVE™ NFP virus filter, or as cassettes (for TFF), such as PELLICON™ cassettes, available from EMD Millipore, Billerica, Mass.

[0085] Filtration operations that can be performed using device (iv) include viral filtration, depth and absolute filtration, ultrafiltration, diafiltration, and microfiltration. In many embodiments, 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 substance is fluidly connected to the feed inlet (i). Processing of the liquid feedstock through the filtration device (iv) is accomplished through a flow-imparting means (v) located upstream of the filtration device (iv), fluidly connected downstream of the multi-inlet flow controller outlet (ii) and the feedstock inlet (i). Filters are often modular and may use configurations known in the field of biomolecule purification.

[0086] 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 performed using commercially available filtration devices. In many embodiments, one or more filtration devices are located between the inlet and outlet of device (iv) to perform filtration as a processing operation. In another embodiment, an additional filter device is located downstream of the device outlet, allowing 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.

[0087] Tangential flow filtration ("TFF") processing operations that can be performed using the devices 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, for example, recirculating tangential flow filtration.

[0088] In one embodiment, the apparatus includes a unit containing a TFF device (iv), for example, a TFF device comprising a flat sheet, hollow fiber, or spiral-wound membrane between the device (iv) inlet and the device (iv) outlet. The retentate from the unit containing the TFF device (iv) can be directed from one of the device outlets to a fluidly connected inlet on a container or bag comprising 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 supplying 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 containing a TFF device (iv) comprising either a flat sheet, hollow fiber, or spiral-wound membrane between the device inlet and the device outlet. The retentate from the unit containing 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. 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 unit containing the TFF device (iv), preferably through one of the inlets of a multi-inlet flow controller. The multi-inlet flow controller (ii) can be used to mix the retentate with at least one other liquid. The operation of recirculating TFF is known in the art and can be controlled by setting the cross-flow rate and transmembrane pressure.

[0089] In certain embodiments, the apparatus includes a unit containing an SPTFF device (iv). The SPTFF device preferably includes 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 is returned to the feed inlet (i) using a variable flow valve downstream of the device (iv).

[0090] Device (iv) preferably optionally includes an outlet for a waste stream. However, this is not required in many embodiments, as the waste stream, if present, may exit the device using the same outlet as the product stream. For example, if device (iv) includes a chromatography column, only one stream exits the column, and typically this stream begins as a waste stream containing impurities until the desired target substance is eluted from the column. Then, for a period of time, the stream exiting the column contains the target substance minus many impurities. Finally, the amount of target substance exiting the column decreases, and the liquid exiting the device typically contains primarily impurities. Processing units and equipment used in the present 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.

[0091] Means for providing a liquid flow (v) 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, means for providing a liquid flow (v) 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 typically depends on the flow capacity and pressure profile appropriate for adjusting and designing the parameters of the device. In certain highly preferred embodiments, pump (v) is a quaternary diaphragm pump.

[0092] 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) means downstream of the device (iv) outlet for controlling the pressure; (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;

[0093] Preferably, one or more units (preferably all units) further comprise 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, and in particular variable position diaphragm valves.

[0094] 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 for simplified communication between the units.

[0095] In one embodiment, at least one (preferably half, more preferably all) of the units of the apparatus divides the liquid feedstocks and / or liquid flows that combine to form the bioprocessing liquid into (a) liquid feedstocks and liquids (2ab), (2cd), (2ef), (2f), (2f). ) and / or (2g) into mixer (iii) for combining and directing the liquid and / or raw material to device (iv) for generating the device feed, or (b) bypassing means (iii) and instead directing the liquid and / or raw material to device (iv) without passing through means (iii). The unit of this embodiment has the advantage that it can be used for the purification of sensitive target materials, where combining the liquid raw material with the bioprocessing liquid in mixer (iii) could damage or degrade the target material.

[0096] The switching bypass (vi) is particularly useful when the device (iv) includes a chromatography column. The switching bypass (vi) can be used to load the target substance 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 serves as an eluent for the target substance already in the column (iv). Furthermore, bypassing the mixing means (iii) can sometimes be useful when the device (iv) performs ultrafiltration and / or diafiltration process steps, where hold-up volume and product stability may otherwise be an issue.

[0097] The diverter bypass assembly (vi) preferably comprises piping and two or three valves that direct the flow of liquid ingredients and liquids (2ab), (2cd), (2ef) and / or (2g) to either mixer (iii) or device (iv) without passing through mixer (iii).

[0098] One embodiment of the apparatus according to the invention is described with reference to Figure 3. The first processing unit includes an inlet (i) for a liquid raw material, inlets (2a) to (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) to (3g), which can be switched on or off to allow flow. In the embodiment shown, buffer feeds from inlets (2a) and (2b), (2c) and (2d), and (2e) and (2f) are combined downstream of 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 water from 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), thereby increasing the operational flexibility of the unit. The outlet (ii) from the multi-inlet flow controller is fluidly connected to a liquid feed containing the target substance at a location (5) upstream of a pump (v), which provides a combined feed flow through a static mixer (iii) (8) equipped with a bubble trap to the inlet of a 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).

[0099] 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 sensors (11). The outlet line from 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 multiwavelength detector, a pH sensor (16), and a variable-position valve (30), which can be used to control pressure and, if desired, apply backpressure. Preferably, operation of pump (v) 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 line passes through a series of valves (17), (19), and (20), which allow flow to be controlled between outlet (18) for a product stream containing the purified monoclonal antibody, outlet (21) for a waste stream containing at least some impurities, or 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 and bypass column (iv) if required during operation, and 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 feed containing the target substance in a second unit for carrying out a second processing operation, configured as shown in Figure 3, although preferably, chromatography column (iv) is replaced by a different means for carrying out a processing operation, such as a different type of chromatographic or non-chromatographic unit operation, where in the second unit for carrying out the second processing operation, the feed supplied to inlet (i) comprises the product stream leaving the previous unit through outlet (18).

[0100] In one method of operation, valves (3a) through (3g) are closed while valve (3) is opened, and a liquid feed containing the monoclonal antibody and impurities is delivered by pump (v) to column (iv), loading the monoclonal antibody and impurities onto a column, e.g., a column containing a Protein A affinity resin, such that the monoclonal antibody selectively binds to the Protein A affinity resin. Once the desired load is complete, valve (3) is closed and one or more valves (3a) through (3g) are opened to pump one or more liquids from inlets (2a) through (2g), forming a bioprocessing liquid, through column (iv). In some embodiments, only valve (3a) is initially opened, and multiple inlet valve (ii) is operated to open the inlet valve through which a buffer, which may be a wash buffer, is delivered from inlet (2a), and the loaded column (iv) is washed with the buffer from inlet (2a). Upon completion of the desired wash step, 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, into 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 supplied to column (iv) can be varied and controlled as desired. For example, when valves (3b), (3c), and (3e) are open, varying the open inlet valves of multi-inlet flow controller (ii) and closing others allows for a stepwise or gradual change in the composition of the bioprocessing liquid. In another example, two or more inlet valves of multi-inlet flow controller (ii) are opened and closed at a given frequency for a selected period of time to supply a given mixture of liquids to column (iv). By adjusting the times and / or frequencies at which the inlet valves of the multiple inlet valve (ii) are open or closed, the composition of the bioprocessing fluid can be altered.When the time and / or frequency are changed stepwise, the composition of the bioprocessing liquid is also changed stepwise. When the time and / or frequency are changed gradually over a period of time, the composition of the bioprocessing liquid is also changed gradually, allowing for the application of a gradient to column (iv). The bioprocessing liquid is fed to column (iv) by any desired method. The liquid composition of the processing liquid can be changed to one that elutes the target substance from column (iv). Prior to elution, the liquid (which may contain impurities) exiting column (iv) 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 target substance reaches valve (17), valves (19) and (20) are closed, valve (17) is opened, and the purified target substance passes through outlet (18) to the second unit and enters the second unit through its inlet (i) as its liquid feed.

[0101] The operation of the second unit and any additional units may be substantially as described above for the first unit. It will be appreciated that the target material exiting the second unit through its outlet (18) may be recovered and used as is, or may be further purified by serving as a feedstock for further processing operations. Such further processing operations may use conventional equipment, or may use additional units in the configuration shown in Figure 3, or may be otherwise in accordance with the present invention.

[0102] The method according to the present invention further comprises the step of mixing the obtained purified monoclonal antibody with one or more pharmaceutically acceptable carriers to form a medicament. The entire subject matter of the claims is incorporated herein by reference thereto.

[0103] The present application is illustrated, but not limited, by the following examples. [Example]

[0104] Example 1 In this example, the composition to be purified includes water, target material, impurities, and cells (obtained from an unclarified cell culture harvest). The apparatus includes two processing units, the first for performing chromatographic purification of the target material, and the second for performing viral inactivation.

[0105] Steps (Ai) and (Aii) The unclarified cell culture harvest, containing water, target material, impurities, and cells (obtained from the unclarified cell culture harvest), can be filtered using a microfilter with an average pore size of 500 nm to remove cells, and then concentrated using a Cadence™ single-pass tangential flow filtration module. The resulting cell-free concentrate can be used as the feedstock for step (B).

[0106] Step (B) Apparatus construction A device can be constructed that includes two of the processing units shown in Figure 3, except that the valve (23b) is replaced by a simple fluid connection. Each unit can include disposable plastic tubing that defines a flow path. Each unit includes substantially identical flow paths.

[0107] Preparation of bioprocessing fluids The stock solutions SS1 to SS7 described below can be used as streams for inlets (2a) to (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 monobasic sodium phosphate (SS5); Inlet (2f)- 1M acetic acid (SS6); Inlet (2g) - Distilled water (SS7).

[0108] Using the multi-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.

[0109] [Table 1]

[0110] 1st processing unit – chromatography The processing unit includes a chromatography column as device (iv) (2.5 L mAbSelect SuRe resin column). Chromatography column (iv) is loaded with the feed containing the mAb and impurities from inlet (i) and chased with 2 L of BPL1 ready-to-use solution.

[0111] Bioprocessing liquids BPL1-BPL5 can be generated by proportionally selecting each stock solution SS1-SS7 using a multi-inlet flow controller (ii), downstream pump (v), and static mixer (iii). While establishing the correct buffer composition as the bioprocessing liquid, column (iv) is bypassed by valve (23a), valves (24) and (25) are closed, and waste buffer is directed to waste (21). Once the buffer is homogeneous, as indicated by a stable reading from the upstream conductivity sensor (11), valves (24) and (25) are opened and the bypass line of valve (23a) is closed, allowing the buffer to be delivered to chromatography column (iv) as the bioprocessing liquid. Process conditions can be monitored using conductivity, UV, and pH sensors (14), (15), and (16) downstream of column (iv). During preconditioning of column (iv) prior to binding of mAb to the column, and during water rinsing after use, the liquid is sent to waste (21). Once preconditioned in BPL1, the chromatography resin is loaded onto column (iv) by the action of pump (v), bypassing static mixer (iii), using a switching bypass assembly (vi). 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 (22). The purified mAb is recovered from column (iv) using elution buffer BPL4 as the bioprocessing fluid and collected through outlet (18). Finally, remaining impurities can be removed from column (iv) using strip buffer BPL5 as the bioprocessing fluid and collected through outlet (22).

[0112] Second Treatment Operation – Viral Inactivation The second processing unit may comprise 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.

[0113] The feedstock from the first operational unit, containing TS and virus (25 L ~7.5 g / L TS in 50 mM sodium acetate pH 4.6), is loaded into the virus inactivation bag (iv) through inlet (i). The feedstock is directed to device (iv) by means of pump (v), bypassing device (iii) using the switching bypass unit (vi) by opening valve (24) and closing the bypass lines of valves (23a) and (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, leaving the switching bypass unit (vi) in bypass mode. The impeller drive is turned on and set to a speed of 450 rpm. The pH of the feedstock is lowered to a target pH of 3.6 by intermittently adding 1 M acetic acid through (3ab) in (2a) connected to the multi-inlet flow controller (ii). During the pH titration to pH 3.6, valve (3a) is opened and the multi-inlet flow controller (ii) is operated to dispense approximately 100 mL of 1 M acetic acid into the virus inactivation bag (iv) every 30 seconds, with each dose being timed by the valve. After eight doses of 1 M acetic acid, the feed 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 feed is then recirculated around the flow path and allowed to mix in the virus inactivation bag (iv) for 60 minutes.

[0114] The pH of the feedstock is raised to a target pH of 5 by intermittently adding 1 M Tris base through valve (2c), which is connected to multi-inlet flow controller (ii) via valve (3cd). During the pH titration to pH 5, valve (3c) is opened and multi-inlet flow controller (ii) is operated to dispense approximately 100 mL of 1 M Tris base into virus inactivation bag (iv) every 30 seconds, each time by valve operation. After eight doses of 1 M Tris base, the feedstock is recirculated around the flow path and allowed to mix within bag (iv) for 15 minutes. During the 15 minutes of recirculation, Tris base dosing is repeated as above until the pH reaches pH 5, as determined by sensor (15). Finally, the virus inactivation target material is recovered by closing outlet (19), opening valve (17), and collecting the contents of virus inactivation bag (iv) through outlet feed (18).

[0115] Example 2 Example 1 can be repeated using a cell-free composition containing water, TS, and impurities, except that step (Ai) is omitted. Additionally, step (Aii) is carried out in an apparatus constructed as shown in Figure 3, including components (i)-(v) in a processing unit, except that valve (23b) is replaced with a simple fluid connection. The resulting product stream containing the concentrate is then subjected to step (B) as described in Example 1.

[0116] Thus, in this example, the apparatus comprises the following units arranged in series in the order listed, each unit comprising components (i) through (v), and the product feed of each unit: The product feed is used as the feedstock for the next unit, or if there is no next unit, the product feed is collected: a. A unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance; c. A unit for performing viral inactivation.

Claims

1. A method for purifying a composition comprising water, a target substance, impurities, and optionally cells, comprising the following steps (A) and (B): (A) Preparing a liquid feedstock by subjecting the composition to steps (Ai) and / or (Aii): (Ai) removing at least some cells from the composition; (Aii) concentrating the composition by removing water from the composition; and (B) passing the liquid feedstock through an apparatus comprising at least two processing units, each such unit producing a product stream comprising a purified target substance and, optionally, a waste stream comprising at least some impurities, each unit comprising the following components (i) through (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 fluid from at least two other fluids combined in a desired ratio; (iii) means for combining a liquid feedstock and a bioprocessing liquid to produce 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, including an inlet for the device feed and / or liquid feed and an outlet for a product stream; (v) means for providing flow 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 a bioprocessing liquid in device (iv), in either case providing a product stream and optionally a waste stream that exits the unit; (II) step (Ai) is performed only if the composition comprises cells; (III) at least one of the units is a unit for performing chromatographic purification of a target substance; (IV) A process in which 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.

2. 10. The method of claim 1, wherein the composition comprises cells and at least some of the cells are removed in step (Ai) by a method comprising centrifugation or filtration.

3. 3. The method of claim 1 or 2, wherein the composition comprises cells and at least some of the cells are removed in step (Ai) by a method comprising recirculating tangential flow filtration.

4. 4. The method of any one of claims 1 to 3, wherein step (Aii) is carried out by a method comprising single-pass tangential flow filtration, ultrafiltration, lyophilization, evaporation, precipitation, crystallization, aqueous two-phase separation, dialysis, or a combination of two or more of the foregoing.

5. 5. The method of any one of claims 1 to 4, wherein step (Aii) is carried out by a method comprising single-pass tangential flow filtration.

6. 2. The method of claim 1, wherein the composition comprises cells, and at least a portion of the cells are removed in step (Ai) by a method comprising recirculation tangential flow filtration, and step (Aii) is performed by a method comprising single-pass tangential flow filtration.

7. 7. The method of claim 1, wherein step (Ai) is performed external to the device.

8. 8. The method of claim 1, wherein step (Aii) is performed external to the device.

9. 9. The method of any one of claims 1 to 6 and 8, wherein step (Ai) is carried out in at least one of at least two processing units of an apparatus comprising components (i) to (v).

10. 10. The method of any one of claims 1 to 7 and 9, wherein step (Aii) is performed in at least one of at least two processing units of an apparatus comprising components (i) to (v).

11. 11. The method of any one of claims 1, 4, 5, 8 and 10, wherein the composition is substantially free of cells and step (Ai) is omitted.

12. 10. The method of any one of claims 1 to 3, 7 and 9, wherein the composition comprises cells and step (Aii) is omitted.

13. 10. The method of claim 1, wherein step (A) is carried out in an apparatus comprising components (i) through (v) in one or more of at least two units.

14. 14. The method of any one of claims 1 to 13, wherein in step (B) the bioprocessing liquid is provided by combining at least three liquid streams, and at least each of the at least three liquid streams is provided by combining at least two additional liquid streams.

15. 15. The method of any one of claims 1 to 14, wherein the apparatus comprises 3, 4, 5, 6, 7, 8, 9 or more processing units.

16. 16. The method of any one of claims 1 to 15, 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.

17. 17. The method of any one of claims 1 to 16, wherein the chromatography is 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.

18. 18. The method of any one of claims 1 to 17, wherein the apparatus comprises at least two units for performing chromatographic purification, and each such chromatographic purification uses different conditions and / or chromatographic columns packed with different resins than all other units for performing the chromatographic purification.

19. 20. The method of claim 18, wherein each chromatographic purification is independently selected from 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, and size exclusion chromatography.

20. 20. The method of any one of claims 1 to 19, wherein during the method each unit prepares its bioprocessing liquid.

21. 21. The method of any one of claims 1 to 20, wherein the apparatus further comprises a unit comprising components (i) to (v) for removing any viruses from the liquid feedstock.

22. 22. The method of any one of claims 1 to 21, 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.

23. 23. The method of any one of claims 1 to 22, wherein the apparatus further comprises a unit comprising components (i) to (v) for diafiltration of the liquid feed to buffer the target material at a pH at which the target material is stable.

24. 24. The method of any one of claims 1 to 23, wherein the means for imparting flow to each unit comprises a single pump, preferably located downstream of component (ii) and upstream of component (iv).

25. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); and b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography. wherein each unit comprises components (i) to (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.

26. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; and c. A unit for the inactivation of any viruses that may be present in the liquid raw material. wherein each unit comprises components (i) to (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.

27. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; and d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography. wherein each unit comprises components (i) to (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.

28. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; and e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography. wherein each unit comprises components (i) to (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.

29. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; and f. The method of any one of claims 1 to 28, including an optional unit for removal of inactivated viruses, each unit comprising components (i) to (v), and the product feed of each unit being used as the feedstock for the next unit, or the product feed being collected if there is no next unit.

30. The apparatus comprises the following units arranged in series: a. optionally a unit for performing step (Ai) and / or a unit for performing step (Aii); b. A unit for carrying out chromatographic purification of the target substance by affinity chromatography; c. A unit for the inactivation of any viruses that may be present in the liquid raw material; d. A unit for carrying out chromatographic purification of the target substance by cation exchange chromatography; e. A unit for carrying out chromatographic purification of the target substance by anion exchange chromatography; f. A unit for the removal of any inactivated viruses; and g. A unit for concentrating the product stream from the preceding unit and / or performing buffer exchange. Each unit comprises components (i) to (v), and the product feed of each unit is used as the raw material for the next unit, or if there is no next unit, the product feed is used as the raw material for the next unit.

30. The method of claim 1, wherein the feed is collected.

31. 31. The method of any one of claims 25 to 30, wherein the units are arranged in series in the order listed.

32. 32. The method of any one of claims 1 to 31, wherein at least one unit further comprises a device for removing air bubbles from one or more liquids passing through the unit.

33. 33. The method of any one of claims 1 to 32, wherein at least one unit further comprises a pressure sensor located upstream of component (iv).

34. 34. The method of any one of claims 1 to 33, wherein at least one unit further comprises a pressure sensor located downstream of component (iv).

35. 35. The method of any one of claims 1 to 34, wherein at least one unit further comprises a UV sensor located downstream of component (iv).

36. 36. The method of any one of claims 1 to 35, wherein at least one unit further comprises a pH sensor located downstream of component (iv).

37. 37. The method of any one of claims 1 to 36, wherein at least one unit further comprises a conductivity sensor located downstream of component (iv).

38. 38. The method of any one of claims 1 to 37, wherein at least one unit further comprises at least one diverting bypass unit (vi) for either (a) directing the flow of liquid feedstock and / or bioprocessing liquid to the 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).

39. 39. The method of claim 38, wherein component (vi) is present in all units.

40. 40. The method of any one of claims 1 to 39, wherein the flow paths used in at least half of the units have substantially the same configuration.

41. 41. A method according to any one of claims 1 to 40, wherein all component parts of each unit, except for component (iv), are identical to all component parts used in all other units of the apparatus.

42. 42. The method of any one of claims 1 to 41, wherein the flow path through each unit is substantially identical to the flow path through every other unit.

43. 43. The method of any one of claims 1 to 42, wherein the composition of the bioprocessing liquid used in at least one unit is changed stepwise or gradually during the method.

44. 44. The method of any one of claims 1 to 43, wherein each unit includes only one pump.

45. The resulting purified target substance is mixed with one or more pharmaceutically acceptable carriers to form a drug.

45. The method of any one of claims 1 to 44, further comprising the step of:

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