Method of treating a fluid

Abstract

Provided herein are methods of treating a fluid comprising a recombinant therapeutic protein, the method comprising the use of a biological treatment system comprising integrated unit operations including cell removal, cell retention, chromatography, viral inactivation, viral filtration, TFF, and formulation. Biological treatment systems comprising these unit operations are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 322,133 filed on March 21, 2022; the entire contents of that patent application are incorporated herein by reference. Technical Field

[0003] The present invention relates to biotechnological methods and the bioproduction of recombinant therapeutic proteins. Background Art

[0004] Mammalian cells containing nucleic acids encoding recombinant therapeutic proteins are routinely used to produce therapeutically or commercially important proteins. In the current environment of diverse product pipelines, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing of therapeutic protein drug substances. Summary of the invention

[0005] The present invention is based at least in part on the discovery that applying a back pressure regulator (BPR) in combination with a booster pump (e.g., a centrifugal pump) to a tangential flow filtration (TFF) unit operation can achieve and maintain a target retentate concentration and maintain the flow rates of retentate and permeate in ultrafiltration operations (UF) and diafiltration (DF) operations.

[0006] Provided herein are methods for treating a fluid containing a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system comprising: (i) a unit operation, (ii) at least one inlet on the unit operation, (iii) at least one outlet on 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 booster pump disposed in the outlet conduit, the booster pump following downstream from the backpressure regulator; (b) flowing a fluid containing a recombinant therapeutic protein through the inlet conduit and into the at least one inlet of the unit operation, treating the fluid within the unit operation to obtain a treated fluid, and then flowing a portion of the treated fluid into the outlet conduit; and (c) maintaining a target flow and / or pressure through the outlet conduit using the backpressure regulator and the booster pump.

[0007] 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 formulation unit operation.

[0008] Also provided herein are methods of treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 flow cell in fluid communication with the TFF unit; The invention relates to a method of treating a TFF unit comprising: providing a recombinant therapeutic protein having an outlet on the retentate side of the unit in a fluidically connected retentate conduit, (vii) a backpressure regulator disposed in the permeate conduit or the retentate conduit, and (viii) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream from the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the booster pump.

[0009] In some embodiments, the TFF unit has a molecular weight cutoff of from about 1 kDa to about 1000 kDa. In some embodiments, the TFF unit has a molecular weight cutoff of from about 10 kDa to about 100 kDa.

[0010] In some embodiments, the TFF unit has an average pore size from about 10 nm to about 100 nm. In some embodiments, the TFF unit has an average pore size from about 15 nm to about 35 nm. In some embodiments, the TFF unit has an average pore size from about 0.1 μm to about 10 μm.

[0011] In some embodiments, the recombinant therapeutic protein in the fluid (also referred to as the feed stream) in the inlet conduit has a concentration of from about 0.1 g / L to about 300 g / L. In some embodiments, the recombinant therapeutic protein in the feed stream in the inlet conduit has a concentration of from about 0.1 g / L to about 150 g / L.

[0012] In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration from about 0.1 g / L to about 300 g / L.

[0013] In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration from about 5 g / L to about 225 g / L.

[0014] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration from about 0 g / L to about 100 g / L. In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration from about 0.1 g / L to about 25 g / L.

[0015] In some embodiments, the TFF unit includes one or more filters, each of which has a diameter of about 50 cm. 2 About 10m 2 In some embodiments, the TFF unit includes one or more filters, each of which has a filter area of ​​about 0.1 m 2 About 5m 2 filter area.

[0016] In some embodiments, the boost pump is a dynamic pump. In some embodiments, the dynamic pump is a centrifugal pump.

[0017] In some embodiments, step (b) comprises continuously flowing the fluid at an average rate of from about 0.1 mL / min to about 10 L / min.

[0018] In some embodiments, step (b) comprises continuously flowing the fluid at an average rate of from about 1 mL / minute to about 1 L / minute.

[0019] In some embodiments, the bioprocessing system further comprises one or more sensors.

[0020] 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.

[0021] In some embodiments, at least one sensor of the one or more sensors is disposed in the retentate conduit.

[0022] 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.

[0023] In some embodiments, recombinant therapeutic protein concentration is measured using UV absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

[0024] In some embodiments, UV absorbance is used to measure the recombinant therapeutic protein concentration, and wherein the UV absorbance is measured between about 200 nm and about 400 nm.

[0025] In some embodiments, UV absorbance is used to measure the recombinant therapeutic protein concentration, and wherein the UV absorbance is measured between about 260 nm and about 320 nm.

[0026] In some embodiments, UV absorbance is measured using a flow cell with a fixed path length.

[0027] In some embodiments, UV absorbance is measured using a flow cell with a variable path length.

[0028] In some embodiments, the method further comprises, prior to step (a), performing one or more second unit operations on the recombinant therapeutic protein, 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.

[0029] 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.

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

[0031] In some embodiments of any of the methods described herein, the method further includes, after step (c), performing one or more second unit operations, wherein the one or more second unit operations are integrated downstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with a retentate conduit of the biological treatment system.

[0032] 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 formulation unit operation.

[0033] Provided herein are bioprocessing systems comprising: (i) a unit operation; (ii) at least one inlet on the unit operation; (iii) at least one outlet on 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 booster pump disposed in the outlet conduit, the booster pump following downstream of the backpressure regulator, wherein: the bioprocessing system is configured to flow a fluid containing a recombinant therapeutic protein through the inlet conduit and into the 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 into the outlet conduit, and the backpressure regulator and the booster pump are configured to maintain a target flow and / or pressure through the outlet conduit.

[0034] 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 formulation unit operation.

[0035] Also provided herein are bioprocessing systems comprising: (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 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 retentate conduit disposed in fluid communication with an outlet on the retentate side of the TFF unit; a backpressure regulator in the permeate conduit or the retentate conduit, and (vi) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream of the backpressure regulator, wherein: the bioprocessing system is configured to flow a fluid comprising a recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit; and the backpressure regulator and the booster pump are configured to maintain a target flow and / or pressure through the permeate conduit or the retentate conduit.

[0036] In some embodiments, the TFF unit has a molecular weight cutoff of from about 1 kDa to about 1000 kDa. In some embodiments, the TFF unit has a molecular weight cutoff of from about 10 kDa to about 100 kDa. In some embodiments, the TFF unit has an average pore size of from about 10 nm to about 100 nm. In some embodiments, the TFF unit has an average pore size of from about 15 nm to about 35 nm. In some embodiments, the TFF unit has an average pore size of from about 0.1 μm to about 10 μm.

[0037] 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 from about 0.1 g / L to about 300 g / L.

[0038] In some embodiments, the recombinant therapeutic protein in the fluid in the inlet conduit has a concentration from about 0.1 g / L to about 150 g / L.

[0039] In some embodiments of any of the bioprocessing systems described herein, the recombinant therapeutic protein in the retentate stream has a concentration from about 0.1 g / L to about 300 g / L. In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration from about 5 g / L to about 225 g / L.

[0040] In some embodiments of any of the bioprocessing systems described herein, the recombinant therapeutic protein in the permeate stream has a concentration from about 0 g / L to about 100 g / L.

[0041] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration from about 0.1 g / L to about 25 g / L.

[0042] In some embodiments of any of the bioprocessing systems described herein, the TFF unit comprises one or more filters, each of which has a diameter of about 50 cm. 2 About 10m 2 filter area.

[0043] In some embodiments, the TFF unit includes one or more filters, each of which has a thickness of about 0.1 m 2 About 5m 2 filter area.

[0044] 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.

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

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

[0047] In some embodiments of any of the bioprocessing systems described herein, at least one sensor of the one or more sensors is disposed in the retentate conduit.

[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 with a sampling time of less than about 2 minutes.

[0049] In some embodiments of any of the bioprocessing systems described herein, one or more sensors are configured to measure the recombinant therapeutic protein concentration using UV absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

[0050] In some embodiments, one or more sensors are configured to measure recombinant therapeutic protein concentration using UV absorbance between about 200 nm to about 400 nm.

[0051] In some embodiments, one or more sensors are configured to measure recombinant therapeutic protein concentration using UV absorbance between about 260 nm and about 320 nm.

[0052] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors comprises a flow cell having a fixed path length.

[0053] In some embodiments of any of the bioprocessing systems described herein, at least one of the one or more sensors comprises a flow cell having a variable path length.

[0054] 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.

[0055] 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.

[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 a permeate conduit of the bioprocessing system.

[0057] 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 a retentate conduit of the bioprocessing system.

[0058] 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 formulation unit operation.

[0059] As used herein, the word "a" before a noun means one or more specific nouns. For example, the phrase "amammalian cell" means "one or more mammalian cells".

[0060] The term "tangential flow filtration unit" or "TFF unit" is known in the art and refers to a device comprising at least one housing (such as a cylinder) and at least one cross-flow (tangential) filter, which is positioned in the housing so that a majority of the surface of the filter is positioned parallel to the flow of a fluid (e.g., a cell culture) through the unit. TFF units are well known in the art and are commercially available. The housing may include a first inlet / outlet and a second inlet / outlet, which are positioned to, for example, allow a fluid to pass through the first inlet / outlet, across at least one cross-flow filter and through the second inlet / outlet. In some examples, a bioprocessing system may include, for example, a plurality of TFF units connected in series and / or in parallel. For example, a bioprocessing system comprising two or more TFF units may include a fluid conduit that fluidically connects adjacent pairs of TFF units in the system. In other examples, a bioprocessing system may include two or more groups of two or more TFF units fluidly connected by a fluid conduit. Any TFF unit described herein or known in the art is capable of receiving fluid in a first flow direction and a second flow direction.

[0061] The term "cross-flow filter" or "tangential filter" is known in the prior art, and refers to a filter designed so that it can be positioned in a TFF unit so that the majority of the surface of the filter is parallel to the flow of the fluid (e.g., the fluid comprising recombinant therapeutic proteins). For example, the cross-flow filter can have any shape that allows tangential flow filtration, such as a tubular shape or a rectangular shape. Particularly useful cross-flow filters are designed to generate a small amount of fluid turbulence or shear stress in the fluid when fluid flows (e.g., unidirectional flow to bidirectional flow) across the surface of the cross-flow filter. Cross-flow filters can be commercially available, for example, from Sartorius, MembraPure, Millipore, and Pall Corporation.

[0062] The terms "ultrafiltration" and "UF" and similar terms refer to the use of synthetic semipermeable membranes with appropriate physical and chemical properties to separate between molecules in a mixture (primarily based on molecular size and shape) and to achieve separation of different molecules or concentration of similar molecules.

[0063] The terms "diafiltration" and "DF" and similar terms refer to the use of ultrafiltration membranes to remove, replace or reduce the concentration of salts or solvents from a solution or mixture containing proteins, peptides, nucleic acids or other biomolecules.

[0064] The term "permeate stream" or "permeate" refers to the flow of fluid leaving the TFF unit after passing through the TFF filter. Fluid components selectively retained by the filter will not be present in the permeate. In some embodiments (e.g., ultrafiltration or diafiltration), the permeate will not contain recombinant therapeutic proteins. In some embodiments (e.g., viral filtration or microfiltration), the permeate will contain recombinant therapeutic proteins but will not contain other impurities (e.g., viral particles or cell debris) present in the TFF feed material.

[0065] The term "retentate stream" or "retentate" refers to the stream of fluid that leaves the TFF unit without passing through the TFF filter. The fluid components selectively retained by the filter will be present in the retentate. In some embodiments (e.g., ultrafiltration), the fluid components selectively retained by the filter will be enriched in the retentate.

[0066] The term "transmembrane pressure" or "TMP" refers to the pressure from the feed to the filtrate side of the membrane and is calculated as TMP [bar] = [(P F +P R ) / 2]-P P The average applied pressure. F is the feed pressure, P R is the retentate pressure, P P is the permeate pressure.

[0067] The term "kinetic pump" or "rotodynamic pump" refers to a type of speed pump in which kinetic energy is continuously transferred to the pumped fluid by a rotating impeller, propeller or rotor.

[0068] The term "centrifugal pump" refers to a dynamic pump that uses an impeller with blades having a substantially radial output to transfer rotational mechanical energy to a fluid primarily by increasing the fluid's kinetic energy (angular momentum) and also increasing potential energy (static pressure).

[0069] The term "mammalian cell" refers to any cell from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cattle, or rabbit). For example, the mammalian cell can 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. Additional examples of mammalian cells are known in the art.

[0070] 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 the process of manufacturing a therapeutic protein drug substance from a liquid culture medium. For example, when used as a verb, a unit operation can be filtration (e.g., removing contaminant bacteria, yeast viruses or mycobacteria and / or particulate matter from a fluid containing a recombinant therapeutic protein), capture, epitope tag removal, purification, holding or storage, polishing, viral inactivation, adjusting the ion concentration and / or pH of a fluid containing a recombinant therapeutic protein, and removing unwanted salts.

[0071] The term "unit operation" is a term of art and, when used as a noun, refers to an instrument that can be used to perform a functional step in the process of manufacturing a therapeutic protein drug substance from a liquid culture medium. For example, when used as a noun, a unit operation can include a chromatography system (e.g., a single column or multi-column chromatography system, a periodic 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 virus inactivation unit operation, a virus removal filtration unit operation, an ultrafiltration unit operation, a diafiltration unit operation, or a formulation unit operation.

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

[0073] The term "continuous process" refers to a process in which a fluid is continuously fed through at least a portion of a system.

[0074] The term "integrated process" refers to a process performed using structural elements that work synergistically to achieve a specific outcome (eg, production of a therapeutic protein drug substance from a liquid culture medium).

[0075] The term "bioprocessing system" refers to a system used to prepare, produce or manufacture biologics from liquid culture medium. A bioprocessing system may have one or more unit operations.

[0076] The term "upstream" refers to performing one or more unit operations before the timing of performing one or more other unit operations. The term "downstream" refers to performing one or more unit operations after the timing of performing one or more other unit operations.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. These 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 the event of a conflict, the present specification, including definitions, shall prevail.

[0078] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figures 1A to 1F An overview and control strategy for a first single pass continuous tangential flow filtration (SP-TFF) or ultrafiltration 1 (UF1) operation, a single pass continuous diafiltration (SP-DF) operation, and a second single pass continuous tangential flow filtration (SP-TFF) or ultrafiltration 2 (UF2) operation are shown, respectively. Figure 1A As shown, the fluid containing the recombinant therapeutic protein is transported from the buffer container to the first ultrafiltration (UF) unit. The online flow meters, pressure sensors and refractive index sensors in the feed stream, retentate stream and permeate stream respectively measure the flow value, pressure value and concentration value in real time. The back pressure regulator (BPR) installed on the retentate stream controls the pressure applied to the filter, thereby concentrating the product. Figure 1B In the process, a booster pump directs the emerging concentrated product stream into the SP-DF unit. Simultaneously, the diafiltration (DF) buffer enters the DF unit. The BPR maintains a constant retentate concentration by adjusting the permeate flux through the filter based on real-time fluid properties measured using online sensors. Figure 1C The final UF2 operation is shown, which involves concentrating the recombinant therapeutic protein to a value above the final DS using a single UF unit. Figure 1DA control strategy for a continuous single pass ultrafiltration (SP-TFF) operation is shown, where a concentration controller is implemented to control the retentate stream concentration based on real-time 1) feed concentration and retentate concentration and 2) feed flux and permeate flux. Figure 1E and Figure 1F A control strategy for continuous single pass diafiltration (SP-DF) operation is shown. Figure 1E A strategy is shown for implementing a concentration controller that controls the retentate stream concentration based on real-time 1) feed concentration and retentate concentration and 2) feed flux and permeate flux. Figure 1F A control strategy is shown for implementing a buffer addition controller that controls fixed volume addition of buffer to a feed based on 1) a calculated buffer flow rate and 2) a real-time buffer flow rate.

[0080] FIG. 2A to FIG. 2D Concerning the first ultrafiltration (UF1) operation. Figure 2A is a graph showing the change in recombinant therapeutic protein concentration (g / L) over time (days) after the first ultrafiltration operation. Figure 2B is a graph showing the flux / TMP value as a function of time (days). Figure 2C is a graph showing the variation of permeate flux (LMH) over time (days). Figure 2D is a graph showing permeate flux as a function of applied transmembrane pressure (psi).

[0081] 3A to 3H is a plot showing the performance of a continuous diafiltration operation. Figure 3A is a graph showing target diafiltration (DF) retentate concentration (g / L) versus time (days). Figure 3B is a graph showing achieved and target diavolume over time (days). Figure 3C is a graph showing the ratio of diafiltration (DF) buffer addition rate to permeate production rate over time (days). Figure 3D is a graph showing flux / TMP values ​​over time (days). Figure 3E The permeate volume flux (LMH) is shown over time (days) during diafiltration. Figure 3F is a graph showing the effect of permeate flux (LMH) on transmembrane pressure (psi). Figure 3G is a graph showing the effect of a boost pump used in combination with a back pressure regulator on retentate concentration (g / L) over time (days). Figure 3H is a graph showing the effect of a boost pump used in combination with a backpressure regulator on retentate pressure (psi) as a function of the pressure (psi) of the backpressure regulator.

[0082] 4A to 4Dis a graph showing the performance of the final SP-TFF ultrafiltration run (UF2) of the integrated manufacturing of recombinant therapeutic proteins. Figure 4A is a graph showing the retentate protein concentration after ultrafiltration over time (days). Figure 4B is a graph showing filter performance (flux / TMP) over time (days) during operation. Figure 4C is a graph showing the variation of permeate flux (LMH) over time (days). Figure 4D is a graph showing permeate flux as a function of transmembrane pressure (psi). DETAILED DESCRIPTION

[0083] Provided herein are methods and bioprocessing systems for treating fluids. Non-limiting aspects of these methods and bioprocessing systems are described herein.

[0084] Methods of handling fluids

[0085] Provided herein are methods for treating a fluid containing a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system comprising: (i) a unit operation, (ii) at least one inlet on the unit operation, (iii) at least one outlet on 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 booster pump disposed in the outlet conduit, the booster pump following downstream from the backpressure regulator; (b) flowing a fluid containing a recombinant therapeutic protein through the inlet conduit and into the at least one inlet of the unit operation, treating the fluid within the unit operation to obtain a treated fluid, and then flowing a portion of the treated fluid into the outlet conduit; and (c) maintaining a target flow and / or pressure through the outlet conduit using the backpressure regulator and the booster pump.

[0086] Provided herein are methods for treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 flow cell in fluid communication with the TFF unit, and (vii) a flow cell in fluid communication with the TFF unit. The invention relates to a method of treating a TFF unit comprising: providing a recombinant therapeutic protein having an outlet on the retentate side of the unit in a fluidically connected retentate conduit, (vii) a backpressure regulator disposed in the permeate conduit or the retentate conduit, and (viii) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream from the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through an inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the booster pump.

[0087] Provided herein are methods for treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 retentate conduit in fluid communication with an outlet on the retentate side of the TFF unit, (vii) a backpressure regulator disposed in the permeate conduit, and (viii) a booster pump disposed in the permeate conduit, the booster pump following downstream of the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit using the backpressure regulator and the booster pump.

[0088] Provided herein are methods for treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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, (viii) a flow cell in fluid communication with the flow cell; i) 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 booster pump disposed in the retentate conduit, the booster pump following downstream of the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the booster pump.

[0089] Provided herein are methods for treating a fluid containing a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 recombinant flow filtration conduit in fluid communication with an outlet on the retentate side of the TFF unit, and (vii) a recombinant flow filtration conduit in fluid communication with an outlet on the retentate side of the TFF unit. The invention relates to a method of manufacturing a TFF unit comprising: providing a retentate conduit connected to a TFF unit, (vii) a backpressure regulator disposed in the retentate conduit, and (viii) a booster pump disposed in the retentate conduit, the booster pump following downstream of the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams 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 conduit or the retentate conduit using the backpressure regulator and the booster pump; wherein the TFF unit has an average pore size of from about 15 nm to about 35 nm or from about 1 μm to about 10 μm.

[0090] Provided herein are methods for treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system, the bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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) providing a a backpressure regulator in the retentate conduit, and (viii) a booster pump disposed in the retentate conduit, the booster pump following downstream of the backpressure regulator; (b) flowing a fluid comprising a recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the booster pump; wherein the TFF unit has a molecular weight cutoff of from about 1 kDa to about 100 kDa, and optionally, the recombinant therapeutic protein concentration in the retentate conduit is maintained constant at about 5 g / L to about 250 g / L.

[0091] Provided herein are methods for treating a fluid comprising a recombinant therapeutic protein, the methods comprising: (a) providing a bioprocessing system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 booster pump disposed in the retentate conduit, the booster pump following downstream of the backpressure regulator, (i x) at least three sensors, wherein one sensor of the at least three sensors is disposed in the inlet conduit and one sensor is disposed in the permeate conduit; one sensor is disposed in the retentate conduit; and (x) a feedback and feedforward controller connected to a backpressure regulator; (b) allowing a fluid containing a recombinant therapeutic protein to flow through the inlet conduit and into at least one inlet of the TFF unit, and 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 allowing those permeate streams and retentate streams to flow into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow 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 value from about 1 kDa to about 100 kDa, and optionally, by controlling the pressure on the backpressure regulator, the concentration of the recombinant therapeutic protein in the retentate conduit is maintained constant at about 5 g / L to 250 g / L in real time.

[0092] 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 formulation unit operation.

[0093] 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 with and in fluid communication with one or more of the inlets on the bioprocessing system described in step (a), and 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 formulation unit.

[0094] In some embodiments, the method further comprises, after step (c), performing one or more second unit operations, wherein the one or more second unit operations are integrated downstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with a permeate conduit of the biological treatment system.

[0095] In some embodiments, the method further comprises, after step (c), performing one or more second unit operations such that one of the one or more second unit operations is in fluid communication with a retentate conduit of the biological treatment system.

[0096] In some embodiments, the method further comprises, after step (c), performing one or more second unit operations, 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 formulation unit operation.

[0097] In some embodiments, the TFF unit includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve tangential filters. In some examples of these methods, the TFF unit may include one or more tangential filters having a diameter of about 50 cm. 2 About 10m 2 , (e.g., about 50cm 2 About 0.1m 2 、Approx. 50cm 2 About 0.5m 2 、About 50cm 2 About 1m 2 、Approx. 50cm 2 About 5m 2 、About 50cm 2 About 10m 2 , about 0.1m 2 About 0.5m 2 , about 0.1m 2 About 1m 2 , about 0.1m 2 About 5m 2 , about 0.1m 2 About 10m 2 , about 0.5m 2 About 1m 2 , about 0.5m 2 About 5m 2 , about 0.5m 2 About 10m 2 , about 1m2 About 5m 2 , about 1m 2 About 10m 2 , or about 5m 2 About 10m 2 ) surface area. In some embodiments, the TFF unit includes one or more filters, each of which has a surface area of ​​about 0.1 m 2 About 5m 2 filter area.

[0098] In some embodiments, the TFF unit (one or more tangential filters in the TFF unit) has an average pore size from 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).

[0099] For example, a TFF unit comprising one or more tangential filters having an average pore size of about 10 nm to about 100 nm can be used to perform virus filtration.

[0100] In some embodiments, the TFF unit (the one or more tangential filters in the TFF unit) has a diameter from 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 0.1 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 In some embodiments, the TFF unit has an average pore size of from about 10 nm to about 100 nm. In some embodiments, the TFF unit has an average pore size of from about 15 nm to about 35 nm.

[0101] For example, a TFF unit comprising one or more tangential filters having an average pore size of about 0.1 μm to about 10 μm can be used to perform microfiltration.

[0102] In some embodiments, one or more tangential filters in a TFF unit can have a diameter of 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 300 kDa, In some embodiments, the TFF unit has a molecular weight cut-off of from about 10 kDa to about 100 kDa. In some embodiments, the TFF unit has a molecular weight cut-off of from about 10 kDa to about 100 kDa. In some embodiments, the TFF unit has a molecular weight cut-off of from about 10 kDa to about 100 kDa.

[0103] For example, a TFF unit comprising 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.

[0104] 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 a TFF unit are commercially available from Millipore (e.g., and ). Additional commercial sources of one or more tangential filters that can be used in a TFF unit are known in the art.

[0105] In some embodiments, the recombinant therapeutic protein in the fluid (also referred to as the feed stream) has 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 300 g / L / 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).

[0106] In some embodiments, the recombinant therapeutic protein in the permeate stream has a concentration from 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 from about 0.1 g / L to about 25 g / L. In some embodiments, the recombinant therapeutic protein is not present in the permeate stream.

[0107] In some embodiments, the recombinant therapeutic protein in the retentate stream has a concentration of from about 0.1 g / L to about 300 g / L (e.g., about 0.1 g / L to about 5 g / L, about 0.1 g / L to about 25 g / L, about 0.1 g / L to about 50 g / L, about 0.1 g / L to about 100 g / L, about 0.1 g / L to about 150 g / L, about 0.1 g / L to about 225 g / L, about 0.1 g / L to about 300 g / L, about 5 g / L to about 25 g / L, about 5 g / L to about 50 g / L, about 5 g / L to about 100 g / L, about 5 g / L to about 150 g / L, about 5 g / L to about 225 g / L, about 5 g / L to about 300 g / L, g / L, about 100 g / L to about 225 g / L, about 100 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 from about 5 g / L to about 225 g / L.

[0108] In some examples of any of the methods described herein, step (b) includes flowing the fluid comprising the recombinant therapeutic protein at a rate of between 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 10 L / min, In some embodiments, step (b) comprises flowing (e.g., continuously) the fluid at an average rate of from about 1 mL / min to about 1 L / min.

[0109] In some embodiments, the fluid comprises a diafiltration medium. Non-limiting examples of diafiltration media can be, for example, a physiologically acceptable buffer (e.g., phosphate buffered saline) or a buffer for formulating a recombinant therapeutic protein (e.g., a buffer comprising 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)).

[0110] 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.

[0111] In some embodiments, where the TFF unit comprises a virus filter, the method can further comprise recovering a permeate stream containing the recombinant therapeutic protein from the permeate conduit.

[0112] In some embodiments, where the TFF unit comprises a microfilter, the method can further comprise recovering a permeate stream containing the recombinant therapeutic protein from the permeate conduit.

[0113] In some embodiments, where the TFF unit is an ultrafiltration or diafiltration filter, the method can further comprise recovering a retentate stream containing the recombinant therapeutic protein from the retentate conduit.

[0114] In some embodiments, the boost pump can be a dynamic pump (eg, a centrifugal pump). In some embodiments, the dynamic pump is a centrifugal pump.

[0115] 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 an inlet conduit. In some embodiments, at least one of the one or more sensors is disposed in a permeate conduit. In some embodiments, at least one of the one or more sensors is disposed in a retentate conduit. In some embodiments, at least one of the one or more sensors is disposed in an outlet conduit.

[0116] 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).

[0117] In some embodiments, therapeutic protein concentration is measured using UV absorbance, refractive index, Fourier transform infrared spectroscopy (FITR), or Raman spectroscopy.

[0118] In some embodiments, the recombinant therapeutic protein concentration is measured using UV absorbance between about 200 nm to about 400 nm. In some embodiments, the recombinant therapeutic protein concentration is measured using UV absorbance, and wherein the UV absorbance is measured between about 260 nm to about 320 nm. In some embodiments, the UV absorbance is measured using a flow cell with a fixed path length. In some embodiments, the UV absorbance is measured using a flow cell with a variable path length.

[0119] In some embodiments, the bioprocessing system achieves from about 5 to about 15 volume exchanges of buffer. In some embodiments, the bioprocessing system achieves about 5-7 volume exchanges of buffer.

[0120] In some embodiments, the flux ratio transmembrane pressure (TMP) is maintained at 0.5 to 1.0 for at least five days (e.g., at least six days, at least seven days, at least eight days, at least nine days, or at least ten days). In some embodiments, the flux ratio transmembrane pressure (TMP) is maintained at 0.6 to 0.7 for at least five days (e.g., at least six days, at least seven days, at least eight days, at least nine days, or at least ten days). In some embodiments, the flux ratio transmembrane pressure (TMP) is maintained at a constant value for at least five days (e.g., at least six days, at least seven days, at least eight days, at least nine days, or at least ten days).

[0121] In some embodiments, the method maintains a target concentration of a 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 a recombinant therapeutic protein in the retentate stream of the TFF unit in real time over about 3 days to about 8 weeks. In some embodiments, the method maintains a target concentration of a recombinant therapeutic protein in the retentate stream of the TFF unit in real time over about 4 weeks.

[0122] Exemplary Ultrafiltration Methods

[0123] In the continuous format, the ultrafiltration operation is divided into two steps: ultrafiltration 1 (UF1) and ultrafiltration 2 (UF2), with an intermediate diafiltration (DF) step. The protein solution is fed as input to UF1. Two 0.5 m 2 The ultrafiltration unit concentrates the protein solution to an intermediate value suitable for diafiltration. This configuration allows for maximum sweep of the inlet flow, thereby preventing concentration polarization and fouling on the membrane surface. In brief, the entire UF1 operation is performed as follows: a quaternary diaphragm pump (Quattroflow Fluid Systems, QF 150S, PSG Germany GmbH) is used to pump the inlet protein solution from a buffer vessel (5 L, Cercell) to a 0.5 m 2The inlet of single pass tangential flow filtration (SP-TFF) unit (30kDa molecular weight cut-off (MWCO), Pellicon capsule with Ultracel membrane, C-screen). The volume flow of feed stream is automatically adjusted using the liquid level controller on the buffer container. Subsequently, the retentate stream from the first UF1 unit is fed to the inlet of the second UF1 unit. In this whole process, the protein concentration in the retentate is maintained at the target value using the proportional integral differential (PID) controller using the combination of feedback and feedforward control. The PID controller uses a back pressure regulator (BPR, Equilibar, Precision Fluid Control) to adjust the transmembrane pressure (TMP) on the retentate stream of the second UF1 unit based on feed and permeate flux and feed and retentate concentration. These fluxes and concentrations are measured independently in real time by online disposable flowmeters (Levitronix Technology Company) and refractive index (RI) sensors (mPath IoR flow tanks, Corporation Life Sciences). Finally, the retentate stream of the second UF1 unit is continuously fed to the downstream diafiltration unit via a booster pump (PuraLev i100SU, Levitronix Technologies), which is essentially a centrifugal pump. The booster pump minimizes the effect of upstream pressure on downstream operations and ensures constant and uninterrupted flow to the downstream unit. Therefore, the combination of BPR and booster pump is essential for the robust control of UF1.

[0124] In the second continuous ultrafiltration step (UF2), the buffer-exchanged protein from the DF step is further concentrated to a value above the desired drug substance (DS). The operating principle and control strategy are the same as in UF1. However, the main difference between them is the use of a 0.5 m 2 A single pass tangential flow filtration (SP-TFF) unit (30 kDa molecular weight cutoff (MWCO), Pellicon capsules with Ultracel membrane, C-Screen) replaced the two units as described in UF1. After the concentration process, a booster pump directed the product to the formulation operation.

[0125] Exemplary Diafiltration Methods

[0126] 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 wash volumes (DV) of DF buffer (10 mM sodium acetate, pH 4.5). To achieve the online buffer exchange, the concentrated protein stream from the second UF1 unit is continuously fed (via the previously mentioned booster pump) to a 1.6 m 2The inlet of single-pass diafiltration box (Hydrosart ultrafilter, 30kDa molecular weight cutoff (MWCO), Sartorius). Meanwhile, a peristaltic pump (Watson-Marlow company) is fed with a volume flow rate equivalent to 5DV (5 times of feed flow) based on the online flow measurement (Levitronix Technology Company) of the feed stream. The continuous DF operation is designed to make the rate at which the DF buffer is added to the system equal to the rate at which the permeate is removed from the system. This condition ensures that the protein concentration does not change during the process. The single-pass continuous DF strategy is implemented by implementing two controllers, i.e. 1) a concentration controller maintaining a consistent retentate concentration and 2) a buffer addition controller, which ensures that a fixed volume of buffer is added to the feed, i.e., the desired wash volume. In order to maintain a consistent retentate concentration, a proportional integral differential (PID) controller using a combination of feedback and feedforward control is implemented to control the TMP on the DF membrane. Similar to UF1, the controller uses a back pressure regulator based on feed and permeate flux (disposable flow meter, Levitronix Technology Company) and feed and retentate concentration (RI sensor, mPath IoR flow tank, Corporation Life Sciences) online measurement to adjust TMP. In order to achieve the desired wash volume, the volume flow of DF buffer is calculated in real time based on the feed flow rate and the permeate flux through the DF box. Subsequently, another PID controller dynamically adjusts to the set point of the peristaltic pump to change the buffer addition rate. The two controllers work together to ensure that the diafiltration is robustly controlled and operated in a continuous single-pass form. For the same reasons outlined in UF1, a booster pump (PuraLev i100SU, Levitronix Technology Company) is used to feed the final outlet flow to the next ultrafiltration unit (UF2) constantly. As in UF1, BPR and booster pumps work together to achieve the robust integrated operation of single-pass DF.

[0127] Biological treatment system

[0128] Also provided herein is a bioprocessing system comprising: (i) a unit operation; (ii) at least one inlet on the unit operation; (iii) at least one outlet on 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 backpressure regulator disposed in the outlet conduit, and (vii) a booster pump disposed in the outlet conduit, the booster pump following downstream of the backpressure regulator, wherein: the bioprocessing system is configured to cause a fluid containing a recombinant therapeutic protein to flow through the inlet conduit and into at least one inlet of the unit operation, to process the fluid within the unit operation to obtain a processed fluid, and then to cause a portion of the processed fluid to flow into the outlet conduit; and the backpressure regulator and the booster pump are configured to maintain a target flow and / or pressure through the outlet conduit.

[0129] Also provided herein is a bioprocessing system, the bioprocessing system comprising: (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 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 fluid communication with an outlet on the retentate side of the TFF unit; a backpressure regulator in the permeate conduit or the retentate conduit, and (vi) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream of the backpressure regulator, wherein: the bioprocessing system is configured to flow a fluid containing a recombinant therapeutic protein through the inlet conduit and into the at least one inlet of the TFF unit, and 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 those permeate streams and retentate streams into the permeate conduit and the retentate conduit; and the backpressure regulator and the booster pump are configured to maintain a target flow and / or pressure through the retentate conduit.

[0130] In some embodiments, the bioprocessing system comprises 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 formulation unit operation.

[0131] In some embodiments, the bioprocessing system further comprises one or more additional unit operations, such that the one or more additional unit operations are integrated with and in fluid communication with one or more of the inlets on the unit operation, and the one or more additional unit operations are 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 formulation unit operation.

[0132] 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.

[0133] In some embodiments, the bioprocessing system comprises 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.

[0134] In some embodiments, the biological treatment system includes a second unit operation integrated downstream of the biological treatment system such that the second unit operation is in fluid communication with a permeate conduit of the biological treatment system.

[0135] In some embodiments, the biological treatment system includes a second unit operation integrated downstream of the biological treatment system such that the second unit operation is in fluid communication with a retentate conduit of the biological treatment system.

[0136] In some embodiments, the bioprocessing system comprises a second unit operation integrated downstream of the bioprocessing system, wherein the second unit operation is 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 formulation operation.

[0137] In some embodiments of any of the bioprocessing systems described herein, the TFF unit comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve tangential filters. In some examples of these bioprocessing systems, the TFF unit may comprise a tangential filter having a diameter of about 50 cm. 2 About 10m 2 (or any of the exemplary sub-ranges of this range described herein).

[0138] Back Pressure Regulator

[0139] In certain embodiments, the methods and bioprocessing systems described herein include a back pressure regulator. Considering that the pressure downstream of the back pressure regulator does not exceed the pressure upstream, the back pressure regulator can be used to maintain a stable and consistent pressure and / or flow, and can be used under a variety of downstream pressure conditions. Various types of back pressure regulators are known in the art, and can be used in any method and / or bioprocessing system described herein (e.g., BP-2080 automatic back pressure regulator (Jasco, DE), ultra-low volume back pressure regulator (IDEX Health & Science LLC), M10BP precision back pressure regulator (Fairchild)). See, for example, Berger and Berger, J. Chromatogr. A1218 (16): 2320-2326, 2011 and Svensson et al., Sci. Rep. 12: 569, 2022.

[0140] In some embodiments, a backpressure regulator is used in a bioprocessing system having a positive displacement pump and a bypass valve.

[0141] Booster pump

[0142] In some embodiments of any one of the methods and bioprocessing systems described herein, the booster pump is a dynamic pump (also referred to as a non-positive displacement pump). In certain embodiments, the dynamic pump is a centrifugal pump or a propeller pump. The centrifugal pump transfers rotational energy from the impeller to the fluid, thereby increasing the speed and pressure of the fluid. Different from a positive displacement pump, a dynamic pump can be operated at multiple 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 method and / or bioprocessing system in the methods and / or bioprocessing systems described herein (e.g., Puralev600MU (Levitronix, Switzerland), LKH UltraPure (Alfa Laval, Sweden)).

[0143] sensor

[0144] 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 associated sensors used in the control algorithm measure flow, pressure and / or recombinant therapeutic protein concentration. The flow sensor is an online flow meter that measures flow in real time. The flow sensor can be installed in, for example, 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.

[0145] An in-line pressure sensor placed downstream of the backpressure regulator and boost pump varies the rpm on the boost pump as pressure changes, ensuring continuous flow to subsequent operations.

[0146] Controllers and Control Strategies

[0147] In some embodiments of any of the methods and bioprocessing systems described herein, one or more controllers are used to manipulate process variables so that the effects of disturbances can be minimized and the process variables are as close to their set points as possible. A variety of control schemes including feedback control, feedforward control, cascade control, and model predictive control-based techniques can be used for robust control of the process.

[0148] In some embodiments, the bioprocessing system includes a buffer addition controller that controls fixed volume addition of buffer to a fluid (feed stream).

[0149] In some embodiments, the method accomplishes buffer exchange at constant volume, constant concentration, or both.

[0150] In some embodiments, the bioprocessing system includes a proportional-integral-derivative (PID) controller for controlling the transmembrane pressure of the TFF unit and a buffer addition controller for controlling fixed volume addition of buffer to the feed.

[0151] In one exemplary case, the control strategy uses a combination of feedback and feedforward control on 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 changing the pressure on the backpressure regulator based on the measured flow rates in any two of the feed (fluid in the inlet conduit), the retentate stream (fluid in the retentate conduit), and the permeate stream (fluid in the permeate conduit) and the measured concentrations of the recombinant therapeutic protein in the feed, retentate, and permeate streams.

[0152] Recombinant therapeutic proteins

[0153] 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 provides a therapeutic effect when administered to a subject in need thereof. Examples of recombinant therapeutic proteins include enzymes (e.g., having one or more amino acid substitutions, deletions, insertions, or additions that result in increased stability and / or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins containing at least one recombinant scaffold sequence.

[0154] Non-limiting examples of recombinant therapeutic proteins that can be treated 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., galactosidase (e.g., α-galactosidase), Myozyme, or imiglucerase), 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 for use in vaccines). The recombinant therapeutic protein can be an engineered antigen-binding polypeptide containing at least one multifunctional recombinant protein scaffold (see, e.g., the recombinant antigen-binding proteins described in Gebauer et al., Current Protocols in Molecular Biology, 2003, 55(5), 15(5), 16(5), 17(6), 18(9), 19(10), 20(11), 21(12), 22(13), 23(14), 24(15), 25(16), 26(17), 27(18), 28(19), 29(20), 30(21), 31(22), 32(23), 33(24), 34(25), 35(26), 36(27), 37(28), 38(29), 39(30), 40(31), 41(32), 42(33), 43(41), 44(45), 47(46), 48(47), 49(5), 50(5), 51(5), 52(6), 53(5), 54(6), 55(7), 56(7), 57(8 Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066 (incorporated herein by reference in its entirety). Non-limiting examples of recombinant therapeutic proteins that are antibodies include: panitumumab, omalizumab, abavoumab, abciximab, academumab, adalimumab, adelimumab, afemotomumab, selegiline ... Antibody, afutuzumab, aracizumab, aracizumab, alemtuzumab, alirocumab, atumomab, ametuximab, ametuximab, anatumomab, anulizumab, apolizumab, acitumomab, atenumab, tocilizumab, basilizimab, betumumab, belimumab, bevacizumab, besozumab, belodulumab, bicizumab, canakinumab, selegiline Tocilizumab, cetuximab, citumumab, daclizumab, denosumab, densumab, eculizumab, edrecolomab, efalizumab, ifengucumab, epratuzumab, ertuinomab, irizumab, fentolimumab, golimumab, ibritumomab tiuxetan, igovomab, ingotozumab, infliximab, inomucomab, inotuzumab, labetuzumab, levofloxacin

[00136] In some embodiments, the present invention relates to recombinant therapeutic antibodies, such as rituximab, tocilizumab, tositumomab, trorocirumab, simoleukin monoclonal antibody, trastuzumab, veltuzumab, zalumab and zatuximab. Additional examples of recombinant therapeutic antibodies that can be produced by the methods described herein are known in the art.Additional non-limiting examples of recombinant therapeutic proteins that can be produced by the methods of the invention include alglucosidase alfa, laronidase, abatacept, galsulase, luteinizing hormone alfa, antihemophilic factor, galactosidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, insulin or an insulin analog, mecasermin, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin-11, laronidase, idursuphase, galsulphase, alpha-1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alfa (e.g., ) and alteplase. Additional examples of recombinant therapeutic proteins that can be produced by the methods of the invention include acid α-glucosidase, alglucosidase α (e.g. and ), α-L-iduronidase (e.g., ), idose sulfatase, heparin N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (e.g., and ), galactosylceramidase, α-galactosidase-A (e.g., ), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A and hexosaminidase B.

[0155] Examples

[0156] In one aspect, UF and DF operations are divided into 3 steps: ultrafiltration 1 (UF1), diafiltration (DF) and final ultrafiltration process (UF2). In ultrafiltration, the recombinant protein solution is concentrated by applying pressure to a semi-permeable membrane, which retains the recombinant protein while allowing the removal of solvent and small molecular weight solutes from the solution. Similar to ultrafiltration, a semi-permeable membrane is used in diafiltration to (i) remove or reduce salt concentration or (ii) exchange the buffer material of the protein solution. The original buffer material is exchanged with the new buffer material by adding the new buffer material to the protein solution at a volume rate equivalent to removing the original material. This condition ensures that the protein solution volume and its concentration remain unchanged in 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. Patent 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, which facilitates diafiltration. The subsequent diafiltration step ensures buffer exchange of the protein solution. The final ultrafiltration step (UF2) concentrates the protein to the desired value. See Figures 1A to 1C The flux and concentration are independently measured in real time by online sensors, and the controller adjusts the transmembrane pressure (TMP) on the retentate stream using a backpressure regulator based on the feed and permeate fluxes and the feed and retentate concentrations to maintain consistent flow and retentate concentration ( Figures 1D to 1F ).

[0157] As an example of UF1 operation, Figure 2A The concentration of the recombinant therapeutic protein after the first ultrafiltration operation is shown as a function of time. The concentration of the recombinant therapeutic protein remains relatively constant over time, approximately 60 g / L ( Figure 2A ).exist Figure 2B The consistent flux / TMP values ​​indicate filter integrity and the absence of fouling throughout the operation. The permeate flux remained consistent throughout the operation ( Figure 2C ), the permeate flux is linearly related to the applied transmembrane pressure ( Figure 2D ). Overall, FIG. 2A to FIG. 2D The data in demonstrate that the bioprocessing system regulates and maintains recombinant therapeutic protein concentration and permeate flux while maintaining the integrity of the UF filter.

[0158] The performance of continuous diafiltration operation is 3A to 3H Shown in. Figure 3A demonstrated the ability to maintain target DF retentate concentrations over time, while Figure 3BThe wash volumes achieved and targeted during the run are shown. Throughout the run, the recombinant therapeutic protein concentration and wash volume remained relatively constant over the course of six days. The ratio of the DF buffer addition rate to the permeate production rate remained relatively constant over the course of six days ( Figure 3C ). DF filter performance, determined by flux / TMP, remains relatively constant over time ( Figure 3D Throughout the operation, the permeate volume flux was maintained between 1.7LMH and 2.5LMH ( Figure 3E The effect of permeate flux through the filter on the transmembrane pressure was modeled and experimentally determined ( Figure 3F ).like Figure 3F As shown, the experimentally determined permeate flux through the filter is related to the transmembrane pressure. Figure 3G and Figure 3H The data in demonstrate that a boost pump used in conjunction with a backpressure regulator decouples pressure effects and allows integration of subsequent operations. Figure 3G It is shown that when the boost pump is offline (~day 1.4 to day 3), the controller is unable to maintain a consistent DF retentate concentration. Figure 3H It is shown that in the case where the boost pump is offline, the back pressure regulator is no longer able to maintain a constant retentate pressure. In this case, the retentate pressure is determined by the flow resistance downstream of the operation.

[0159] Data from UF2 operations are FIG. 4A to FIG. 4D As shown in Figure 4A As shown, the concentration of the recombinant therapeutic protein remained relatively constant over time, at approximately 175 g / L. Figure 4B The consistent flux / TMP values ​​indicate filter integrity and the absence of fouling throughout the operation. The permeate flux remained consistent throughout the operation ( Figure 4C ). Figure 4D It shows that when the permeate flux increases, the transmembrane pressure also increases. Overall, 4A to 4D The data in demonstrate that the bioprocessing system regulates and maintains recombinant therapeutic protein concentration (at higher levels than UF1 or DF) and permeate flux during UF2 operation.

[0160] As mentioned above, it is demonstrated that the combination of back pressure regulator and booster pump promotes a) single pass continuous ultrafiltration and diafiltration of recombinant therapeutic protein solution, b) integration of multiple UF and / or DF operations and filter configurations (in series and / or in parallel) with other purification processes, and c) robust, independent and integrated control of each UF and DF step. As shown for DF operation, a combination of back pressure regulator and booster pump is needed to maintain control state. The implementation of both feedback and feedforward control provides the ability to maintain consistent retentate concentration, retentate flow rate and to achieve desired wash volume in continuous diafiltration in UF / DF.

[0161] The combined use of back pressure regulator and booster pump described herein has applications beyond TFF. By decoupling the pressure at the outlet of a unit operation from the pressure downstream of the unit operation, this method allows the unit operation to be integrated in a continuous processing chain without the need for an intermediate break tank or cascade pressure in the entire system. This method can be applied between each unit operation in a traditional bioprocessing system. These key features facilitate the conversion of current biomanufacturing processes into end-to-end integrated and continuous architectures.

[0162] Other embodiments

[0163] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A method for treating a fluid containing a recombinant therapeutic protein, the method comprising: (a) providing a bioprocessing system comprising: (i) a unit operation, (ii) at least one inlet on the unit operation, (iii) at least one outlet on 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 booster pump disposed in the outlet conduit, the booster pump following downstream from the backpressure regulator; (b) flowing the fluid comprising the recombinant therapeutic protein through the inlet conduit and into at least one inlet of the unit operation, treating the fluid within the unit operation to obtain a treated fluid, and then flowing a portion of the treated fluid into the outlet conduit; and (c) maintaining a target flow and / or pressure through the outlet conduit using the back pressure regulator and the boost pump.

2. The method of claim 1, wherein: 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 formulation unit operation.

3. A method of treating a fluid containing a recombinant therapeutic protein, the method comprising: (a) providing a biotreatment system, the biotreatment system comprising: (i) a tangential flow filtration (TFF) unit having a permeate side and a retentate side, (ii) at least one inlet located on the TFF unit, (iii) at least two outlets located on the TFF unit, 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 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 permeate conduit or the retentate conduit, and (viii) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream from the backpressure regulator; (b) flowing the fluid comprising the recombinant therapeutic protein through the inlet conduit and into at least one inlet of the TFF unit, and 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 those permeate and retentate streams into the permeate conduit and the retentate conduit, respectively; and (c) maintaining a target flow and / or pressure through the permeate conduit or the retentate conduit using the backpressure regulator and the boost pump.

4. The method of claim 3, wherein: The TFF unit has a molecular weight cutoff of from about 1 kDa to about 1000 kDa.

5. The method of claim 4, wherein: The TFF unit has a molecular weight cutoff of from about 10 kDa to about 100 kDa.

6. The method of claim 3, wherein: The TFF unit has an average pore size from about 10 nm to about 100 nm.

7. The method of claim 6, wherein: The TFF unit has an average pore size of from about 15 nm to about 35 nm.

8. The method of claim 3, wherein: The TFF unit has an average pore size of from about 0.1 μm to about 10 μm.

9. The method according to any one of claims 3 to 8, wherein: The recombinant therapeutic protein in the fluid in the inlet conduit has a concentration from about 0.1 g / L to about 300 g / L.

10. The method of claim 9, wherein: The recombinant therapeutic protein in the feed stream in the inlet conduit has a concentration from about 0.1 g / L to about 150 g / L.

11. The method according to any one of claims 3 to 10, wherein: The recombinant therapeutic protein in the retentate stream in the retentate conduit has a concentration from about 0.1 g / L to about 300 g / L.

12. The method of claim 11, wherein: The recombinant therapeutic protein in the retentate stream in the retentate conduit has a concentration from about 5 g / L to about 225 g / L.

13. The method according to any one of claims 3 to 12, wherein: The recombinant therapeutic protein in the permeate stream in the permeate conduit has a concentration from about 0 g / L to about 100 g / L.

14. The method of claim 13, wherein: The recombinant therapeutic protein in the permeate stream in the permeate conduit has a concentration from about 0.1 g / L to about 25 g / L.

15. The method according to any one of claims 3 to 14, wherein: The TFF unit includes one or more filters, each having a size of about 50 cm 2 About 10m 2 filter area.

16. The method of claim 15, wherein: The TFF unit includes one or more filters, each of which has a diameter of about 0.1 m 2 About 5m 2 filter area.

17. The method according to any one of claims 1 to 16, wherein: The booster pump is a dynamic pump.

18. The method of claim 17, wherein: The dynamic pump is a centrifugal pump.

19. The method according to any one of claims 3 to 18, wherein: Step (b) comprises continuously flowing the fluid at an average rate of about 0.1 mL / min to about 10 L / min.

20. The method of claim 19, wherein: Step (b) comprises continuously flowing the fluid at an average rate of about 1 mL / minute to about 1 L / minute.

21. The method according to any one of claims 3 to 20, wherein: The bioprocessing system further includes one or more sensors.

22. The method of claim 21, wherein: At least one sensor of the one or more sensors is disposed in the inlet conduit.

23. The method of claim 21 or 22, wherein: At least one sensor of the one or more sensors is disposed in the permeate conduit.

24. The method of any one of claims 21 to 23, wherein: At least one sensor of the one or more sensors is disposed in the retentate conduit.

25. The method of any one of claims 21 to 24, wherein: The recombinant therapeutic protein concentration is measured using the one or more sensors with a sampling time of less than about 2 minutes.

26. The method of any one of claims 3 to 25, wherein: The recombinant therapeutic protein concentration is measured using UV absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

27. The method of any one of claims 3 to 26, wherein: The recombinant therapeutic protein concentration is measured using UV absorbance, and wherein the UV absorbance is measured between about 200 nm to about 400 nm.

28. The method of claim 27, wherein: The recombinant therapeutic protein concentration is measured using UV absorbance, and wherein the UV absorbance is measured between about 260 nm to about 320 nm.

29. The method of any one of claims 26 to 28, wherein: The UV absorbance is measured using a flow cell with a fixed path length.

30. The method of any one of claims 26 to 28, wherein: The UV absorbance was measured using a flow cell with a variable path length.

31. The method of any one of claims 1 to 30, further comprising, prior to step (a), performing one or more second unit operations on the recombinant therapeutic protein, wherein: The one or more second unit operations are integrated upstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with an inlet conduit of the biological treatment system.

32. The method of claim 31, wherein: 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.

33. The method of any one of claims 3 to 32, further comprising, after step (c), performing one or more second unit operations such that one of the one or more second unit operations is in fluid communication with the permeate conduit of the biological treatment system.

34. The method of any one of claims 3 to 32, further comprising, after step (c), performing one or more second unit operations such that one of the one or more second unit operations is in fluid communication with the retentate conduit of the biological treatment system.

35. The method of any one of claims 33 or 34, wherein: The one or more second unit operations are a chromatography unit operation, a viral inactivation unit operation, a viral removal unit operation, an ultrafiltration unit operation, a diafiltration unit operation, or a formulation unit operation.

36. A biological treatment system comprising: (i) unit operation; (ii) at least one inlet on the unit operation; (iii) at least one outlet from 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 disposed in the outlet conduit, and (vii) a boost pump disposed in the outlet conduit, the boost pump following downstream of the back pressure regulator, in: The bioprocessing system is configured to flow the fluid comprising the recombinant therapeutic protein through the inlet conduit and into at least one inlet of the unit operation, to process the fluid within the unit operation to obtain a processed fluid, and then to flow a portion of the processed fluid into the outlet conduit, and The backpressure regulator and the boost pump are configured to maintain a target flow and / or pressure through the outlet conduit.

37. The bioprocessing system of claim 36, wherein: 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 formulation unit operation.

38. A biological treatment system, include: (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 retentate side of the TFF unit; (ii) an inlet conduit in fluid communication with the 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 back pressure regulator disposed in the permeate conduit or the retentate conduit, and (vii) a booster pump disposed in the permeate conduit or the retentate conduit, the booster pump following downstream of the backpressure regulator, in: The bioprocessing system is configured to flow a fluid comprising a recombinant therapeutic protein through the inlet conduit and into the at least one inlet of the TFF unit, and to 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 those permeate and retentate streams into the permeate conduit and the retentate conduit; and The backpressure regulator and the boost pump are configured to maintain a target flow rate and / or pressure through the permeate conduit and the retentate conduit.

39. The bioprocessing system of claim 38, wherein: The TFF unit has a molecular weight cutoff of from about 1 kDa to about 1000 kDa.

40. The bioprocessing system of claim 39, wherein: The TFF unit has a molecular weight cutoff of from about 10 kDa to about 100 kDa.

41. The bioprocessing system of claim 38, wherein: The TFF unit has an average pore size from about 10 nm to about 100 nm.

42. The bioprocessing system of claim 41, wherein: The TFF unit has an average pore size of from about 15 nm to about 35 nm.

43. The bioprocessing system of claim 38, wherein: The TFF unit has an average pore size of from about 0.1 μm to about 10 μm.

44. A biological treatment system as claimed in any one of claims 38 to 43, wherein: The recombinant therapeutic protein in the fluid in the inlet conduit has a concentration from about 0.1 g / L to about 300 g / L.

45. The bioprocessing system of claim 44, wherein: The recombinant therapeutic protein in the fluid in the inlet conduit has a concentration from about 0.1 g / L to about 150 g / L.

46. ​​A biological treatment system as claimed in any one of claims 38 to 45, wherein: The recombinant therapeutic protein in the retentate stream in the retentate conduit has a concentration from about 0.1 g / L to about 300 g / L.

47. The bioprocessing system of claim 46, wherein: The recombinant therapeutic protein in the retentate stream in the retentate conduit has a concentration from about 5 g / L to about 225 g / L.

48. The bioprocessing system of any one of claims 38 to 47, wherein: The recombinant therapeutic protein in the permeate stream in the permeate conduit has a concentration from about 0 g / L to about 100 g / L.

49. The bioprocessing system of claim 48, wherein: The recombinant therapeutic protein in the permeate stream in the permeate conduit has a concentration from about 0.1 g / L to about 25 g / L.

50. The bioprocessing system of any one of claims 38 to 49, wherein: The TFF unit includes one or more filters, each having a size of about 50 cm 2 About 10m 2 filter area.

51. The bioprocessing system of claim 50, wherein: The TFF unit includes one or more filters, each of which has a diameter of about 0.1 m 2 About 5m 2 filter area.

52. The bioprocessing system of any one of claims 36 to 51, wherein: The booster pump is a dynamic pump.

53. The bioprocessing system of claim 52, wherein: The dynamic pump is a centrifugal pump.

54. A biological treatment system as claimed in any one of claims 38 to 53, wherein: The bioprocessing system further includes one or more sensors.

55. The bioprocessing system of claim 54, wherein: At least one sensor of the one or more sensors is disposed in the inlet conduit.

56. The bioprocessing system of claim 54 or 55, wherein: At least one sensor of the one or more sensors is disposed in the permeate conduit.

57. A biological treatment system as claimed in any one of claims 54 to 56, wherein: At least one sensor of the one or more sensors is disposed in the retentate conduit.

58. A bioprocessing system as claimed in any one of claims 54 to 57, wherein: The one or more sensors are configured to measure the recombinant therapeutic protein concentration with a sampling time of less than about 2 minutes.

59. The bioprocessing system of any one of claims 54 to 58, wherein: The one or more sensors are configured to measure the recombinant therapeutic protein concentration using UV absorbance, refractive index, Fourier transform infrared spectroscopy (FTIR), or Raman spectroscopy.

60. The bioprocessing system of claim 59, wherein: The one or more sensors are configured to measure the recombinant therapeutic protein concentration using UV absorbance between about 200 nm to about 400 nm.

61. The bioprocessing system of claim 60, wherein: The one or more sensors are configured to measure the recombinant therapeutic protein concentration using UV absorbance between about 260 nm to about 320 nm.

62. The bioprocessing system of any one of claims 60 to 61, wherein: At least one sensor of the one or more sensors includes a flow channel having a fixed path length.

63. The bioprocessing system of any one of claims 60 to 61, wherein: At least one sensor of the one or more sensors includes a flow cell having a variable path length.

64. The bioprocessing system of any one of claims 36 to 63, wherein: One or more second unit operations are integrated upstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with an inlet conduit of the biological treatment system.

65. The bioprocessing system of claim 64, wherein: 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.

66. The bioprocessing system of any one of claims 38 to 63, wherein: One or more second unit operations are integrated downstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with a permeate conduit of the biological treatment system.

67. The bioprocessing system of any one of claims 38 to 63, wherein: One or more second unit operations are integrated downstream of the biological treatment system such that one of the one or more second unit operations is in fluid communication with a retentate conduit of the biological treatment system.

68. The bioprocessing system of claim 66 or 67, 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 formulation unit operation.

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