RU2824133C2 - Apparatus for treating liquid containing target substance - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to treatment of liquids containing a target substance, namely liquids containing a biomolecule, for example, a recombinant polypeptide. Invention discloses an apparatus for treating a liquid, an apparatus for preparing a liquid mixture, a method for preparing a buffer solution and a method for producing a biomolecule. Liquid treatment apparatus includes one pump located below the liquid connection between the connection between the starting material and an outlet from the flow controller for imparting flow through the flow rate controller and from the liquid feed system through the processing device through the inlet of the device.

EFFECT: simplifying the design of the liquid treatment apparatus and performing several different stages of treatment using a common flow line.

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Description

The present invention relates to methods and apparatus for processing liquids containing a target substance, in particular liquids containing a biomolecule and especially recombinant polypeptides.

Many biomolecules, especially recombinant polypeptides and nucleic acids such as plasmids (pDNA), have attracted much attention, particularly for therapeutic applications. Such biomolecules are typically produced by culturing recombinant host cells that have been engineered to express the desired biomolecule. The biomolecule is then recovered from the culture medium by methods typically involving a number of unit processes. These unit processes typically include one or more of chromatographic purifications, viral inactivation, filtration (including viral, depth, and absolute filtration), refolding, and ultrafiltration/diafiltration.

An apparatus for processing a solution containing a target substance is known in the art. However, an apparatus for use in industrial production of such compounds is extremely cumbersome and requires a large area and infrastructure. In addition, although some unification of the apparatus can be achieved for several apparatus operations, the apparatus designs for certain apparatus operations, such as viral inactivation and/or ultrafiltration, differ significantly from those for, for example, chromatographic purification. This means that either more space is required to accommodate two or more sets of apparatus, or that the interoperability and control of the apparatus at the stages is excessively complex. In addition, training of operators on each of the different types of apparatus used is required. Accordingly, it would be desirable to have a simplified and widely applicable apparatus. It would also be desirable to identify an apparatus that allows several processing steps to be performed using a common flow line.

According to a first aspect of the present invention, an apparatus for processing a liquid containing a target substance is proposed, wherein said apparatus comprises at least a first and a second device for performing a standard process, wherein each device for performing a standard process comprises:

(i) a system for supplying a liquid to a liquid connection using an inlet of a flow regulator comprising an inlet valve with a variable flow and an outlet, where at least one of the means for performing a unit process includes systems for supplying at least two liquids, wherein the supply systems are in a liquid connection using inlets of a flow regulator with multiple inlets comprising two or more inlet valves with a variable flow for dosing at least two liquids, wherein the flow regulator also comprises an outlet;

(ii) a feed system for a liquid feedstock containing a target substance in fluid communication with an outlet from the flow regulator, thereby allowing the feed system for the liquid containing the target substance to be combined with the mixed bioprocessing fluids to form a feed system for the device;

(iii) a device for performing a treatment process, comprising a device inlet and a device outlet, wherein the device inlet is in fluid communication with the feed device; and

(iv) a means for imparting a flow motion through the flow controller and from the feed of liquid feedstock through the treatment device via an inlet of the device;

wherein the system for supplying a liquid feedstock containing a target substance to the second device for performing a standard process comprises an outlet from the first device for performing a standard process.

In some embodiments, the apparatus comprises two devices for performing a unit process. In other embodiments, the apparatus comprises three, four, five, six, seven, eight, nine or more devices for performing a unit process, preferably each having the features described above for the first and second devices. In many preferred embodiments, the devices for performing a unit process are connected in series with a supply system for a liquid feedstock containing a target substance for a subsequent device for performing a unit process, comprising an outlet from the previous device for performing a unit process. In many highly preferred embodiments, each unit process is different from the other unit processes.

In some embodiments, each device for performing a unit process comprises substantially the same flow line.

Devices for a liquid supply system are well known in the art and include applying gas pressure to a liquid, especially an inert gas such as nitrogen or helium. Preferably, the device for imparting movement to the liquid flow comprises one or more pumps. Pumps that can be used include peristaltic, diaphragm, vane and centrifugal pumps. Both single-use and reusable pumps can be used. In many preferred embodiments, one pump is used for each device for performing a unit process, located downstream of the fluid connection between the connection between the feedstock and the outlet of the flow controller. Most preferably, the pump is located upstream of the biological treatment device. The type and size of the pump selected typically depends on the throughput and pressure profile corresponding to the scale and design parameters of the device. In some particularly preferred embodiments, the pump is a quaternary membrane pump.

The flow regulator comprises variable flow, preferably intermittent flow, inlet valves that regulate the flow of liquid through the flow regulator. The flow regulator with multiple inlets comprises at least 2 inlet valves and in many cases includes up to 8, such as 3, 4, 5, 6 or 7 inlet valves. Each of the inlet valves may have the same dimensions, or one or more inlet valves may have different dimensions. In some preferred embodiments, the volume measured from each inlet valve to the outlet of the flow regulator is the same for each inlet, and it is highly preferred that both the volume and the path length measured from each inlet valve to the outlet of the flow regulator are the same for each inlet. In many embodiments, each device for performing a unit process preferably comprises a flow regulator with multiple inlets.

The flow regulator used in the present invention also comprises at least one outlet, and although two or more outlets may be present, it is preferable to use one outlet.

Variable flow valves can regulate the flow between a first, relatively low flow rate, at which the fluid remains able to flow, and at least a second, higher flow rate. In preferred embodiments, the variable flow valve is an intermittent flow valve that prevents flow in the first position, but allows flow in at least the second position. Most preferably, all valves are intermittent flow valves. The valves can comprise actuators known in the art, such as pneumatic or, preferably, solenoid actuators.

Preferably, the variable flow valves are controlled, most preferably by a programmable control unit, to regulate the opening and closing of the valves to achieve the desired relative quantities of the inlet liquids flowing through the flow controller with several inlets. This is preferably achieved by cyclically repeating, with a predetermined period of time or cycle rate, through the inlet valves in the flow controller and regulating the opening or closing of the valve in accordance with the desired proportion of the cycle time to create the desired composition. The cycle rate can be constant or variable. Most preferably, intermittent flow valves are used and controlled so that during operation only one valve is open at any one time. In many embodiments, the cycle rate of the flow controller with several inlets is maintained constant and the desired relative quantities of the inlet liquids remain constant.

In many embodiments, multiple cycles are used. The number of cycles used will depend on many factors, such as the duration of the process, the volume of liquid being processed, the flow rate, and the maximum operating pressure of the apparatus. In some embodiments, at least 10 cycles can be used, such as at least 50, 100, 500, 750, 1000, 1500, 2000, 3000, 5000, 7500, 10000 or more cycles.

It is understood that a range of cycle frequencies can be used. In many cases, the frequency is less than 100 Hz, typically less than 50 Hz, typically less than 10 Hz, and preferably less than 5 Hz. In some preferred embodiments, the frequency is 2 Hz or less, most preferably 1 Hz or less, such as from 0.05 to 0.5 Hz.

Although mixing can be achieved by simply combining the flows of the target feed system and the outlet of the flow controller, optionally in combination with the action of a pump, in many preferred embodiments the apparatus further comprises an in-line mixer, preferably a mixing chamber, preferably comprises a static mixer, most preferably a static mixer with a split flow with a time delay. In many embodiments, the in-line mixer is located downstream of the pump and upstream of the device for performing the bioprocessing process. In some preferred embodiments, the in-line mixer also comprises a bubble trap.

Typical processes that can be performed by the device for performing the processing operation are most preferably bioprocessing operations. Bioprocessing operations include chromatography, viral inactivation, filtration, refolding, ultrafiltration, diafiltration, microfiltration, in-line conditioning, and refolding.

Chromatographic operations that can be performed using the device of the present invention include affinity chromatography, ion exchange (either anion or cation exchange) chromatography, hydrophobic interaction chromatography (HIC), reverse phase chromatography, expanded bed chromatography, mixed chromatography, membrane chromatography and size exclusion chromatography (SEC). In many embodiments, protein A affinity chromatography includes at least one of the unit processes. Devices for performing chromatographic operations include an appropriate chromatographic apparatus, such as a membrane, a fiber support or a column. The number and sequence of unit chromatographic processes will be selected in accordance with the nature of the target biomolecule.

The operations with the viral inactivation unit that can be performed using the device of the present invention typically include a storage tank in which the liquid containing the target biomolecule can be stored under conditions where the residence time is sufficient to inactivate viruses. In some embodiments, the outlet and inlet openings of the device can be in fluid communication to create a recirculation loop. In one such embodiment, the device is provided with a container or bag that is in fluid communication between the inlet opening of the "device" and the outlet opening of the "device", and one of the outlet openings of the device is in fluid communication with one of the inlets of the flow regulator with several inlets. A container or a bag between the "inlet" and "outlet" of the device, which are in fluid communication with the inlet of the liquid feedstock, are filled with a means for communicating the flow, typically a pump, or are conditioned with at least one other liquid through at least one of the other inlets of the flow controller with multiple inlets. In certain embodiments, the container or bag is a mixing container or bag. The process liquid is recirculated through the inlet of the flow controller with multiple inlets into the container or bag and back to the inlet of the flow controller with multiple inlets, since the solution containing the target substance is conditioned with at least one additional liquid in fluid communication with at least one other inlet on the flow controller with multiple inlets.

Typical filtration processes that can be performed include viral, depth and absolute filtration, ultrafiltration, diafiltration and microfiltration. In many embodiments, a typical filtration process comprises a filter module between an inlet of the device and an outlet of the device. The filtration module is flushed and displaced using at least two fluid delivery systems attached to the inlets of a flow controller with multiple inlets and a solution containing a target molecule, which is in fluid communication with the feed inlet. Processing of the fluid through the filter is achieved using a flow imparting device, which is in fluid communication and is located downstream of the outlet of the flow controller with multiple inlets and the feed inlet, and upstream of the filter module. Filter modules typically use configurations that are well known in the field of bioprocessing.

Viral filtration, depth filtration and absolute filtration are unit processes that are well known in the art and can be performed using the apparatus of the present invention, typically using filter devices that are well known in the field of bioprocessing. In many embodiments, the filter device or devices are located between the inlet and outlet of the device to perform a particular unit process. In other embodiments, the filter device is located downstream of the outlet of the apparatus, which in some embodiments allows the apparatus to perform a primary unit process such as chromatography, viral inactivation, tangential flow filtration, viral filtration or depth filtration, followed by a secondary filtration operation.

Typical tangential flow filtration ("TFF") processes that can be performed using the apparatus of the present invention include conventional TFF with recirculation and single-pass TFF. In some embodiments, the outlet and inlet ports of the apparatus may be in fluid communication to create a recirculation loop, an example of which is tangential flow filtration with recirculation. In one embodiment, as known in the art, the apparatus is equipped with a TFF module containing membranes of a flat sheet, hollow fiber or spiral membranes between the inlet port of the device and the outlet port of the device, and the retentate from the TFF module is directed from one of the outlet ports of the device to the inlet port in a fluid communication on a container or package containing at least one inlet port and one outlet port. The outlet port of the container or package is in fluid communication with the inlet port for the liquid feedstock. The container or the bag is maintained at a constant level by means of auxiliary means for feeding the feedstock or liquid into the container or the bag by means of a liquid connection with a second inlet on the container or in the bag. In another embodiment, the apparatus is equipped with a TFF module comprising either membranes in the form of flat sheets, hollow fibers or spiral membranes between the inlet of the device and the outlet of the device, and the retentate from the TFF module is in a liquid connection from one of the outlets of the apparatus back to one of the inlet valves of the flow controller with several inlets. In some embodiments, the recirculation loop from the outlet of the device to its inlet comprises a separating container or bag. The solution containing the target substance or liquid is drawn into the recirculation loop through the inlet for the liquid feedstock by means of a means for communicating the flow motion, usually a pump. The retentate is recirculated through the TFF module, preferably through one of the inlets of the flow controller with several inlets. A flow controller with multiple inlets may be used to mix the retentate with at least one other liquid. The TFF process with recirculation is well known in the art and is controlled by setting the cross-flow rate and the transmembrane pressure.

In some embodiments, the single-pass TFF may be configured with a TFF module comprising, for example, a flat sheet, hollow fiber, or helical membrane between the device inlet and the device outlet, as in the case of the single-pass TFF described in WO2017/118835.

In some embodiments, a hybrid of single-pass and recirculating TFF may be used, where retentate generated using a variable flow valve downstream of the TFF module is returned to a feed tank.

The apparatus according to the present invention typically further comprises one or more bubble traps, pressure sensors, temperature sensors, pH sensors, flow sensors, conductivity sensors, air sensors and ultraviolet radiation sensors, such as a multi-wavelength UV/visible light sensor. One or more of the above sensors may be present.

In one particular embodiment of the present invention, an apparatus for processing a liquid containing a target substance is provided, said apparatus comprising:

(i) systems for supplying at least two liquids;

(ii) a flow controller with multiple inlets comprising two or more variable flow inlet valves for mixing at least two fluids, wherein the flow controller also comprises one outlet;

(iii) a system for feeding a liquid feedstock containing the target substance into the liquid connection via an outlet from the flow controller;

(iv) a means for imparting a flow, typically by use of a pump, through a flow controller and from a feed system for liquid feed material through the treatment apparatus by means of inlets of the feed system;

(v) a device for mixing at least two liquids;

(vii) a device for trapping air bubbles from process liquids;

(vii) a common flow line for performing the processing process, comprising an inlet opening of the device and an outlet opening of the device, wherein the inlet opening of the device is in fluid communication with the inlets of the feed system;

(viii) the inlet and outlet ports of the device are in fluid communication with modules used for chromatography, filtration (including viral filtration), tangential flow filtration, single-pass tangential flow filtration, refolding, and viral inactivation;

(ix) a device for bypassing the inlet and outlet opening of the device;

(x) a device downstream of the outlet of the pressure regulating device;

(xi) a series of sensors suitable for monitoring the processing operation above and below the inlet and outlet of the device; and

(xii) at least one outlet in fluid communication with the inlet openings of the feed system.

The apparatus according to the present invention preferably comprises a device for further increasing the pressure of the processing device located downstream of the device. Devices for creating pressure are known in the art and include shut-off valves, diaphragm valves, especially diaphragm valves with variable position.

In many embodiments, the liquid mixing device and the air bubble trapping device include a single device.

In some embodiments, the apparatus comprises a universal flow line constructed from materials, such as stainless steel, that allow for a significant number of reuses before requiring replacement.

In some embodiments, the apparatus comprises a disposable flow line made from materials, such as plastics, that have a limited life and are used as disposable consumables.

In many embodiments, each typical process is performed under the control of a programmable control unit, preferably a computer. In some embodiments, one control unit controls two or more typical processes. In other embodiments, each typical process is under the control of a separate control unit. In these other embodiments, preferably, each control unit uses a common programming language that provides simplified communication between control units.

According to a second aspect of the present invention, an apparatus for preparing a liquid mixture is provided, said apparatus comprising:

(i) systems for supplying at least two liquids;

(ii) a flow regulator with multiple inlets comprising two or more variable flow inlet valves for metering at least two fluids, wherein the flow regulator also comprises an outlet;

(iii) a pump for imparting flow through the flow controller and from the feed system for liquid feedstock through the bioprocessing device through the feed system inlets;

(iv) a static mixer for in-line mixing of at least two liquids; and

(v) outlet for liquid mixture;

where the pump is located upstream of the inline mixing device and the pump and inline mixing device are located downstream of the plurality of flow inlets.

Liquid feed devices, multi-inlet flow controllers, pumps and static mixers that can be used in the second aspect are as described above in relation to the first aspect of the present invention.

The apparatus according to the second aspect can be used for preparing liquid mixtures, preferably solutions, such as buffer solutions, for example by mixing acid and/or salt solutions, typically for use in treatment processes, and especially in biotreatment processes. The solutions can be stored before use, for example during a treatment process, especially in a biotreatment process, or can be used directly, for example by liquidly connecting the outlet for the liquid mixture to a device using the mixture.

The apparatus of the second aspect of the present invention may comprise additional elements as described above for the first aspect of the present invention, in particular one or more bubble traps, pressure sensors, temperature sensors, pH sensors, flow sensors, conductivity sensors, air sensors and UV sensors, such as a multi-wavelength UV/visible light sensor. One or more of the above sensors may be present.

The apparatus of the second aspect of the present invention preferably comprises one pump. In some particularly preferred embodiments, the pump is a quaternary membrane pump.

The static mixer used in the apparatus according to the second aspect of the present invention is preferably a split-flow static mixer with a time delay.

In many embodiments, the apparatus according to the second aspect of the present invention is operated under the control of a programmable control unit, preferably a computer.

The apparatus of the second aspect of the present invention can advantageously operate without the need to include complex feedback mechanisms to regulate the composition of the liquid produced.

Liquids that can be used with the apparatus of the present invention include those known in the art for carrying out the corresponding unit process. Examples include acidic, neutral and basic solutions, such as solutions having a pH in the range from 2.5 to 14, for example sodium, potassium or ammonium hydroxide solutions, phosphoric, sulfuric, hydrochloric or acetic acid solutions; salt solutions, such as solutions having a salt concentration of up to about 3 M, including sodium, calcium, potassium and ammonium salts, for example phosphate, chloride, acetate, citrate and sulfate salts; buffer solutions, examples of which are well known in the art; reducing agents (for example DTT and TCEP); amino acids (for example histidine, arginine, glycine); detergents (for example Tween 20, Triton-X100) and water-soluble solvents such as polyols, for example glycerol and PEG; and mixtures of one or more of the above.

Biomolecules that can be processed using the present invention include, for example, pDNA; cell therapy, vaccines such as viral vaccines, gene therapy products, sugars, inclusion bodies, in particular inclusion bodies containing polypeptides; and especially recombinant polypeptides.

pDNA may exist in one or more of a variety of forms, such as supercoiled, linear, and uncoiled circular (i.e., single-cut or free-form) DNA isoforms. The supercoiled pDNA isoform has a covalently closed circular form, and the pDNA is negatively supercoiled in the host cell by the action of host enzymatic systems. In the uncoiled circular isoform, one strand of the pDNA duplex is nicked at one or more sites.

Methods for producing pDNA are well known in the art. The pDNA may be natural or artificial, such as cloning vectors carrying foreign DNA inserts. In many embodiments, the pDNA is in the range of 1 to 50 kb in size. For example, the pDNA encoding the expressed interfering RNA is typically in the range of 3 to 4 kb in size.

Polypeptides, especially 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.

Antibodies include monoclonal antibodies, polyclonal antibodies, and antibody fragments having biological activity, including polyvalent and/or polyspecific forms of any of the above.

Natural antibodies typically comprise four polypeptide chains, two identical heavy (H) and two identical light (L) chains, linked together by disulfide bonds. Each heavy chain contains a variable region (VH) and a constant region (CH), the CH region containing in its native form three domains, CH1, CH2, and CH3. Each light chain contains a variable region (VL) and a constant region containing a single domain, CL.

The VH and VL regions can be further divided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Antibody fragments that can be expressed comprise a portion of an intact antibody, wherein the portion has a target biological activity. Antibody fragments typically comprise at least one antigen-binding site. Examples of antibody fragments include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab derivatives, such as a Fab' fragment having one or more cysteine residues at the C-terminus of the CH1 domain, which can form divalent fragments by a disulfide bridge between two Fab derivatives; (iii) an Fd fragment having VH and CH1 domains; (iv) Fd derivatives, such as Fd derivatives having one or more cysteine residues at the C-terminus of the CH1 domain; (v) Fv fragments having the VL and VH domains of one arm of an antibody; (vi) single-chain antibody molecules, such as single-chain Fv antibodies (scFv), in which the VL and VH domains are covalently linked; (vii) a VH or VL domain polypeptide without constant region domains linked to another variable domain (VH or VL domain polypeptide), i.e. with or without constant region domains (e.g. VH-VH, VH-VL or VL-VL) (viii) domain antibody fragments, such as those consisting of a VH domain or a VL domain, and antigen-binding fragments of the VH or VL domains, such as isolated CDR regions; (ix) so-called "diabodies" comprising two antigen-binding sites, such as a heavy chain variable domain (VH) linked to a light chain variable domain (VL), in the same polypeptide chain; and (x) so-called linear antibodies, which contain a pair of tandem Fd segments that, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

Inclusion bodies include insoluble aggregates formed in the cytoplasm of bacterial cells such as E. coli, most often containing a polypeptide and especially a recombinant polypeptide.

Methods for processing a biomolecule, such as a recombinant polypeptide, and most preferably purifying or isolating the recombinant polypeptide, constitute further aspects of the present invention.

One embodiment of an apparatus according to the present invention is described with reference to Fig. 1. A first device for performing a bioprocessing process comprises a supply system for a liquid containing a target biomolecule, 1, and supply systems for six different buffers, 2a-2f, and water for injection 2g is also provided. Each supply system is provided with a valve, such as a direct-acting diaphragm valve, 3 and 3a-3g, so that the flow can be turned on or off. In the embodiment shown, the buffer supply systems 2a and 2b, 2c and 2b and 2d and 2e are combined downstream of the valves, 3a-3f respectively, to form three buffer supply lines that are in fluid communication with the supply system for water for injection, 2g, with different inlets on a flow regulator with several inlets, 4, comprising a four-valve distributor with one outlet, having a fast-acting solenoid actuator. Thanks to this configuration and the corresponding opening and closing of valves 3a and 3b, 3c and 3d, as well as 3e and 3f, it is possible to choose between buffers 2a and 2b, 2c and 2d or 2e and 2f, thereby increasing the flexibility of the device. The outlet of the flow controller 4 with multiple inlets is in fluid communication with the feed system of the liquid containing the target biomolecule, 1, 5, above the pump 6, which imparts movement to the flow of the combined feed systems through the static mixer equipped with a bubble trap, 8, and to the inlet of the first chromatographic column, 12. The feed line at the outlet of the pump 6 to the chromatographic column 12 is equipped with a pressure sensor 7, an air sensor 9, a flow meter 10, such as an ultrasonic flow meter and a combined temperature and conductivity sensor, 11. In some embodiments, the pump 6 is controlled via a programmable control unit in response to a feedback signal 29 from the flow meter 10. In some embodiments, optionally, the flow controller 4 with multiple inlets is controlled via a programmable control unit in response to a feedback signal 28 from the conductivity and temperature sensor 11. The outlet line from the chromatographic column 12 is provided with a pressure sensor 13, a combined temperature and conductivity sensor 14, a UV detector, such as a UV/visible detector, 15, a pH meter, 16, and a variable position valve 30, which can be used to regulate the pressure and to create a back pressure, if necessary. Preferably, the operation of the pump 6 and the variable position valve 30 and, therefore, the regulation of the pressure in the device 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 regulated between the outlet feed line 18, the waste feed line 21 or the outlet feed system 22, for example, to allow collection or sampling. The device is further provided with valves 23a and 23b, which allow the flow to be diverted around the column 12, if this is required during operation, and additional valves 24 and 25, which allow the flow through the column to be stopped. The outlet feed system 18 can then be used as a feed system containing the target biomolecule in a second device for performing a bioprocessing process, configured as shown in Fig. 1, but where preferably the chromatographic column 12 is replaced by another device for performing a unit process, such as another type of chromatography or a non-chromatographic unit process, and where in the second device for performing a unit process the feed system 1 comprises the outlet feed system 18 from the first device for performing a unit process.

In one method of the process, valve 3 is opened while valves 3a-3g are closed and a liquid containing a biomolecule is fed by pump 6 to column 12 to load the column with the biomolecule, for example, where the biomolecule is a monoclonal antibody, a column containing a protein A affinity resin, so that the monoclonal antibody selectively binds to the protein A resin. Upon completion of the required loading, valve 3 is closed and one or more valves 3a-3g are opened to allow one or more biological treatment liquids 2a-2g to be pumped through column 12. In some embodiments, only valve 3a is initially opened and valve 4 with multiple inlet valves is operated to open an inlet valve into which buffer 2a is fed, which may be a wash buffer, so that the loaded column is washed with buffer, 2a. Upon completion of the desired washing step, one or more valves 3b-3g may be opened, with valve 3a remaining either open or closed. The inlet valves on the multi-inlet valve 4 are opened to allow the biotreatment fluids 2b-2g or mixtures thereof to be pumped through the column 12. By controlling the opening and closing of the valves on the multi-inlet valve 4 and/or the valves 3a-3g, the composition of the biotreatment fluid fed to the column may be varied and controlled as needed. For example, when valves 3b, 3c and 3e are open, changing the inlet valve that is open in the multi-inlet flow controller 4 and closing the others allows for a stepwise change in the composition of the feed fluid. In another example, two or more inlet valves of the flow controller 4 with multiple inlets may be opened and closed at a given frequency and for a selected period of time to allow a given mixture of bioprocessing fluids to be fed to the column 12. Adjusting the time and/or frequency that the inlet valves on the valve 4 with multiple inlets are opened or closed allows the composition of the fluid fed to the column to be changed. When the time and/or frequency are changed in steps, the composition also changes in steps. If the time and/or frequency are changed gradually over a period of time, the composition also changes gradually, allowing a gradient to be applied to the column 12. In any desired manner, the composition of the fluid fed to the column is changed to a composition that causes the biomolecule to elute from the column. Before elution, the liquids leaving the column 12 are either collected through the outlet 22 or sent to the discharge 21, and the valves 17, 19 and 20 are set accordingly. For the elution of the biomolecule, the valves 19 and 20 are closed, and the valve 17 is open, which allows the biomolecule to proceed to the second typical process 18.

The operation of the second unit process device may be substantially the same as described above with respect to the first unit process. It should be understood that the biomolecule exiting the second unit process device via an exit line equivalent to the exit line of the first unit process device 18 may either be recovered and used as is, or may be subjected to one or more additional unit processes. Such additional unit processes may use a conventional device or an additional device in accordance with the configuration shown in Fig. 1, or otherwise in accordance with the present invention.

This application is illustrated without limitation by the following example.

During the chromatography process, the protein is bound to the chromatography resin, washed with buffers of varying salt concentrations, and then removed (eluted) using a high salt concentration buffer. As an example, recombinant lactoferrin was bound and eluted from a 2.3 L POROS-XS cation exchange resin column using pH 7.5 sodium phosphate buffers with sodium chloride concentrations ranging from 0 to 1 M. This was accomplished on a single, self-contained unit with a completely disposable flow line that contained the elements described in Fig. 1, except that valve 23b was replaced by a simple fluid connection. The feed solutions were attached to the inlets in the following order: 2 M sodium chloride was attached to inlet 2a; 0.1 M dibasic sodium phosphate was attached to inlet 2c; 0.01 M monobasic sodium phosphate was attached to inlet 2e; water was attached to inlet 2g; and a protein feed line was attached to sample inlet 1. Buffers were generated by proportionally selecting each of the feed solutions to obtain the desired buffer composition through the action of multiple inlet flow controller 4 and downstream pump 6 and static mixer 8. During creation of the desired buffer composition, column 12 was bypassed through valve 23a with valves 24 and 25 closed, with unwanted buffer directed to waste 21. Once the buffer became homogeneous, as evidenced by a continuous reading from the upstream conductivity sensor 11, the buffer was fed to chromatography column 12 through open valves 24 and 25 and closing the bypass line at valve 23a. The process conditions were controlled using conductivity, UV and pH sensors 14, 15 and 16 downstream of the column 12. During column conditioning before protein binding to the column and washing with water after use, the liquid was sent to waste 21. After conditioning, the chromatography resin was loaded with protein aspirated through the sample inlet 1 by a pump 6, pushing it through a static mixer 8 onto the column 12. The eluate from the column was collected through the outlet feed line 18, while the first wash buffer with a low salt content was collected through the outlet feed line 22 and the second wash buffer with a medium salt content was collected through the outlet feed line 18. Finally, the protein was extracted from the column using the elution buffer with a high salt content and collected through the outlet feed line 22.

Three processing buffers were prepared from four stock solutions attached to the system: 25 mM sodium phosphate, pH 7.5 ± 0.1 (low salt); 25 mM sodium phosphate, 0.5 M sodium chloride, pH 7.5 ± 0.1 (medium salt); and 25 mM sodium phosphate, 1.0 M sodium chloride, pH 7.5 ± 0.1 (high salt) using specific ratios of salt, acid, base, and water as listed in Table 1. The chromatography column was loaded (high salt), equilibrated (low salt), washed (low salt and medium salt), and eluted (high salt) at 31 L/h using the four stock concentrates. 3 L of 0.8 g/L lactoferrin in 25 mM sodium phosphate, pH 7.5 were loaded onto the column and washed with 2 L of pre-mixed 25 mM sodium phosphate at 15 L/h. The results of the run are shown in Figs. 2-4. Fig. 2 shows the sensor readings for conductivity, pH, and A280 absorbance for the entire run including the primary buffer blocks when the column was placed in parallel. Fig. 3 shows the conductivity of the buffers pumped onto the column, indicating successful operation of the in-line buffer conditioning. Fig. 4 shows the conductivity, pH, and A280 absorbance after the column. Fig. 4 shows that there is the expected residual buffer leachate from the bypass line to the sensors and a delay before new buffer passes through the column, caused by the sharp rise in conductivity and step drop. The critical step is the elution step, where the column goes from wash to high salt and successfully removes the bound protein. This demonstrates the advantage of the system; it can generate the correct buffers using on-line conditioning in real time from the feed solutions to the batch process. In this example, the system successfully performed a typical BEC run used for protein purification using stock solutions, without the need to pre-prepare all the different processing buffers.

Table 1. Conductivity and pH values for built-in conditioned buffers

Buffer 2 M sodium chloride (%) 0.1 M dibasic sodium phosphate (%) 0.01 M sodium phosphate monobasic (%) Water (%) Conductivity (mS/cm) pH 25 mM sodium phosphate pH 7.5 0 20 53 27 2.5 7.55 25 mM sodium phosphate, 0.5 M sodium chloride pH 7.5 25 23 22 30 37.2 7.48 25 mM sodium phosphate, 0.5 M sodium chloride pH 7.5 50 24 14 12 62.3 7.42

Claims (36)

1. An apparatus for processing a liquid containing a target substance, wherein said apparatus comprises at least a first and second device for performing a standard process, wherein each device for performing a standard process includes: (i) a system for supplying a liquid to a liquid connection using an inlet of a flow regulator comprising an inlet valve with a variable flow and an outlet, where at least one of the means for performing a unit process includes systems for supplying at least two liquids, wherein the supply systems are in a liquid connection using inlets of a flow regulator with multiple inlets comprising two or more inlet valves with a variable flow for dosing at least two liquids, wherein the flow regulator also comprises an outlet; (ii) a system for feeding a liquid feedstock containing a target substance into the liquid connection using an outlet from the flow regulator, thereby allowing the feed system for the liquid containing the target substance to be combined with the liquid flows specified in (i) to form the feed system of the device; (iii) a device for performing a treatment process, comprising a device inlet and a device outlet, wherein the device inlet is in fluid communication with the feed system of the device; and (iv) one pump located below the fluid connection between the connection between the feedstock and the outlet of the flow controller for communicating flow through the flow controller and from the feedstock liquid system through the processing device via the inlet of the device; wherein the supply of liquid feedstock containing the target substance to the second device for performing the unit process comprises an outlet from the first device for performing the unit process, where each typical process differs from other typical processes, and where each fixture for performing a typical process comprises essentially the same flow line. 2. The apparatus of claim 1, wherein the typical processes are selected from chromatography, viral inactivation, filtration, ultrafiltration, diafiltration, microfiltration, in-line conditioning, and refolding, in each case where the target substance contains a recombinant polypeptide. 3. An apparatus according to any of the preceding paragraphs, characterized in that the pump is located above the device for carrying out the bioprocessing process. 4. An apparatus according to any of the preceding claims, characterized in that the devices for performing a typical process are connected in series with a system for feeding a liquid source material containing a target substance to a subsequent device for performing a typical process, containing an outlet from the previous device for performing a typical process. 5. The apparatus according to item 1, comprising: (i) systems for supplying at least two liquids; (ii) a flow controller with multiple inlets comprising two or more variable flow inlet valves for mixing at least two fluids, wherein the flow controller also comprises one outlet; (iii) a system for feeding a liquid feedstock containing the target substance into the liquid connection using an outlet from the flow controller; (iv) a means for imparting a flow, typically by means of a pump, through a flow controller and from a feed system for liquid feed material through the bioprocessing apparatus via inlets of the feed system; (v) a device for ensuring homogeneous mixing of at least two liquids; (vii) a device for trapping air bubbles from process liquids; (vii) a common flow line for performing a bioprocessing operation, comprising an inlet port of the device and an outlet port of the device, wherein the inlet port of the device is in fluid communication with the inlets of the feed system; (viii) the inlet and outlet ports of the device that are in fluid communication with modules used for chromatography, filtration (including viral filtration), tangential flow filtration, single-pass tangential flow filtration, refolding, and viral inactivation; (ix) a device for bypassing the inlet and outlet opening of the device; (x) a device downstream of the outlet of the pressure regulating device; (xi) a series of sensors suitable for monitoring the bioprocessing process upstream and downstream of the inlet and outlet ports of the device; and (xii) at least one outlet in fluid communication with the inlet openings of the feed system. 6. An apparatus for preparing a liquid mixture, said apparatus comprising: (i) a system for supplying at least two liquids; (ii) a flow regulator with multiple inlets comprising two or more variable flow inlet valves for metering at least two fluids, wherein the flow regulator also comprises an outlet; (iii) one pump for imparting flow through the flow controller and from the liquid supply system through the apparatus via the inlet ports of the supply system; (iv) a static mixer for in-line mixing of at least two liquids; and (v) outlet for liquid mixture; where the pump is located upstream of the static mixer, and the pump and static mixer are located downstream of the multiple inlet flow controller, and the static mixer also serves as a bubble trap. 7. The apparatus according to claim 6, characterized in that the static mixer is a static mixer with a divided flow. 8. A method for preparing a buffer solution which includes mixing liquids using the apparatus according to item 6 or 7. 9. The method according to claim 8, which includes mixing liquids that are acidic and/or salt solutions. 10. A method for obtaining a biomolecule, including processing the biomolecule using an apparatus according to any of paragraphs 1-7. 11. The method according to claim 10, characterized in that the biomolecule is a recombinant polypeptide.