Method for processing fluids

Abstract

Provided herein are methods for treating fluids containing recombinant therapeutic proteins, including the use of a bioprocessing system comprising integrated unit operations including cell removal, cell retention, chromatography, viral inactivation, viral filtration, TFF, and compounding. Also provided are bioprocessing systems comprising these unit operations.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 322,133, filed March 21, 2022, the entire contents of which are incorporated by reference herein.

[0002] The present invention relates to methods of biotechnology and biomanufacturing of recombinant therapeutic proteins. [Background technology]

[0003] Mammalian cells containing nucleic acids encoding recombinant therapeutic proteins are often used to produce therapeutically or commercially important proteins. In the current environment of diverse product pipelines, biotechnology companies are increasingly challenged to develop innovative solutions that are highly flexible and cost-effective for manufacturing therapeutic protein drug substances. Summary of the Invention [Means for solving the problem]

[0004] The present invention is based, at least in part, on the discovery that the combined application of a back pressure regulator (BPR) and a boost pump (e.g., a centrifugal pump) in a tangential flow filtration (TFF) unit operation can achieve and maintain target retentate concentrations and maintain retentate and permeate flow rates in ultrafiltration (UF) and diafiltration (DF) operations.

[0005] Provided herein is a method of processing a fluid comprising a recombinant therapeutic protein, the method comprising: (a) providing a bioprocessing system comprising: (i) a unit operation; (ii) at least one inlet in the unit operation; (iii) at least one outlet in the unit operation; (iv) an inlet conduit in fluid communication with the at least one inlet of the unit operation; (v) an outlet conduit in fluid communication with the at least one outlet of the unit operation; (vi) a backpressure regulator disposed in the outlet conduit; and (vii) a boost pump disposed in the outlet conduit downstream of the backpressure regulator; b) flowing the fluid comprising the recombinant therapeutic protein through the inlet conduit to at least one inlet of the unit operation, processing the fluid in the unit operation to obtain a processed fluid, and then flowing a portion of the processed fluid to the outlet conduit; and (c) maintaining a target flow rate and / or pressure through the outlet conduit using the backpressure regulator and the boost pump.

[0006] In some embodiments the unit operation is selected from the group consisting of a cell removal unit operation, a cell retention unit operation, a tangential flow filtration unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal filtration unit operation and a blending unit operation.

[0007] Also provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with the outlet on the permeate side of the TFF unit; (vi) a permeate conduit in fluid communication with the outlet on the retentate side of the TFF unit; In one embodiment, the method includes providing a bioprocessing system comprising: (a) a retentate conduit disposed in a permeate conduit or retentate conduit; (b) a backpressure regulator disposed in the permeate conduit or retentate conduit; (c) a backpressure regulator and a boost pump disposed in the permeate conduit or retentate conduit downstream of the backpressure regulator; (d) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into the permeate conduit and the retentate conduit, respectively; and (e) maintaining a target flow rate and / or pressure through the permeate conduit or retentate conduit using the backpressure regulator and the boost pump.

[0008] In some embodiments, the TFF unit has a molecular weight cutoff of about 1 kDa to about 1000 kDa, hi some embodiments, the TFF unit has a molecular weight cutoff of about 10 kDa to about 100 kDa.

[0009] In some embodiments, the TFF unit has an average pore size of about 10 nm to about 100 nm. In some embodiments, the TFF unit has an average pore size of about 15 nm to about 35 nm. In some embodiments, the TFF unit has an average pore size of about 0.1 μm to about 10 μm.

[0010] In some embodiments, the recombinant therapeutic protein in the fluid in the inlet conduit (also referred to as the feed stream) has a concentration of about 0.1 g / L to about 300 g / L. In some embodiments, the recombinant therapeutic protein in the fluid in the inlet conduit (also referred to as the feed stream) has a concentration of about 0.1 g / L to about 150 g / L.

[0011] In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration of about 0.1 g / L to about 300 g / L.

[0012] In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration of about 5 g / L to about 225 g / L.

[0013] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration of about 0 g / L to about 100 g / L. In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration of about 0.1 g / L to about 25 g / L.

[0014] In some embodiments, the TFF units are each about 50 cm 2 ~about 10m 2 In some embodiments, the TFF units each include one or more filters having a filter area of ​​about 0.1 m. 2 ~about 5m 2 The filter includes one or more filters having a filter area of

[0015] In some embodiments, the booster pump is a dynamic pump. In some embodiments, the dynamic pump is a centrifugal pump.

[0016] In some embodiments, step (b) comprises continuously flowing the fluid at an average rate of about 0.1 mL / min to about 10 L / min.

[0017] In some embodiments, step (b) comprises continuously flowing the fluid at an average rate of about 1 mL / min to about 1 L / min.

[0018] In some embodiments, the bioprocessing system further comprises one or more sensors.

[0019] In some embodiments, at least one of the one or more sensors is disposed in the inlet conduit. In some embodiments, at least one of the one or more sensors is disposed in the permeate conduit.

[0020] In some embodiments, at least one of the one or more sensors is disposed in the retentate conduit.

[0021] In some embodiments, the recombinant therapeutic protein concentration is measured using one or more sensors having a sampling time of less than about 2 minutes.

[0022] In some embodiments, the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

[0023] In some embodiments, the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, where the ultraviolet absorbance is measured at about 200 nm to about 400 nm.

[0024] In some embodiments, the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, where the ultraviolet absorbance is measured at about 260 nm to about 320 nm.

[0025] In some embodiments, UV absorbance is measured in a fixed pathlength flow cell.

[0026] In some embodiments, the ultraviolet absorbance is measured in a variable pathlength flow cell.

[0027] In some embodiments, the method further comprises performing one or more second unit operations on the recombinant therapeutic protein prior to step (a), wherein the one or more second unit operations are integrated upstream of the bioprocessing system such that one of the one or more second unit operations is in fluid communication with an inlet conduit of the bioprocessing system.

[0028] In some embodiments, the one or more second unit operations are selected from the group consisting of a bioreactor unit operation, a cell removal unit operation, a cell retention unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, and a diafiltration unit operation.

[0029] In some embodiments, the method further comprises performing one or more second unit operations after step (c), wherein the one or more second unit operations are integrated downstream of the biotreatment system such that one of the one or more second unit operations is in fluid communication with a permeate conduit of the biotreatment system.

[0030] In some embodiments of any of the methods described herein, the method further comprises performing one or more second unit operations after step (c), wherein the one or more second unit operations are integrated downstream of the bioprocessing system such that one of the one or more second unit operations is in fluid communication with a retentate conduit of the bioprocessing system.

[0031] In some embodiments, the one or more second unit operations are selected from the group consisting of a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, or a blending unit operation.

[0032] Provided herein is a bioprocessing system comprising: (i) a unit operation; (ii) at least one inlet in the unit operation; (iii) at least one outlet in the unit operation; (iv) an inlet conduit in fluid communication with the at least one inlet of the unit operation; (v) an outlet conduit in fluid communication with the at least one outlet of the unit operation; (vi) a backpressure regulator disposed in the outlet conduit; and (vii) a boost pump disposed in the outlet conduit downstream of the backpressure regulator; the bioprocessing system is configured to flow a fluid comprising a recombinant therapeutic protein through the inlet conduit to at least one inlet of the unit operation, process the fluid in the unit operation to obtain a processed fluid, and then flow a portion of the processed fluid to the outlet conduit, wherein the backpressure regulator and the boost pump are configured to maintain a target flow rate and / or pressure through the outlet conduit.

[0033] In some embodiments, the unit operation is selected from the group consisting of a cell removal unit operation, a cell retention unit operation, a tangential flow filtration unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, and a blending unit operation.

[0034] Also provided herein is a tangential flow filtration (TFF) unit having: (i) at least one inlet and at least two outlets, where one of the at least two outlets is on a permeate side of the TFF unit and one of the at least two outlets is on a retentate side of the TFF unit; (ii) an inlet conduit in fluid communication with at least one inlet of the TFF unit; (iii) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; (iv) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; (v) a backflow filtration unit disposed in the permeate conduit or the retentate conduit; A bioprocessing system is provided that includes: (vi) a backpressure regulator; and (vi) a boost pump disposed in a permeate or retentate conduit downstream of the backpressure regulator; the bioprocessing system is configured to flow a fluid containing the recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separate the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flow the retentate and permeate streams into the permeate and retentate conduits; the backpressure regulator and the boost pump are configured to maintain a target flow rate and / or pressure through the permeate or retentate conduit.

[0035] In some embodiments, the TFF units have a molecular weight cutoff of about 1 kDa to about 1000 kDa. In some embodiments, the TFF units have a molecular weight cutoff of about 10 kDa to about 100 kDa. In some embodiments, the TFF units have an average pore size of about 10 nm to about 100 nm. In some embodiments, the TFF units have an average pore size of about 15 nm to about 35 nm. In some embodiments, the TFF units have an average pore size of about 0.1 μm to about 10 μm.

[0036] In some embodiments of any of the bioprocessing systems described herein, the recombinant therapeutic protein in the fluid in the inlet conduit has a concentration of about 0.1 g / L to about 300 g / L.

[0037] In some embodiments, the recombinant therapeutic protein in the fluid in the inlet conduit has a concentration of about 0.1 g / L to about 150 g / L.

[0038] In some embodiments of any of the bioprocessing systems described herein, the recombinant therapeutic protein in the retentate stream has a concentration of about 0.1 g / L to about 300 g / L. In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration of about 5 g / L to about 225 g / L.

[0039] In some embodiments of any of the bioprocessing systems described herein, the recombinant therapeutic protein in the permeate stream has a concentration of about 0 g / L to about 100 g / L.

[0040] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration of about 0.1 g / L to about 25 g / L.

[0041] In some embodiments of any of the bioprocessing systems described herein, the TFF units each have a volume of about 50 cm 2 ~about 10m 2 The filter includes one or more filters having a filter area of

[0042] In some embodiments, the TFF units are each about 0.1 m 2 ~about 5m 2 The filter includes one or more filters having a filter area of

[0043] In some embodiments of any of the bioprocessing systems described herein, the booster pump is a dynamic pump. In some embodiments, the dynamic pump is a centrifugal pump.

[0044] In some embodiments of any of the bioprocessing systems described herein, the bioprocessing system further comprises one or more sensors, hi some embodiments, at least one of the one or more sensors is disposed in the inlet conduit.

[0045] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors is disposed in the permeate conduit.

[0046] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors is disposed in the retentate conduit.

[0047] In some embodiments of any of the bioprocessing systems described herein, the one or more sensors are configured to measure the recombinant therapeutic protein concentration with a sampling time of less than about 2 minutes.

[0048] In some embodiments of any of the bioprocessing systems described herein, the one or more sensors are configured to measure the recombinant therapeutic protein concentration using ultraviolet absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

[0049] In some embodiments, the one or more sensors are configured to measure recombinant therapeutic protein concentration using ultraviolet absorbance from about 200 nm to about 400 nm.

[0050] In some embodiments, the one or more sensors are configured to measure recombinant therapeutic protein concentration using ultraviolet absorbance from about 260 nm to about 320 nm.

[0051] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors includes a fixed pathlength flow cell.

[0052] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors includes a variable pathlength flow cell.

[0053] In some embodiments of any of the bioprocessing systems described herein, a second unit operation is integrated upstream of the bioprocessing system such that the second unit operation is in fluid communication with an inlet conduit of the bioprocessing system.

[0054] In some embodiments, the second unit operation is selected from the group consisting of a bioreactor unit operation, a cell removal unit operation, a cell retention unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, and a diafiltration unit operation.

[0055] In some embodiments of any of the bioprocessing systems described herein, a second unit operation is integrated downstream of the bioprocessing system such that the second unit operation is in fluid communication with the permeate conduit of the bioprocessing system.

[0056] In some embodiments of any of the bioprocessing systems described herein, a second unit operation is integrated downstream of the bioprocessing system such that the second unit operation is in fluid communication with the retentate conduit of the bioprocessing system.

[0057] In some embodiments of any of the bioprocessing systems described herein, the second unit operation is selected from the group consisting of a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, and a blending unit operation.

[0058] As used herein, the word "a" before a noun refers to one or more of that particular noun. For example, the phrase "a mammalian cell" refers to "one or more mammalian cells."

[0059] The term "tangential flow filtration unit" or "TFF unit" is known in the art and refers to an apparatus that includes at least one housing (such as a cylinder) and at least one cross-flow (tangential) filter disposed within the housing such that a majority of the filter surface is disposed parallel to the flow of fluid (e.g., cell culture fluid) through the unit. TFF units are well known in the art and are commercially available. The housing may include, for example, a first inlet / outlet and a second inlet / outlet disposed such that fluid can pass through the first inlet / outlet, traverse the at least one cross-flow filter, and pass through the second inlet / outlet. In some examples, a bioprocessing system may include multiple TFF units, for example, connected in series and / or in parallel. For example, a bioprocessing system including two or more TFF units may include fluid conduits that fluidically connect pairs of adjacent TFF units in the system. In other examples, a bioprocessing system may include two or more sets of two or more TFF units fluidly connected by fluid conduits. Any of the TFF units described herein or known in the art are capable of receiving fluid in a first flow direction and a second flow direction.

[0060] The term "cross-flow filter" or "tangential filter" is known in the art and refers to a filter designed to be placed in a TFF unit such that the majority of the filter surface is parallel to the flow of a fluid (e.g., a fluid containing a recombinant therapeutic protein). For example, a cross-flow filter can be of any shape that allows for tangential flow filtration, e.g., cylindrical or rectangular. Particularly useful cross-flow filters are designed to create slight fluid turbulence or shear stress in the fluid as it flows across the surface of the cross-flow filter (e.g., flowing from one direction to another). Cross-flow filters are commercially available, e.g., from Sartorius, MembraPure, Millipore, and Pall Corporation.

[0061] The terms "ultrafiltration" and "UF" and similar terms refer to the use of synthetic semipermeable membranes having appropriate physical and chemical properties to separate molecules in a mixture primarily on the basis of molecular size and shape, thereby allowing the separation of different molecules or the concentration of similar molecules.

[0062] The terms "diafiltration" and "DF" and similar terms refer to the use of ultrafiltration membranes to remove, displace, or reduce the concentration of salts or solvents from a solution or mixture containing proteins, peptides, nucleic acids, or other biomolecules.

[0063] The term "permeate stream" or "permeate" refers to the fluid stream exiting the TFF unit after passing through a TFF filter. Fluid components selectively retained by the filter are not present in the permeate. In some embodiments (e.g., ultrafiltration or diafiltration), the permeate does not contain recombinant therapeutic protein. In some embodiments (e.g., viral filtration or microfiltration), the permeate contains recombinant therapeutic protein but does not contain other impurities (e.g., viral particles or cellular debris) present in the TFF feed material.

[0064] The term "retentate stream" or "retentate" refers to the stream of fluid that is discharged from a TFF unit without passing through a TFF filter. Fluid components that are selectively retained by the filter are present in the retentate. In some embodiments (e.g., ultrafiltration), fluid components that are selectively retained by the filter are concentrated in the retentate.

[0065] The term "transmembrane pressure" or "TMP" refers to the pressure averaged from the feed side to the filtrate side of a membrane, and TMP [bar] = [(P F +P R ) / 2]-P P (In the formula, P F is the supply pressure, P R is the holding pressure, and P P is the permeate pressure).

[0066] The term "dynamic pump" or "turbo pump" refers to a type of velocity pump in which kinetic energy is continuously imparted to the pumped fluid by a rotating impeller, propeller, or rotor.

[0067] The term "centrifugal pump" refers to a dynamic pump that uses an essentially radially-driven bladed impeller to transfer rotational mechanical energy to a fluid primarily by increasing the fluid's kinetic energy (angular momentum) and also increasing its potential energy (static pressure).

[0068] The term "mammalian cell" refers to any cell derived from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, the mammalian cell may be an immortalized cell. In some embodiments, the mammalian cell is a differentiated cell. In some embodiments, the mammalian cell is an undifferentiated cell. Non-limiting examples of mammalian cells are described herein. Further examples of mammalian cells are known in the art.

[0069] The term "unit operation" is a term of art and, when used as a verb, refers to a functional step that can be performed in a process for producing a therapeutic protein drug substance from a liquid medium. For example, a unit operation, when used as a verb, can be filtering (e.g., removing contaminating bacteria, yeast viruses, or mycobacteria, and / or specific substances from a fluid containing a recombinant therapeutic protein), capturing, removing an epitope tag, purifying, retaining or preserving, polishing, viral inactivation, adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein, and removing unwanted salts.

[0070] The term "unit operation" is a term of art and, when used as a noun, refers to the use of equipment that can be used to perform a functional step in the process of producing a therapeutic protein drug substance from a liquid medium. For example, a unit operation, when used as a noun, can include a chromatography system (e.g., a single-column or multi-column chromatography system, a cyclic countercurrent chromatography system), a filtration unit (e.g., a tangential flow filtration (TFF) unit), and / or a holding tank. Such unit operations can include, but are not limited to, a bioreactor unit operation, a cell removal unit operation, a cell retention unit operation, a tangential flow filtration unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal filtration unit operation, an ultrafiltration unit operation, a diafiltration unit, or a compounding unit operation.

[0071] The term "continuous unit operation" means a unit operation that continuously supplies fluid through at least a portion of a system.

[0072] The term "continuous process" refers to a process in which a fluid is continuously fed through at least a portion of a system.

[0073] The term "integrated process" refers to a process carried out using structural elements that work in concert to achieve a particular result (e.g., producing a therapeutic protein drug substance from a liquid medium).

[0074] The term "bioprocessing system" refers to a system for creating, producing, or manufacturing a biological product from a liquid medium. A bioprocessing system can have one or more unit operations.

[0075] The term "upstream" refers to one or more unit operations being performed prior to the timing of one or more other unit operations being performed. The term "downstream" refers to one or more unit operations being performed after the timing of one or more other unit operations being performed.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art can also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0077] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0078] [Fig. 1A-1F]The operation overview and control method of the first single-pass continuous tangential flow filtration (SP-TFF) or ultrafiltration 1 (UF1), the single-pass continuous diafiltration (SP-DF), and the second single-pass continuous tangential flow filtration (SP-TFF) or ultrafiltration 2 (UF2) are shown, respectively. As can be seen in FIG. 1A, a fluid containing a recombinant therapeutic protein is delivered from a surge vessel to the first ultrafiltration (UF) unit. In-line flow meters, pressure sensors, and refractive index sensors in the feed, retentate, and permeate streams measure flow rates, pressures, and concentration values ​​in real time, respectively. A back pressure regulator (BPR) installed in the retentate stream controls the pressure on the filter, thereby concentrating the product. In FIG. 1B, a boost pump directs the emerging concentrated product stream to the SP-DF unit. At the same time, diafiltration (DF) buffer flows into the DF unit. The BPR maintains a uniform retentate concentration by adjusting the permeate flux through the filter based on real-time fluid properties measured using an in-line sensor. Figure 1C shows the final UF2 operation, which involves concentrating a recombinant therapeutic protein to a value higher than the final DS using a single UF unit. Figure 1D shows a control scheme for a continuous single-pass ultrafiltration (SP-TFF) operation, where a concentration controller is implemented to control the concentration of the retentate stream based on real-time 1) feed and retentate concentrations, and 2) feed and permeate fluxes. Figures 1E and 1F show control schemes for a continuous single-pass diafiltration (SP-DF) operation. Figure 1E shows a scheme for the implementation of a concentration controller that controls the concentration of the retentate stream based on real-time 1) feed and retentate concentrations, and 2) feed and permeate fluxes. Figure 1F shows a control scheme for the implementation of a buffer addition controller that controls the addition of a fixed volume of buffer to the feed based on 1) a calculated buffer flow rate, and 2) real-time buffer flow rate. [Figures 2A-2D]For the first ultrafiltration (UF1) run: Figure 2A is a graph showing recombinant therapeutic protein concentration (g / L) as a function of time (days) after the first ultrafiltration run; Figure 2B is a graph showing flux / TMP values ​​as a function of time (days); Figure 2C is a graph showing permeate flux (LMH) as a function of time (days); and Figure 2D is a graph showing permeate flux as a function of applied transmembrane pressure (psi). [Figures 3A-3H] Plots showing the performance of continuous diafiltration operations. FIG. 3A is a graph showing the target diafiltration (DF) retentate concentration (g / L) over time (days). FIG. 3B is a graph showing the target diavolume achieved over time (days). FIG. 3C is a graph showing the ratio of diafiltration (DF) buffer addition rate to permeate production rate over time (days). FIG. 3D is a graph showing the flux / TMP values ​​over time (days). FIG. 3E is a graph showing the permeate volumetric flux (LMH) during diafiltration over time (days). FIG. 3F is a graph showing the effect of permeate flux (LMH) on transmembrane pressure (psi). FIG. 3G is a graph showing the effect of a boost pump used in combination with a backpressure regulator on the retentate concentration (g / L) over time (days). FIG. 3H is a graph showing the effect of a boost pump used in combination with a backpressure regulator on retentate pressure (psi) versus backpressure regulator pressure (psi). [Figure 4A-4D] 4A and 4B are graphs showing the performance of the final SP-TFF ultrafiltration run (UF2) for integrated recombinant therapeutic protein manufacturing. FIG. 4A is a graph showing the protein concentration of the retentate after ultrafiltration over time (days). FIG. 4B is a graph showing the filter performance (flux / TMP) over time (days) during the run. FIG. 4C is a graph showing the permeate flux (LMH) as a function of time (days). FIG. 4D is a graph showing the permeate flux as a function of transmembrane pressure (psi). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Provided herein are methods and bioprocessing systems for treating a fluid. Non-limiting aspects of these methods and bioprocessing systems are described herein.

[0080] Method for treating fluids Provided herein is a method of processing a fluid comprising a recombinant therapeutic protein, the method comprising: (a) providing a bioprocessing system comprising: (i) a unit operation; (ii) at least one inlet in the unit operation; (iii) at least one outlet in the unit operation; (iv) an inlet conduit in fluid communication with the at least one inlet of the unit operation; (v) an outlet conduit in fluid communication with the at least one outlet of the unit operation; (vi) a backpressure regulator disposed in the outlet conduit; and (vii) a boost pump disposed in the outlet conduit downstream of the backpressure regulator; b) flowing the fluid comprising the recombinant therapeutic protein through the inlet conduit to at least one inlet of the unit operation, processing the fluid in the unit operation to obtain a processed fluid, and then flowing a portion of the processed fluid to the outlet conduit; and (c) maintaining a target flow rate and / or pressure through the outlet conduit using the backpressure regulator and the boost pump.

[0081] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, the method comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with the outlet on the permeate side of the TFF unit; (vi) a permeate conduit in fluid communication with the outlet on the retentate side of the TFF unit; The method includes providing a bioprocessing system including a retentate conduit, (vii) a backpressure regulator disposed in the permeate or retentate conduit, and (viii) a boost pump disposed in the permeate or retentate conduit downstream of the backpressure regulator; (b) flowing a fluid including a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate or retentate conduit using the backpressure regulator and the boost pump.

[0082] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; (vi) a retentate conduit in fluid communication with the outlet on the permeate side of the TFF unit; (vii) a retentate conduit in fluid communication with the outlet on the permeate side of the TFF unit; In one embodiment, the method includes providing a bioprocessing system comprising: (a) a retentate conduit in fluid communication with an outlet of the permeate conduit downstream of the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into a permeate conduit and a retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate conduit using the backpressure regulator and the boost pump.

[0083] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; and (vi) an outlet on the retentate side of the TFF unit. (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on a pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into a permeate conduit and a retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the boost pump.

[0084] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; (vi) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; (vii) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; The method includes providing a bioprocessing system including (viii) a backpressure regulator disposed in a fluid conduit, and (b) a boost pump disposed in a retentate conduit downstream of the backpressure regulator; (b) flowing a fluid including a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into a permeate conduit and a retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the boost pump, wherein the TFF unit has an average pore size of about 15 nm to about 35 nm or about 1 μm to about 10 μm.

[0085] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, the method comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; (vi) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; (vii) a backpressure regulator disposed in the retentate conduit; and (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into a permeate conduit and a retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the intensifier pump, wherein the TFF unit has a molecular weight cutoff of about 1 kDa to about 100 kDa, and optionally the recombinant therapeutic protein concentration in the retentate conduit is maintained constant at about 5 to about 250 g / L.

[0086] Provided herein is a method for treating a fluid comprising a recombinant therapeutic protein, the method comprising: (a) (i) a tangential flow filtration (TFF) unit comprising a permeate side and a retentate side; (ii) at least one inlet in the TFF unit; (iii) at least two outlets in the TFF unit, where one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retentate side of the TFF unit; (iv) an inlet conduit in fluid communication with the at least one inlet of the TFF unit; (v) a permeate conduit in fluid communication with an outlet on the permeate side of the TFF unit; (vi) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; (vii) a backpressure regulator disposed in the retentate conduit; and (viii) a boost pump disposed in the retentate conduit downstream of the backpressure regulator; (ix) at least three sensors, where one of the at least three sensors is disposed in the inlet conduit and one and (x) a feedback controller and a feedforward controller connected to a backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separating the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flowing the permeate stream and the retentate stream into a permeate conduit and a retentate conduit, respectively; and (c) maintaining a target flow rate and / or pressure through the permeate conduit or the retentate conduit using a backpressure regulator and a boost pump, wherein the TFF unit has a molecular weight cutoff of about 1 kDa to about 100 kDa, and optionally, the recombinant therapeutic protein concentration in the retentate conduit is maintained constant in real time at about 5 to 250 g / L by controlling the pressure with the backpressure regulator.

[0087] In some embodiments, the bioprocessing system further comprises a cell removal unit operation, a cell retention unit operation, a tangential flow filtration unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, or a blending unit operation.

[0088] In some embodiments, the method further comprises, prior to step (a), performing one or more unit operations on the recombinant therapeutic protein such that the one or more unit operations are integrated and in fluid communication with one or more of the inlets of the bioprocessing system described in step (a), wherein the one or more unit operations are selected from the group consisting of a bioreactor unit operation, a cell removal unit operation, a cell retention unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, and a blending unit.

[0089] In some embodiments, the method further comprises performing one or more second unit operations after step (c), wherein the one or more second unit operations are integrated downstream of the biotreatment system such that one of the one or more second unit operations is in fluid communication with a permeate conduit of the biotreatment system.

[0090] In some embodiments, the method further comprises, after step (c), performing one or more second unit operations, one of the one or more second unit operations in fluid communication with the retentate conduit of the bioprocessing system.

[0091] In some embodiments, the method further comprises performing one or more second unit operations after step c), wherein the one or more second unit operations are integrated downstream of the bioprocessing system, wherein the one or more second unit operations are selected from the group consisting of a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, and a blending unit operation.

[0092] In some embodiments, the TFF unit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 tangential filters. In some examples of these methods, the TFF unit comprises about 50 cm 2 ~about 10m 2, (e.g., about 50 cm 2 ~ approx. 0.1m 2 , about 50cm 2 ~about 0.5m 2 , about 50cm 2 ~ approx. 1m 2 , about 50cm 2 ~about 5m 2 , about 50cm 2 ~about 10m 2 , about 0.1m 2 ~about 0.5m 2 , about 0.1m 2 ~ approx. 1m 2 , about 0.1m 2 ~about 5m 2 , about 0.1m 2 ~about 10m 2 , about 0.5m 2 ~ approx. 1m 2 , about 0.5m 2 ~about 5m 2 , about 0.5m 2 ~about 10m 2 , about 1m 2 ~about 5m 2 , about 1m 2 ~about 10m 2 , or about 5m 2 ~about 10m 2 In some embodiments, the TFF units may each include one or more tangential filters having a surface area of ​​about 0.1 m. 2 ~about 5m 2 The filter is composed of one or more filters having a filter area of

[0093] In some embodiments, the TFF unit (one or more tangential filters within the TFF unit) has an average pore size of about 10 nm to about 100 nm, e.g., about 10 nm to about 15 nm, about 10 nm to about 20 nm, about 10 nm to about 35 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 15 nm to about 20 nm, about 15 nm to about 35 nm, about 15 nm to about 50 nm, about 15 nm to about 100 nm, about 20 nm to about 35 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 35 nm to about 50 nm, about 35 nm to about 100 nm, or about 50 nm to about 100 nm.

[0094] For example, to perform viral filtration, a TFF unit containing one or more tangential filters having an average pore size of about 10 nm to about 100 nm can be used.

[0095] In some embodiments, the TFF unit (one or more tangential filters in the TFF unit) has a tangential filter diameter of about 0.1 μm to about 10 μm (e.g., about 0.1 μm to about 0.2 μm, about 0.1 μm to about 0.45 μm, about 0.1 μm to about 0.65 μm, about 0.1 μm to about 1 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 10 μm, about 0.2 μm to about 0.45 μm, about 0.2 μm to about 0.65 μm, , about 0.2 μm to about 1 μm, about 0.2 μm to about 5 μm, about 0.2 μm to about 10 μm, about 0.45 μm to about 0.65 μm, about 0.45 μm to about 1 μm, about 0.45 μm to about 5 μm, about 0.45 μm to about 10 μm, about 0.65 μm to about 1 μm, about 0.65 μm to about 5 μm, about 0.65 μm to about 10 μm, about 1 μm to about 5 μm, about 1 μm to about 10 μm, or about 5 μm to about 10 μm). In some embodiments, the TFF unit has an average pore size of about 10 nm to about 100 nm. In some embodiments, the TFF unit has an average pore size of about 15 nm to about 35 nm.

[0096] For example, to perform microfiltration, a TFF unit containing one or more tangential filters having an average pore size of about 0.1 μm to about 10 μm can be used.

[0097] In some embodiments, one or more tangential filters in the TFF unit have a diameter of about 1 kDa to about 1000 kDa (e.g., about 1 kDa to about 10 kDa, about 1 kDa to about 30 kDa, about 1 kDa to about 50 kDa, about 1 kDa to about 100 kDa, about 1 kDa to about 300 kDa, about 1 kDa to about 1000 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 3 The TFF unit may have a molecular weight cutoff of about 100 kDa, about 10 kDa to about 1000 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 100 kDa, about 30 kDa to about 300 kDa, about 30 kDa to about 1000 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 1000 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 1000 kDa, or about 300 kDa to about 1000 kDa). In some embodiments, the TFF unit has a molecular weight cutoff of about 10 kDa to about 100 kDa.

[0098] For example, a TFF unit containing one or more tangential filters having a molecular weight cutoff of about 1 kDa to about 1000 kDa can be used to perform ultrafiltration and / or diafiltration.

[0099] In some embodiments, the TFF unit includes one or more tangential filters made of, for example, cellulose (e.g., regenerated cellulose or cuprammonium regenerated cellulose), polyethersulfone, polysulfone, polyvinylidene fluoride, or mixed cellulose esters. Non-limiting examples of one or more tangential filters that can be used in the TFF unit are commercially available from Millipore (e.g., Ultracel® and Biomax®). Additional commercial sources of one or more tangential filters that can be used in the TFF unit are known in the art.

[0100] In some embodiments, the recombinant therapeutic protein in the fluid (also referred to as the feed stream) is present at a concentration of from about 0.1 g / L to about 300 g / L (e.g., from about 0.1 g / L to about 5 g / L, from about 0.1 g / L to about 25 g / L, from about 0.1 g / L to about 50 g / L, from about 0.1 g / L to about 100 g / L, from about 0.1 g / L to about 150 g / L, from about 0.1 g / L to about 225 g / L, from about 0.1 g / L to about 300 g / L, from about 5 g / L to about 25 g / L, from about 5 g / L to about 50 g / L, from about 5 g / L to about 100 g / L, from about 5 g / L to about 150 g / L, from about 5 g / L to about 225 g / L, from about 5 g / L to about 3 00g / L, about 25g / L to about 50g / L, about 25g / L to about 100g / L, about 25g / L to about 150g / L, about 25g / L to about 225g / L, about 25g / L to about 300g / L, about 50g / L to about 100g / L, about 50g / L to about 150g / L, about 50g / L to about 225g / L, about 50g / L to about 300g / L, about 100g / L to about 150g / L, about 100g / L to about 225g / L, about 100g / L to about 300g / L, about 150g / L to about 225g / L, about 150g / L to about 300g / L, or about 225g / L to about 300g / L).

[0101] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration of about 0.1 g / L to about 100 g / L (e.g., about 0.1 g / L to about 1 g / L, about 0.1 g / L to about 5 g / L, about 0.1 g / L to about 10 g / L, about 0.1 g / L to about 25 g / L, about 0.1 g / L to about 100 g / L, about 1 g / L to about 5 g / L, about 1 g / L to about 10 g / L, about 1 g / L to about 25 g / L, about 1 g / L to about 100 g / L, about 5 g / L to about 10 g / L, about 5 g / L to about 25 g / L, about 5 g / L to about 100 g / L, about 10 g / L to about 25 g / L, about 10 g / L to about 100 g / L, or about 25 g / L to about 100 g / L). In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration of about 0.1 g / L to about 25 g / L. In some embodiments, there is no recombinant therapeutic protein in the permeate stream.

[0102] In some embodiments, the recombinant therapeutic protein in the retentate stream is present in an amount of from about 0.1 g / L to about 300 g / L (e.g., from about 0.1 g / L to about 5 g / L, from about 0.1 g / L to about 25 g / L, from about 0.1 g / L to about 50 g / L, from about 0.1 g / L to about 100 g / L, from about 0.1 g / L to about 150 g / L, from about 0.1 g / L to about 225 g / L, from about 0.1 g / L to about 300 g / L, from about 5 g / L to about 25 g / L, from about 5 g / L to about 50 g / L, from about 5 g / L to about 100 g / L, from about 5 g / L to about 150 g / L, from about 5 g / L to about 225 g / L, from about 5 g / L to about 300 g / L). , about 25 g / L to about 50 g / L, about 25 g / L to about 100 g / L, about 25 g / L to about 150 g / L, about 25 g / L to about 225 g / L, about 25 g / L to about 300 g / L, about 50 g / L to about 100 g / L, about 50 g / L to about 150 g / L, about 50 g / L to about 225 g / L, about 50 g / L to about 300 g / L, about 100 g / L to about 150 g / L, about 100 g / L to about 225 g / L, about 100 g / L to about 300 g / L, about 150 g / L to about 225 g / L, about 150 g / L to about 300 g / L, or about 225 g / L to about 300 g / L). In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration of about 5 g / L to about 225 g / L.

[0103] In some examples of any of the methods described herein, step (b) comprises pumping a fluid comprising the recombinant therapeutic protein through an inlet conduit at a rate of about 0.1 mL / min to about 10 L / min (e.g., about 0.1 mL / min to about 1 mL / min, about 0.1 mL / min to about 5 mL / min, about 0.1 mL / min to about 25 mL / min, about 0.1 mL / min to about 100 mL / min, about 0.1 mL / min to about 500 mL / min, about 0.1 mL / min to about 1 L / min, about 0.1 mL / min to about 10 L / min, about 1 mL / min to about 5 mL / min, about 1 mL / min to about 25 mL / min, about 1 mL / min to about 100 mL / min, about 1 mL / min to about 500 mL / min, about 1 mL / min to about 1 L / min, about 1 mL / min to about 1 The present invention includes flowing (e.g., continuously flowing) the liquid into at least one inlet of the TFF unit at a rate of about 0 L / min, about 5 mL / min to about 25 mL / min, about 5 mL / min to about 100 mL / min, about 5 mL / min to about 500 mL / min, about 5 mL / min to about 1 L / min, about 5 mL / min to about 10 L / min, about 25 mL / min to about 100 mL / min, about 25 mL / min to about 500 mL / min, about 25 mL / min to about 1 L / min, about 25 mL / min to about 10 L / min, about 100 mL / min to about 500 mL / min, about 100 mL / min to about 1 L / min, about 100 mL / min to about 1 L / min, about 500 mL / min to about 1 L / min, about 500 mL / min to about 10 L / min, or about 1 L / min to about 10 L / min). In some embodiments, step (b) comprises continuously flowing the fluid at an average rate of about 1 mL / min to about 1 L / min.

[0104] In some embodiments, the fluid comprises a diafiltration medium. Non-limiting examples of diafiltration medium can be, for example, a physiologically acceptable buffer (e.g., phosphate buffered saline) or a buffer used to formulate recombinant therapeutic proteins, such as a buffer containing one or more of glycine, phosphate, acetate, citrate, histidine, arginine, a bulking agent (e.g., a non-reducing carbohydrate, such as sucrose), or a surfactant (e.g., polysorbate 80, Triton X-100, and Tween).

[0105] In some embodiments, the methods described herein include the use of a holding tank fluidly connected to the inlet conduit, the permeate conduit, and / or the retentate conduit. In some embodiments, the methods described herein do not include the use of a holding tank fluidly connected to the inlet conduit, the permeate conduit, and / or the retentate conduit. In some embodiments, the methods described herein do not include the use of a holding tank at all.

[0106] In some embodiments, where the TFF unit includes a viral filter, the method can further include receiving a permeate stream from the permeate conduit containing the recombinant therapeutic protein.

[0107] In some embodiments, where the TFF unit includes a microfilter, the method can further include receiving a permeate stream containing the recombinant therapeutic protein from the permeate conduit.

[0108] In some embodiments, when the TFF unit is an ultrafiltration filter or a diafiltration filter, the method can further include receiving a retentate stream containing the recombinant therapeutic protein from the retentate conduit.

[0109] In some embodiments, the booster pump can be a dynamic pump (e.g., a centrifugal pump). In some embodiments, the dynamic pump is a centrifugal pump.

[0110] In some embodiments, the bioprocessing system further comprises one or more sensors. In some embodiments, at least one of the one or more sensors is disposed in the inlet conduit. In some embodiments, at least one of the one or more sensors is disposed in the permeate conduit. In some embodiments, at least one of the one or more sensors is disposed in the retentate conduit. In some embodiments, at least one of the one or more sensors is disposed in the outlet conduit.

[0111] In some embodiments, the recombinant therapeutic protein concentration is measured using one or more sensors having a sampling time of less than about 2 minutes (e.g., less than about 90 seconds, less than about 60 seconds, less than about 45 seconds, less than about 30 seconds, less than about 25 seconds, less than about 20 seconds, less than about 15 seconds, less than about 10 seconds, or less than about 5 seconds).

[0112] In some embodiments, the therapeutic protein concentration is measured using ultraviolet absorbance, refractive index, Fourier transform infrared spectroscopy (FITR), or Raman spectroscopy.

[0113] In some embodiments, the recombinant therapeutic protein concentration is measured using ultraviolet absorbance from about 200 nm to about 400 nm. In some embodiments, the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, where the ultraviolet absorbance is measured from about 260 nm to about 320 nm. In some embodiments, the ultraviolet absorbance is measured by a fixed path length flow cell. In some embodiments, the ultraviolet absorbance is measured by a variable path length flow cell.

[0114] In some embodiments, the biotreatment system achieves about 5 to about 15 times volumetric buffer exchange, hi some embodiments, the biotreatment system achieves about 5 to about 7 times volumetric buffer exchange.

[0115] In some embodiments, the flux versus transmembrane pressure (TMP) is maintained between 0.5 and 1.0 for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days). In some embodiments, the flux versus transmembrane pressure (TMP) is maintained between 0.6 and 0.7 for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days). In some embodiments, the flux versus transmembrane pressure (TMP) is maintained at a constant value for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days).

[0116] In some embodiments, the method maintains a target concentration of the recombinant therapeutic protein in the retentate stream of the TFF unit in real time over a period of time. In some embodiments, the method maintains a target concentration of the recombinant therapeutic protein in the retentate stream of the TFF unit in real time for about 3 days to about 8 weeks. In some embodiments, the method maintains a target concentration of the recombinant therapeutic protein in the retentate stream of the TFF unit in real time for about 4 weeks.

[0117] Exemplary Ultrafiltration Methods In the continuous mode, the ultrafiltration operation is divided into two steps with an intermediate diafiltration (DF) step: Ultrafiltration 1 (UF1) and Ultrafiltration 2 (UF2). The protein solution is fed as input to UF1. The protein solution is fed into two 0.5 ml diafiltration filters configured in series. 2 Two ultrafiltration units can be used to concentrate the protein solution to an intermediate value suitable for diafiltration. Such a configuration allows for maximum inlet flow, thereby preventing concentration polarization and membrane surface fouling. Briefly, the overall operation of UF1 is carried out as follows: A quad diaphragm pump (Quattroflow Fluid Systems, QF 150S, PSG Germany GmbH) is used to pump the inlet protein solution from a surge vessel (5 L, Cercell) to 0.5 ml. 2The feed stream is then fed to the inlet of a single-pass tangential flow filtration (SP-TFF) unit (30 kDa molecular weight cut-off (MWCO), Pellicon capsule with Ultracel membrane, C-screen). The volumetric flow rate of the feed stream is automatically adjusted using a surge vessel level controller. The retentate stream from the first UF1 unit is then fed to the inlet of the second UF1 unit. This entire process employs a proportional-integral-derivative (PID) controller that uses a combination of feedback and feedforward control to maintain the protein concentration in the retentate at the target value. The PID controller adjusts the transmembrane pressure (TMP) of the retentate stream of the second UF1 unit using a backpressure regulator (BPR, Equilibar, Precision Fluid Control) based on the feed and permeate fluxes, and the feed and retentate concentrations. These fluxes and concentrations are measured independently in real time by in-line single-use flowmeters (Levitronix Technologies Inc.) and refractive index (RI) sensors (mPath IoR flow cell, Corporation Life Sciences), respectively. Finally, the retentate flow of the second UF1 unit is continuously fed to the downstream diafiltration unit by a booster pump (PuraLev i100SU, Levitronix Technologies Inc.), which is essentially a centrifugal pump. The booster pump minimizes the effect of upstream pressure on downstream operation and ensures a constant and uninterrupted flow to the downstream unit. Therefore, the combination of BPR and booster pump is essential for robust control of UF1.

[0118] In the second continuous ultrafiltration step (UF2), the buffer exchanged protein from the DF step is further concentrated to a value higher than the desired drug substance (DS). The operating principle and control scheme are the same as UF1. However, the main difference between them is that UF2 uses one 0.5 ml filter instead of two units as described in UF1. 2The main focus of the concentration process is to use a single-pass tangential flow filtration (SP-TFF) unit (30 kDa molecular weight cut-off (MWCO), Pellicon capsule with Ultracel membrane, C-screen). After the concentration process, the product is driven to the compounding operation by a booster pump.

[0119] Exemplary Diafiltration Methods The diafiltration operation involves exchanging the starting buffer (300 mM sodium acetate, pH 4.5) of the concentrated protein solution from UF1 with 5-7 diavolumes (DV) of DF buffer (10 mM sodium acetate, pH 4.5). To achieve in-line buffer exchange, the concentrated protein stream from the second UF1 unit is passed through a 1.6 ml 2The inlet of a single-pass diafiltration cassette (Hydrosart ultrafiltration filter, 30 kDa molecular weight cut-off (MWCO), Sartorius) is continuously fed (by the boost pump mentioned previously). Simultaneously, a peristaltic pump (Watson-Marlow Inc.) feeds DF buffer at a volumetric flow rate equal to 5 DV (5 times the feed flow rate) based on an in-line flow measurement of the feed stream (Levitronix Technologies Inc.). This continuous DF operation is designed such that the rate at which DF buffer is added to the system is equal to the rate at which permeate is removed from the system. This condition ensures that the protein concentration does not change during the process. The single-pass continuous DF method is achieved by implementing two controllers: 1) a concentration controller that maintains a uniform retentate concentration, and 2) a buffer addition controller that ensures the addition of a fixed volume of buffer to the feed, i.e., the desired diavolume. To maintain a uniform retentate concentration, a proportional-integral-derivative (PID) controller using a combination of feedback and feedforward control is implemented to control the TMP of the DF membrane. Similar to UF1, the controller adjusts the TMP using a backpressure regulator based on in-line measurements of feed and permeate flux (single-use flowmeter, Levitronix Technologies Inc.) and feed and retentate concentrations (RI sensor, mPath IoR flow cell, Corporation Life Sciences). To achieve the desired diavolume, the volumetric flow rate of the DF buffer is calculated in real time based on the feed flow rate and permeate flux through the DF cassette. Another PID controller then dynamically adjusts the peristaltic pump setpoint and varies the buffer addition rate. Both controllers work synergistically to ensure robust control and operation of diafiltration in a continuous single-pass format. The final outlet flow is continuously fed to the next ultrafiltration unit (UF2) using a booster pump (PuraLev i100SU, Levitronix Technologies Inc.) for the same reasons outlined for UF1.As with UF1, the BPR and booster pump work together to enable robust, integrated operation of the single-pass DF.

[0120] Biotreatment System Also provided herein is a bioprocessing system comprising: (i) a unit operation; (ii) at least one inlet in the unit operation; (iii) at least one outlet in the unit operation; (iv) an inlet conduit in fluid communication with the at least one inlet of the unit operation; (v) an outlet conduit in fluid communication with the at least one outlet of the unit operation; (vi) a backpressure regulator disposed in the outlet conduit; and (vii) a boost pump disposed in the outlet conduit downstream of the backpressure regulator; the bioprocessing system is configured to flow a fluid comprising a recombinant therapeutic protein through the inlet conduit to the at least one inlet of the unit operation, process the fluid in the unit operation to obtain a processed fluid, and then flow a portion of the processed fluid to the outlet conduit; the backpressure regulator and the boost pump are configured to maintain a target flow rate and / or pressure through the outlet conduit.

[0121] Also provided herein are: (i) a tangential flow filtration (TFF) unit having at least one inlet and at least two outlets, where one of the at least two outlets is on a permeate side of the TFF unit and one of the at least two outlets is on a retentate side of the TFF unit; (ii) an inlet conduit in fluid communication with at least one inlet of the TFF unit; (iii) a permeate outlet conduit in fluid communication with an outlet on the permeate side of the TFF unit; (iv) a retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit; (v) a retentate conduit disposed in the permeate conduit or the retentate conduit; and (vi) a boost pump disposed in a permeate or retentate conduit downstream of the backpressure regulator; the bioprocessing system is configured to flow a fluid comprising a recombinant therapeutic protein through an inlet conduit to at least one inlet of a TFF unit, separate the fluid into a retentate stream and a permeate stream based on the pore size or molecular weight cutoff of the TFF unit, and then flow the retentate and permeate streams into the permeate and retentate conduits; the backpressure regulator and boost pump are configured to maintain a target flow rate and / or pressure through the retentate conduit.

[0122] In some embodiments, the bioprocessing system includes a cell removal unit operation, a cell retention unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, or a blending unit operation.

[0123] In some embodiments, the bioprocessing system further comprises one or more additional unit operations integrated with and in fluid communication with one or more of the inlets of the unit operations, the one or more additional unit operations being selected from the group consisting of a cell removal unit operation, a cell retention unit operation, a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, or a blending unit operation.

[0124] In some embodiments, the bioprocessing system includes a second unit operation integrated upstream of the bioprocessing system such that the second unit operation is in fluid communication with an inlet conduit of the bioprocessing system.

[0125] In some embodiments, the bioprocessing system includes a second unit operation selected from the group consisting of a bioreactor, a cell removal unit operation, a cell retention unit operation, a chromatography operation, a viral inactivation operation, a viral removal filtration operation, an ultrafiltration operation, and a diafiltration operation.

[0126] In some embodiments, the bioprocessing system includes a second unit operation integrated downstream of the bioprocessing system such that the second unit operation is in fluid communication with a permeate conduit of the bioprocessing system.

[0127] In some embodiments, the bioprocessing system includes a second unit operation integrated downstream of the bioprocessing system such that the second unit operation is in fluid communication with a retentate conduit of the bioprocessing system.

[0128] In some embodiments, the bioprocessing system includes a second unit operation integrated downstream of the bioprocessing system, the second unit operation selected from the group consisting of a chromatography operation, a viral inactivation operation, a viral removal filtration operation, an ultrafiltration operation, a diafiltration operation, and a blending operation.

[0129] In some embodiments of any of the bioprocessing systems described herein, the TFF unit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 tangential filters. In some examples of these bioprocessing systems, the TFF unit comprises about 50 cm 2 ~about 10m 2 (or any of the exemplary subranges of this range described herein).

[0130] back pressure regulator In some embodiments, the methods and bioprocessing systems described herein include a backpressure regulator. A backpressure regulator can be used to maintain a stable and uniform pressure and / or flow rate and can be used under various downstream pressure conditions, provided that the pressure downstream of the backpressure regulator does not exceed the upstream pressure. Various types of backpressure regulators are known in the art and can be used in any of the methods and / or bioprocessing systems described herein (e.g., BP-2080 Automated Backpressure Regulator (Jasco, DE), Ultratrace Backpressure Regulator (IDEX Health&Science LLC), M10BP Precision Backpressure Regulator (Fairchild)). See, e.g., Berger and Berger, J. Chromatogr. A 1218(16);2320-2326, 2011, and Svensson et al., Sci. Rep. 12:569, 2022.

[0131] In some embodiments, a backpressure regulator is used in a bioprocessing system in conjunction with a positive displacement pump and a bypass valve.

[0132] Booster Pump In some embodiments of any of the methods and bioprocessing systems described herein, the booster pump is a dynamic pump (also known as a non-positive displacement pump). In some embodiments, the dynamic pump is a centrifugal pump or a propeller pump. A centrifugal pump transfers rotational energy from an impeller to a fluid, increasing the velocity and pressure of the fluid. Unlike a positive displacement pump, a dynamic pump can be operated at a variety of pressures for a given flow rate and pump speed. Various types of dynamic pumps are known in the art and can be used in any of the methods and / or bioprocessing systems described herein (e.g., Puralev 600 MU (Levitronix, Switzerland), LKH UltraPure (Alfa Laval, Sweden)).

[0133] Sensors In some embodiments of any of the methods and bioprocessing systems described herein, one or more sensors can be used to control the TFF unit. The relevant sensors used in the control algorithm measure flow rate, pressure, and / or recombinant therapeutic protein concentration. The flow sensor is an in-line flow meter that measures the flow rate in real time. The flow sensors can be installed, for example, at the inlet of the TFF unit, at least two outlets of the TFF unit, the inlet conduit, the permeate conduit, and / or the retentate conduit.

[0134] An in-line pressure sensor located downstream of the back pressure regulator and intensifier pump varies the rpm of the intensifier pump as pressure varies, thereby ensuring continuous flow to subsequent runs.

[0135] Controller and control method In some embodiments of any of the methods and bioprocessing systems described herein, one or more controllers are used to manipulate the process variables so that the effects of disturbances can be minimized and the process variables are as close as possible to their set points. For robust control of the process, various control schemes can be used, including techniques based on feedback control, feedforward control, cascade control, and model predictive control.

[0136] In some embodiments, the bioprocessing system includes a buffer addition controller, which controls the addition of a fixed volume of buffer to the fluid (feed stream).

[0137] In some embodiments, the methods achieve buffer exchange at a constant value, a constant concentration, or both.

[0138] In some embodiments, the bioprocessing system includes a proportional-integral-derivative (PID) controller to control the transmembrane pressure and buffer addition controller of the TFF unit and to control the addition of a fixed volume of buffer to the feed solution.

[0139] In one exemplary case, the control scheme uses a combination of feedback and feedforward control in two PID controller blocks to control the recombinant protein concentration in the retentate stream. The control algorithm adjusts the transmembrane pressure (TMP) of the TFF unit by dynamically varying the backpressure regulator pressure based on the measured flow rates of any two of the feed (fluid in the inlet conduit), retentate stream (fluid in the retentate conduit), and permeate stream (fluid in the permeate conduit), and the measured recombinant therapeutic protein concentrations in the feed, retentate, and permeate streams.

[0140] Recombinant Therapeutic Proteins The term "recombinant therapeutic protein" refers to a polypeptide that is not naturally encoded by an endogenous nucleic acid present in an organism (e.g., a mammal) and that provides a therapeutic effect when administered to a subject in need thereof. Examples of recombinant therapeutic proteins include enzymes (e.g., containing one or more amino acid substitutions, deletions, insertions, or additions that result in increased stability and / or catalytic activity of the modified enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins that contain at least one recombinant scaffold sequence.

[0141] Non-limiting examples of recombinant therapeutic proteins that can be processed using the methods provided herein include immunoglobulins (including light and heavy chain immunoglobulins, antibodies, or antibody fragments (e.g., any of the antibody fragments described herein), enzymes (e.g., galactosidases (e.g., α-galactosidase), Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or interferon α or β), or immunogenic or antigenic proteins or protein fragments (e.g., proteins used in vaccines). The recombinant therapeutic protein may be an engineered antigen-binding polypeptide that contains at least one multifunctional recombinant protein scaffold (see, e.g., the recombinant antigen-binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Publication No. 2012 / 0164066, which is incorporated by reference in its entirety).Non-limiting examples of recombinant therapeutic proteins that are antibodies include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatuximab, anatubomab, anrukinzumab, apolizumab, arcitumomab, atinumab, tocilizumab, basilizumab, bectumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, bicilomab, canakinumab, certolizumab, cetuximab, cixutumumab, Daclizumab, denosumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab, imgatuzumab, infliximab, inolimomab, inotuzumab, Labetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab. Further examples of recombinant therapeutic antibodies that can be produced by the methods described herein are known in the art.Further non-limiting examples of recombinant therapeutic proteins that can be produced by the present methods include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, insulin or insulin. Phosphoanalogs, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin-11, laronidase, idursulfase, galsulfase, alpha-1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alpha (e.g., Thyrogen®), and alteplase. Further examples of recombinant therapeutic proteins that can be produced by the present methods include acid α-glucosidase, alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase (e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, N-acetylglucosamine-1-phosphotran, and acetylglucosamine-1-phosphotrans. Examples of enzymes that may be used include spherase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase, α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B. EXAMPLES

[0142] In one aspect, the UF and DF operations are divided into three steps: ultrafiltration 1 (UF1), diafiltration (DF), and the final ultrafiltration process (UF2). Ultrafiltration concentrates the recombinant protein by removing solvent and low molecular weight solutes from the solution while applying pressure to a semipermeable membrane that retains the recombinant protein. Similar to ultrafiltration, semipermeable membranes are used in diafiltration to (i) remove or reduce salt concentration or (ii) exchange buffer species of the protein solution. The original buffer species is exchanged with a new buffer species by adding the new buffer species to the protein solution in a volumetric proportion equal to that of the original buffer species removed. This condition ensures that the volume of the protein solution and its concentration remain unchanged during the process. For integrated biomanufacturing of recombinant therapeutic proteins, UF and DF can be operated in a single-pass tangential flow filtration (SP-TFF) format (Casey et al., Journal of Membrane Science, 384(2); 82-88, 2011; U.S. Pat. No. 7,384,549; and Arunkumar et al., Journal of Membrane Science, 524; 20-32, 2017). The first ultrafiltration step (UF1) involves concentrating the protein to an intermediate value suitable for diafiltration. The subsequent diafiltration step ensures buffer exchange of the protein solution. In the final ultrafiltration step (UF2), the protein is concentrated to the desired value. See Figures 1A-1C. Flux and concentration are independently measured in real time by in-line sensors, and a controller adjusts the transmembrane pressure (TMP) of the retentate stream using a backpressure regulator based on the feed and permeate fluxes and the feed and retentate concentrations to maintain uniform flow rate and retentate concentration (Figures 1D-1F).

[0143] As an example of a UF1 run, Figure 2A shows the recombinant therapeutic protein concentration as a function of time after the first ultrafiltration run. The recombinant therapeutic protein concentration remained relatively constant over time at approximately 60 g / L (Figure 2A). Figure 2B shows uniform flux / TMP values, indicating filter integrity and the absence of fouling over the course of the run. Over the course of the run, the permeate flux remained uniform (Figure 2C), and the permeate flux correlated linearly with the applied transmembrane pressure (Figure 2D). Taken together, the data in Figures 2A-2D demonstrate that the biotreatment system controlled and maintained recombinant therapeutic protein concentration and permeate flux while maintaining UF filter integrity.

[0144] The performance of consecutive diafiltration runs is shown in Figures 3A-3H. Figure 3A shows that the target DF retentate concentration can be maintained over time, while Figure 3B shows the target diavolume achieved during the run. Over the course of the run, the recombinant therapeutic protein concentration and diavolume remained relatively constant over six days. The ratio of DF buffer addition rate to permeate production rate remained relatively constant over six days (Figure 3C). The performance of the DF filter, as measured by flux / TMP, remained relatively constant over time (Figure 3D). Over the course of the run, the volumetric flux of permeate was maintained at 1.7-2.5 LMH (Figure 3E). The effect of permeate flux through the filter on transmembrane pressure was modeled and experimentally measured (Figure 3F). As shown in Figure 3F, the experimentally measured permeate flux through the filter correlated with the transmembrane pressure. The data in Figures 3G and 3H show that the boost pump used in combination with the back pressure regulator isolates pressure effects and allows subsequent runs to be integrated. Figure 3G shows that when the boost pump is taken offline (approximately days 1.4-3), the controller is unable to maintain a uniform DF retentate concentration. Figure 3H shows that with the boost pump offline, the back pressure regulator is no longer able to maintain a uniform retentate pressure. In this case, the retentate pressure is measured by the resistance to flow downstream of the run.

[0145] Data from the UF2 run are shown in Figures 4A-4D. As shown in Figure 4A, the recombinant therapeutic protein concentration remained relatively constant over time at approximately 175 g / L. In Figure 4B, the uniform flux / TMP values ​​indicate filter integrity and the absence of fouling over the course of the run. The permeate flux remained uniform over the course of the run (Figure 4C). Figure 4D shows that as the permeate flux increased, the transmembrane pressure also increased. Taken together, the data in Figures 4A-4D demonstrate that the biotreatment system controlled and maintained recombinant therapeutic protein concentration and permeate flux (at higher levels than UF1 or DF) during UF2 run.

[0146] As explained above, the combined use of a backpressure regulator and boost pump has been demonstrated to facilitate a) single-pass sequential UF and DF of recombinant therapeutic protein solutions, b) integration with other purification processes in multiple UF and / or DF operations and filter configurations (series and / or parallel), and c) robust, independent, and integrated control of each UF and DF step. As shown in the DF operation, the combined backpressure regulator and boost pump are required to maintain control. By implementing both feedback and feedforward control, it is possible to maintain uniform retentate concentration in UF / DF, retentate flow rate, and achieve the desired diavolume in the sequential diafiltration.

[0147] The combined use of the backpressure regulator and boost pump described herein has applications outside of TFF. By isolating the pressure at the outlet of a unit operation from the pressure downstream of that unit operation, the method allows for the integration of unit operations in a continuous processing train without the need for intermediate break tanks or cascade pressures throughout the system. The method can be applied between any unit operations in a conventional bioprocessing system. These key features facilitate the transition of current biomanufacturing processes to an end-to-end, integrated, and continuous framework.

[0148] Other embodiments Although the present disclosure has been described with reference to its detailed description, it will be understood that the foregoing description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for processing a fluid containing recombinant therapeutic proteins, (a) To provide a bioprocessing system comprising (i) a unit operation, (ii) at least one inlet in the unit operation, (iii) at least one outlet in the unit operation, (iv) an inlet conduit in fluid communication with at least one inlet of the unit operation, (v) an outlet conduit in fluid communication with at least one outlet of the unit operation, (vi) a back pressure regulator located in the outlet conduit, and (vii) a pressure boosting pump located in the outlet conduit downstream of the back pressure regulator; (b) flowing the fluid containing the recombinant therapeutic protein through the inlet conduit to at least one inlet of the unit operation, processing the fluid within the unit operation to obtain a processed fluid, and then flowing a portion of the processed fluid through the outlet conduit; and (c) Using the back pressure regulator and the booster pump, maintain the target flow rate and / or pressure through the outlet conduit. A method that includes this.

2. The method according to claim 1, wherein the unit operation is selected from the group consisting of cell removal unit operation, cell retention unit operation, tangential flow filtration unit operation, chromatography unit operation, virus inactivation unit operation, virus removal filtration unit operation, and compounding unit operation.

3. A method for processing a fluid containing recombinant therapeutic proteins, (a) (i) a tangential flow filtration (TFF) unit including a permeate side and a retaining liquid side, (ii) at least one inlet in the TFF unit, (iii) at least two outlets in the TFF unit, one of the at least two outlets being on the permeate side of the TFF unit and one of the at least two outlets being on the retaining liquid side of the TFF unit, (iv) an inlet conduit in fluid communication with at least one inlet of the TFF unit, (v) a permeate conduit in fluid communication with the outlet on the permeate side of the TFF unit, (vi) a retaining liquid conduit in fluid communication with the outlet on the retaining liquid side of the TFF unit, (vii) a back pressure regulator positioned in the permeate conduit or the retaining liquid conduit, and (viiii) the permeate conduit continuing downstream of the back pressure regulator To provide a bioprocessing system including a pressure boosting pump located in the peroxide conduit or the retaining liquid conduit; (b) to flow the fluid containing the recombinant therapeutic protein through the inlet conduit to at least one inlet of the TFF unit, to separate the fluid into a retaining fluid flow and a permeate flow based on the pore size or molecular weight cutoff of the TFF unit, and then to flow the permeate flow and retaining fluid flow into the permeate conduit and the retaining fluid conduit, respectively; and (c) A method comprising using the back pressure regulator and the pressure boosting pump to maintain a target flow rate and / or pressure through the permeate conduit or the retaining conduit.

4. The method according to claim 3, wherein the TFF unit has a molecular weight cutoff of about 1 kDa to about 1000 kDa or about 10 kDa to about 100 kDa.

5. The method according to claim 3, wherein the TFF unit has an average pore size of about 10 nm to about 100 nm, about 15 nm to about 35 nm, or about 0.1 μm to about 10 μm.

6. (i) The recombinant therapeutic protein in the fluid in the inlet conduit has a concentration of about 0.1 g / L to about 300 g / L, or about 0.1 g / L to about 150 g / L; and / or (ii) The recombinant therapeutic protein in the retaining fluid flow within the retaining fluid conduit has a concentration of about 0.1 g / L to about 300 g / L or about 5 g / L to about 225 g / L; and / or (iii) The recombinant therapeutic protein in the permeate flow within the permeate conduit has a concentration of about 0 g / L to about 100 g / L or about 0.1 g / L to about 25 g / L; and / or (iv) The TFF units are each approximately 50 cm 2 ~about 10m 2 Or approximately 0.1 m 2 ~about 5m 2 The method according to claim 3, comprising one or more filters having a filter area.

7. The method according to any one of claims 1 to 6, wherein the pressure boosting pump is a dynamic pump.

8. The method according to claim 7, wherein the dynamic pump is a centrifugal pump.

9. The method according to any one of claims 3 to 6, wherein step (b) comprises continuously flowing the fluid at an average rate of about 0.1 mL / min to about 10 L / min or about 1 mL / min to about 1 L / min.

10. The method according to any one of claims 3 to 6, wherein the bioprocessing system further includes one or more sensors.

11. The method according to claim 10, wherein at least one of the one or more sensors is disposed in the inlet conduit, the permeate conduit, or the retaining conduit.

12. The method according to claim 10, wherein the recombinant therapeutic protein concentration is measured using one or more sensors having a sampling time of less than approximately two minutes.

13. The method according to any one of claims 3 to 6, wherein the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

14. Claim 3, wherein the recombinant therapeutic protein concentration is measured using ultraviolet absorbance, and the ultraviolet absorbance is measured at approximately 200 nm to approximately 400 nm or approximately 260 nm to approximately 320 nm. The method described in any one of items ~6.

15. The method according to claim 13, wherein the ultraviolet absorbance is measured by a flow cell with a fixed or variable path length.

16. The method according to any one of claims 1 to 6, wherein the back pressure regulator and the booster pump function in combination to maintain a target flow rate.

17. The method according to any one of claims 1 to 6, further comprising performing one or more second unit operations on the recombinant therapeutic protein prior to step (a), wherein the one or more second unit operations are integrated upstream of the bioprocessing system such that one of the one or more second unit operations is in fluid communication with the inlet conduit of the bioprocessing system.

18. The method according to claim 17, wherein the one or more second unit operations are selected from the group consisting of bioreactor unit operations, cell removal unit operations, cell retention unit operations, chromatography unit operations, virus inactivation unit operations, virus removal unit operations, ultrafiltration unit operations, and dialysis filtration unit operations.

19. The method according to any one of claims 3 to 6, further comprising, after step (c), performing one or more second unit operations such that one of the second unit operations is in fluid communication with the permeate conduit or the retaining fluid conduit of the bioprocessing system.

20. The method according to claim 19, wherein the one or more second unit operations are chromatography unit operations, virus inactivation unit operations, virus removal unit operations, ultrafiltration unit operations, dialysis filtration unit operations, or compounding unit operations.

21. A bioprocessing system, (i) Unit operation; (ii) at least one inlet in the operation of the unit; (iii) at least one outlet in the operation of the unit; (iv) an inlet conduit that is in fluid communication with at least one inlet of the unit operation; (v) an outlet conduit that is in fluid communication with at least one outlet of the unit operation; (vi) A back pressure regulator located in the outlet conduit, and (vii) A pressure boosting pump located in the outlet conduit downstream of the back pressure regulator. Includes, The bioprocessing system is configured to flow a fluid containing the recombinant therapeutic protein through the inlet conduit to at least one inlet of the unit operation, process the fluid within the unit operation to obtain a processed fluid, and then flow a portion of the processed fluid through the outlet conduit. A bioprocessing system configured such that the back pressure regulator and the pressure boosting pump maintain a target flow rate and / or pressure through the outlet conduit.

22. A bioprocessing system, (i) A tangential flow filtration (TFF) unit having at least one inlet and at least two outlets, wherein one of the at least two outlets is on the permeate side of the TFF unit and one of the at least two outlets is on the retaining liquid side of the TFF unit; (ii) an inlet conduit that is in fluid communication with the at least one inlet of the TFF unit; (iii) A permeate conduit that is in fluid communication with the outlet on the permeate side of the TFF unit; (iv) A retaining fluid conduit that is in fluid communication with the outlet on the retaining fluid side of the TFF unit; (v) Back pressure regulators positioned in the permeate conduit or the retaining conduit; and (vi) A pressure boosting pump located in the permeate conduit or retaining conduit downstream of the back pressure regulator. Includes, The bioprocessing system is configured to flow a fluid containing recombinant therapeutic protein through the inlet conduit to at least one inlet of the TFF unit, separate the fluid into a retaining fluid flow and a permeate flow based on the pore size or molecular weight cutoff of the TFF unit, and then flow the retaining fluid flow and the permeate flow into the permeate conduit and the retaining fluid conduit; A bioprocessing system configured to maintain a target flow rate and / or pressure through the back pressure regulator and the pressure boosting pump via the permeate conduit or the retaining conduit.

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