Methods for concentrating protein

Abstract

Provided herein is an optimized method for concentrating large volumes of antibody feedstock to produce concentrated drug substance by ultrafiltration in a batch-like manner using a fed-batch setup.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 087,719, filed on 5 October 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of Disclosure Antibody therapies are shifting to subcutaneous delivery for greater patient convenience and compliance. To enable subcutaneous delivery, therapeutic proteins, such as antibodies, must be delivered via high-dose, low-volume injections, requiring formulation into high-concentration final formulations. In the protein manufacturing process, the burden of producing high-concentration active pharmaceutical ingredients (APIs) primarily falls on the ultrafiltration / diafiltration (UF / DF) process, where the purified protein feedstream is typically concentrated to a moderate concentration in the first ultrafiltration step, then the buffer is replaced with the desired formulation, and it is concentrated to a high final concentration in the second ultrafiltration step. This presents unique challenges due to the significant increase in solution viscosity and protein aggregation tendency under prolonged exposure to shear and interfacial stress. Higher viscosity leads to higher system pressure, which limits the maximum achievable protein concentration due to safety constraints related to system pressure. Increased viscosity can correlate with a significant decrease in permeate flow, which prolongs process time and thus amplifies the risk of protein aggregation. The generation of high-concentration substances significantly reduces the volume of the packing material, which not only raises issues regarding equipment suitability and capacity, but can also amplify the problem of aggregation caused by long-term exposure to shear and interfacial stress. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] There is a need for improved methods to produce high-concentration active pharmaceutical ingredients. [Means for solving the problem]

[0004] Summary of Disclosure This disclosure relates to a method for reducing the filtration process time of a target protein, comprising continuously filling a feed tank with a protein mixture containing the target protein that has been filtered at least once ("retaining solution"), wherein the feed tank is separated from a main reservoir ("retaining solution") tank. This disclosure also relates to a method for concentrating a target protein, comprising continuously filling a feed tank with a protein mixture containing the target protein that has been filtered at least once ("retaining solution"), wherein the feed tank is separated from a main reservoir ("retaining solution") tank. In some embodiments, the feed tank further comprises an initial protein mixture containing the target protein that has been filtered at least once. In some embodiments, the initial protein mixture and the retaining solution are mixed. In some embodiments, the protein mixture and / or the retaining solution are filtered. In some embodiments, the filtered protein mixture and retaining solution ("retaining solution") are filled into the feed tank. In some embodiments, the filling is continued until the protein of interest is concentrated to at least about 1 mg / mL, at least about 10 mg / mL, at least about 20 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, or at least about 80 mg / mL. In some embodiments, the filling is continued until the target protein is concentrated to approximately 1 mg / mL to 80 mg / mL, approximately 5 mg / mL to 70 mg / mL, approximately 10 mg / mL to 60 mg / mL, approximately 10 mg / mL to 50 mg / mL, approximately 10 mg / mL to 40 mg / mL, approximately 10 mg / mL to 30 mg / mL, approximately 10 mg / mL to 20 mg / mL, approximately 20 mg / mL to 70 mg / mL, approximately 20 mg / mL to 60 mg / mL, approximately 20 mg / mL to 50 mg / mL, approximately 20 mg / mL to 40 mg / mL, or approximately 20 mg / mL to 30 mg / mL.In some embodiments, the filling of the retaining fluid is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 times. In some embodiments, the method further includes stopping the filling of the retaining fluid into the supply tank. In some embodiments, the method further includes directing the retaining fluid to a reservoir tank.

[0005] The disclosure also relates to a method for reducing the filtration process time of a target protein, comprising filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve connecting the filter to the supply tank and a reservoir tank valve connecting the filter to the reservoir tank, and a reservoir input connecting the supply tank and the reservoir tank, with a protein mixture containing the target protein. The disclosure also relates to a method for concentrating a target protein, which comprises filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve connecting the filter to the supply tank and a reservoir tank valve connecting the filter to the reservoir tank, and a reservoir input connecting the supply tank and the reservoir tank, with a protein mixture containing the target protein. In some embodiments, the reservoir tank valve is kept closed until the target protein is sufficiently concentrated. In some embodiments, the method further includes frequently adding the protein mixture to the supply tank. In some embodiments, the protein mixture is directed from the supply tank to the reservoir tank. In some embodiments, the reservoir tank is connected to the filter. In some embodiments, the filter includes an inline filtration membrane. In some embodiments, the inline filtration membrane is an ultrafiltration membrane.In some embodiments, the inline filtration membrane is polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultra-nanocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrenedivinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose, or a combination thereof. In some embodiments, the filtration membrane has a molecular weight cutoff (MWCO) of approximately 50kD to approximately 5kD, approximately 50kD, approximately 40kD, approximately 30kD, approximately 20kD, approximately 10kD, or less than approximately 5kD. In some embodiments, the MWCO is less than approximately 5kD.

[0006] In some embodiments, the mixture is allowed to flow until a desired filtration protein concentration is reached. In some embodiments, the desired filtration protein concentration is about 10 mg / mL to about 300 mg / mL, for example, about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 250 mg / mL, or about 300 mg / mL. In some embodiments, the desired filtration protein concentration is about 150 mg / mL. In some embodiments, the protein viscosity is about 0 cP to about 200 cP. In some embodiments, the protein viscosity is about 20 cP to about 60 cP. In some embodiments, the volume ratio between the supply tank capacity and the reservoir capacity is approximately 1:2 to 10:1, 1:2 to 1:1, 1:1 to 1:2, 1:1 to 1:3, 1:1 to 1:4, 1:1 to 1:5, 1:1 to 1:6, 1:1 to 1:7, 1:1 to 1:8, 1:1 to 1:9, or 1:1 to 1:10. In some embodiments, the volume ratio between the supply tank capacity and the reservoir capacity is approximately 1:1, 2:1, or 5:1. In some embodiments, the protein mixture is directed to the reservoir tank and / or filter using a diaphragm pump, rotary lobe pump, or peristaltic pump.

[0007] In some embodiments, the method further includes filling the feed tank with an initial protein mixture containing the target protein that has not been filtered at all, prior to the continuous filling of the feed tank with a protein mixture containing the target protein that has been filtered at least once ("retaining solution"). In some embodiments, the initial protein mixture is added to the feed tank at a concentration of about 1 mg / mL to about 30 mg / mL. In some embodiments, the initial protein mixture is added to the feed tank at a concentration of about 5 mg / mL.

[0008] In some embodiments, the process time is reduced by approximately 1%, 5%, 10%, 20%, 30%, 40%, or 50% compared to the process time of the Fed batch enrichment process. In some embodiments, the process time is reduced by approximately 40% compared to the process time of the Fed batch enrichment process.

[0009] In some embodiments, the process time is reduced by approximately 0.2 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.8 hours, or 1.0 hour compared to the process time of the Fed batch enrichment process. In some embodiments, the process time is reduced by approximately 0.5 hours compared to the process time of the Fed batch enrichment process.

[0010] In some embodiments, the number of 1-2 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process. In some embodiments, the number of 5-10 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process. In some embodiments, the number of 10-25 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process.

[0011] In some embodiments, the protein mixture comprises antibodies, antibody fragments, antigen-binding fragments, fusion proteins, naturally occurring proteins, chimeric proteins, or any combination thereof. In some embodiments, the protein mixture comprises antibodies selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the protein mixture comprises antibodies, which are IgG antibodies selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a bivalent variable domain immunoglobulin. In some embodiments, the antibody comprises a trivalent antibody. In some embodiments, the antibody or antibody fragment includes an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-8 antibody, or a fragment thereof.

[0012] In some embodiments, the protein mixture is derived from bacterial, yeast, insect, or mammalian cell cultures. In some embodiments, the mammalian cell culture is Chinese hamster ovary (CHO) cell culture.

[0013] In some embodiments, the protein mixture is obtained from batch cell cultures. In some embodiments, the protein mixture is obtained from fed batch cell cultures. In some embodiments, the protein mixture is produced in a bioreactor. In some embodiments, the protein mixture is produced in a disposable bioreactor. In some embodiments, the protein mixture is obtained from perfused cell cultures. In some embodiments, the protein mixture is produced in a perfusing or TFF perfusing bioreactor. In some embodiments, the protein mixture is produced from cell cultures that persist for approximately 1 to 60 days. In some embodiments, the protein mixture is produced from cell cultures that persist for approximately 25 days.

[0014] In some aspects, the protein mixture is added to a feed tank together with a loading buffer. In some aspects, the loading buffer comprises an amino acid, a weak acid, a weak base and / or a sugar.

[0015] In some aspects, the method further comprises formulating the protein into a pharmaceutical composition. In some aspects, the protein is prepared by the method disclosed herein. In some aspects, the pharmaceutical composition comprises the protein prepared herein.

[0016] The present disclosure also relates to a method of administering the pharmaceutical composition disclosed herein. The present disclosure also relates to a method of treating a disease or condition in a subject in need thereof, comprising administering the pharmaceutical composition to the subject.

[0017] The present disclosure also provides (a) a feed tank, (b) a reservoir tank connected to the feed tank via a first fluid path, (c) a filtration membrane connected to the reservoir tank via a second fluid path, and (d) a three-way valve connected to the filtration membrane via a third fluid path, connected to the reservoir tank via a fourth fluid path, and connected to the feed tank via a fifth fluid path A system for concentrating a target protein, comprising: the reservoir tank receives a protein mixture comprising the target protein from the feed tank via the first fluid path, the filtration membrane receives the protein mixture comprising the target protein from the reservoir tank via the second fluid path, and filters the protein mixture, the three-way valve receives retentate from the filter via the third fluid path, and directs the retentate to either the reservoir tank via the fourth fluid path or the feed tank via the fifth fluid path.

[0018] In some aspects, the three-way valve directs retentate to the reservoir tank when the total volume of the protein mixture in the system is less than the capacity of the reservoir tank, and directs retentate to the feed tank when the total volume of the protein mixture in the system is greater than the capacity of the reservoir tank.

[0019] In some aspects, the system further comprises a sensor configured to determine the total volume and / or concentration of the protein mixture in the system, wherein the three-way valve automatically directs retentate to either the reservoir tank or the feed tank based on feedback from the sensor. In some aspects, the system further comprises one or more diaphragm pumps, rotary lobe pumps or peristaltic pumps. In some aspects, the filter comprises an in-line filtration membrane. In some aspects, the in-line filtration membrane is an ultrafiltration membrane. In some aspects, the in-line filtration membrane is made of polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultra-nanocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrene divinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [Figure 1A]Figures 1A to 1C show schematic diagrams of tangential flow filtration (TFF) systems. Figure 1A shows a schematic diagram of the TFF ultrafiltration / dialysis filtration system settings in a fed-batch configuration. Figure 1B shows a schematic diagram of the TFF ultrafiltration / dialysis filtration system settings in a pseudo-batch configuration. Figure 1C shows a schematic diagram of the TFF ultrafiltration / dialysis filtration system settings in a batch configuration. [Figure 1B] Same as above. [Figure 1C] Same as above.

[0021] [Figure 2A] Figures 2A and 2B show the retaining solution mAb concentration as a function of elapsed process time in batch, fed-batch, and pseudo-batch filling configurations. Figure 2A shows the calculated retaining solution concentration of mAb A (g / L) as a function of elapsed process time (hours) for batch, fed-batch, and pseudo-batch filling strategies. Figure 2B shows the corresponding process times at each process stage of ultrafiltration 1 (UF1), diafiltration (DF), and ultrafiltration 2 (UF2), from left to right. [Figure 2B] Same as above.

[0022] [Figure 3] Figure 3 shows the permeate flow rate (LHM) as a function of the calculated retention solution concentration of mAb A during ultrafiltration / diafiltration. The runs were performed using batch, fed-batch, or pseudo-batch filling (UF1), but all three runs were operated using batch configurations in the DF and UF2 steps.

[0023] [Figure 4] Figure 4 shows the levels of high molecular weight (HMW) species of mAb A at the midpoint of the UF / DF process for batch, fed-batch (hybrid), and pseudo-batch filling strategies.

[0024] [Figure 5A]Figures 5A–5D show the number of mAb A particles in UF / DF pools during the process generated using batch, fed-batch (hybrid), and pseudo-batch packing strategies. Figure 5A shows the number of 1–2 μm particles. Figure 5B shows the number of 5–10 μm particles. Figure 5C shows the number of 10–25 μm particles. Figure 5D shows the number of 50–100 μm particles. Particle counts were quantified using microfluidic imaging. Particle counts in the 2–5 μm and 25–50 μm size ranges were excluded for brevity, but were consistent with the trends observed in the 5–10 μm and 50–100 μm size ranges, respectively. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above.

[0025] [Figure 6A] Figure 6A shows the retaining mAb concentration as a function of process time in pseudo-batch execution using diaphragm pumps and peristaltic pumps. Figure 6B shows the corresponding process time at each process stage (e.g., UF1, DF, and UF2) in Figure 6A. [Figure 6B] Same as above.

[0026] [Figure 7] Figure 7 shows the permeate flow as a function of retained fluid concentration in pseudo-batch execution using diaphragm pumps and peristaltic pumps.

[0027] [Figure 8] Figure 8 shows the levels of high molecular weight (HMW) species in pseudobatch processes using diaphragm pumps and peristaltic pumps.

[0028] [Figure 9A]Figures 9A–9D show the mAb A particle count of particles in a UF / DF pool during a process generated using a pseudo-batch filling strategy with either a diaphragm pump or a peristaltic pump. Figure 9A shows the particle count for 1–2 μm particles. Figure 9B shows the particle count for 5–10 μm particles. Figure 9C shows the particle count for 10–25 μm particles. Figure 9D shows the particle count for 50–100 μm particles. Particle counts were quantified using microfluidic imaging. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above.

[0029] [Figure 10A] Figure 10A shows the retaining mAb concentration as a function of process time in pseudo-batch execution. Here, the liquid volume of the retaining tank during the filling process was kept constant at a low volume (e.g., 10% and 20%) relative to the total filling volume. Figure 10B shows the corresponding process times at each process stage (e.g., UF1, DF, and UF2) in Figure 10A. [Figure 10B] Same as above.

[0030] [Figure 11] Figure 11 shows the permeate flow as a function of the retaining fluid concentration during pseudobatch execution. Here, the liquid volume of the retaining fluid tank during the filling process was kept constant at a low volume relative to the total filling volume.

[0031] [Figure 12] Figure 12 shows the levels of high molecular weight (HMW) species in pseudobatch execution. Here, the liquid volume of the holding liquid tank during the filling process was kept constant at a low volume relative to the total filling volume.

[0032] [Figure 13A]Figures 13A–13D show the mAb A particle count of particles in a UF / DF pool during the process generated using pseudo-batch filling. Here, the liquid volume of the holding fluid tank during the filling process was kept constant at a low volume relative to the total filling volume. Figure 13A shows the particle count of 1–2 μm particles. Figure 13B shows the particle count of 5–10 μm particles. Figure 13C shows the particle count of 10–25 μm particles. Figure 13D shows the particle count of 50–100 μm particles. Particle counts were quantified using microfluidic imaging. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above.

[0033] [Figure 14] Figure 14 shows the retention solution protein concentration as a function of process time during pseudobatch and fedbatch process executions for non-mAb therapeutic proteins (MW approximately 20 Da).

[0034] [Figure 15] Figure 15 shows the permeate flow as a function of retaining fluid concentration during the execution of the pseudobatch and fedbatch processes shown in Figure 14.

[0035] [Figure 16] Figure 16 shows the levels of high molecular weight (HMW) species in the pseudo-batch and fed-batch process executions shown in Figure 14.

[0036] [Figure 17A] Figures 17A–17D show the particle counts in pseudobatch and fed-batch process executions for non-mAb therapeutic proteins shown in Figure 14. Figure 17A shows the particle count for 1–2 μm particles. Figure 17B shows the particle count for 5–10 μm particles. Figure 17C shows the particle count for 10–25 μm particles. Figure 17D shows the particle count for 50–100 μm particles. Particle counts were quantified using microfluidic imaging. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Modes for carrying out the invention]

[0037] Detailed explanation of disclosure This disclosure relates to a method for reducing the filtration process time of a target protein. It also relates to a method for concentrating a target protein.

[0038] I. Definition To make this disclosure easier to understand, certain terms are defined first. Where used herein, unless otherwise expressly provided herein, each of the following terms shall have the meanings set forth below. Further definitions are provided throughout this specification.

[0039] It should be noted that the terms "a" or "an" refer to one or more entities. For example, "nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably in this specification.

[0040] As used herein, the term "and / or" should be interpreted as disclosing each of the two features or components being identified in detail, with or without the other. Thus, when used herein in a phrase such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, when used in a phrase such as "A, B and / or C," the term "and / or" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] Whenever an aspect is described in this specification using the word “comprising,” it is understood that similar aspects described using the terms “consisting of” and / or “consisting essentially of” are also to be presented.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which this disclosure relates. For example, general dictionaries of many of the terms used herein are provided to those skilled in the art by the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press.

[0043] Units, prefixes, and symbols are expressed in the form recognized by the International System of Units (SI). Numerical ranges include the number defining the range. The headings presented herein are not limitations on the various aspects of this disclosure, but may be found by referring to this specification as a whole. Accordingly, the terms defined immediately below are further fully defined by referring to this specification as a whole.

[0044] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof. Where used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any enumerated or counted components.

[0045] The terms “approximately” or “essentially from” refer to a value or composition within the acceptable margin of error of a particular value or composition, as determined by those skilled in the art, which depends in part on the limitations of the method of measuring or determining the value or composition, i.e., the measuring system. For example, “approximately” or “essentially from” may mean one or more standard deviations per practice in the art. Or, “approximately” or “essentially from” may mean a range of up to 20%. Moreover, particularly with respect to biological systems or processes, the terms may mean a value of up to one order of magnitude or up to five times the value. Where a particular value or composition is presented in this application and claims, unless otherwise stated, the meaning of “approximately” or “essentially from” should be assumed to be within the acceptable margin of error of that particular value or composition.

[0046] Where described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0047] The term "ultrafiltration" refers to a membrane-based separation process that separates molecules in a solution based on their size, thereby achieving either the separation of different molecules or the concentration of the same molecules.

[0048] The term "tangential flow filtration" refers to a specific filtration method in which a solute-containing solution passes tangentially through an ultrafiltration membrane, and pressurization is used to allow low molecular weight solutes to pass through the membrane. The high molecular weight solute-containing solution passing tangentially through the ultrafiltration membrane is retained, and this solution is referred to herein as the "retaining solution." The low molecular weight solute passing through the ultrafiltration membrane is referred to herein as the "permeate." Therefore, the retaining solution is concentrated by flowing, for example, tangentially across the surface of the ultrafiltration membrane under pressure. The ultrafiltration membrane has a pore size with a specific cutoff value. In some embodiments, the cutoff value is about 50 kDa or less, for example, 50 kDa, 40 kDa, 30 kDa, 20 kDa, or 10 Da. In some embodiments, the cutoff value is 30 kDa or less.

[0049] The term "diafiltration" or "DF" refers, for example, to the removal, substitution, or reduction of the concentration of solvents, buffers, and / or salts from a solution or mixture containing proteins, peptides, nucleic acids, or other biomolecules using an ultrafiltration membrane.

[0050] The terms “Fed batch,” “Fed batch filtration,” or “Fed batch filtration process,” as used herein, refer to a tangential flow filtration (e.g., ultrafiltration) method in which a feedstock containing the protein of interest is filled into a feed tank and then directed into a reservoir tank, where the feedstock is concentrated in a TFF and the retained liquid is directed back into a retained liquid tank. The terms “batch,” “batch filtration,” or “batch filtration process” refer to a filtration (e.g., ultrafiltration) configuration in which a protein mixture is filled into a reservoir tank to generate a retained liquid from which the retained liquid is directed back into the reservoir tank, while the permeate is directed into a drainpipe for disposal.

[0051] In this specification, the terms “polypeptide” and “protein” as used interchangeably refer to polymers of amino acids of any length. These polymers may be linear or branched, may contain modified amino acids, or may be fragmented by non-amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other modification operations, such as conjugation with labeling components. For example, polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included in the definition. As used herein, the terms “polypeptide” and “protein” particularly encompass antibodies and Fc domain-containing polypeptides (e.g., immunoadhesins).

[0052] As used herein, the term “protein of interest” is used to include any protein (either native or recombinant) present in the mixture to be purified. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or fusion proteins. In some embodiments, the protein of interest refers to any protein that can be purified and / or concentrated using the tangential flow filtration (TFF) method described herein. In some embodiments, the protein of interest is an antibody. In some embodiments, the protein of interest is a recombinant protein.

[0053] The terms “fed batch culture” or “fed batch culture process,” as used herein, refer to a method of culturing cells in which additional components are provided to the culture at some point after the start of the culture process. Fed batch culture can be initiated using a basic medium. The culture medium to which additional components are provided at some point after the start of the culture process is called a feed medium. Fed batch culture is typically stopped at some point, and the cells and / or components in the medium are harvested and purified as appropriate.

[0054] As used herein, “perfusion,” “perfusion culture,” or “perfusion culture process” refers to a sustained flow of physiological nutrients at a stable rate through or over a cell population. Because perfusion systems generally involve the retention of cells in culture units, perfusion cultures are characterized by relatively high cell densities, but maintaining and regulating culture conditions is difficult. In addition, because cells proliferate and are retained at high density in culture units, the growth rate typically declines steadily over time, thereby slowing even the logarithmic or quiescent phase of cell proliferation. This sustained culture strategy generally involves culturing mammalian cells expressing the polypeptide and / or virus of interest, e.g., non-anchorage-dependent cells, during the generational stage of the sustained cell culture system.

[0055] As used herein, “setpoint” refers, unless otherwise specified, to the initial setting of conditions in the TFF system or any other upstream processing vessel used for the concentration and / or production of protein products. The setpoint is established at the start of the UF / DF process described herein. Because UF / DF conditions in the TFF fluctuate, conditions in the UF / DF after the setpoint may subsequently change. For example, the setpoint may be a mass setpoint. In some embodiments, the setpoint is a temperature setpoint. In some embodiments, the setpoint can be maintained throughout the cell culture method. In other embodiments, the setpoint can be maintained until a different setpoint is established. In other embodiments, the setpoint can be changed to a different setpoint.

[0056] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain has a heavy chain variable region (V in this specification). H It includes a heavy chain constant region (abbreviated as V in this specification). The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain has a light chain variable region (V in this specification). L It includes the light chain constant region (abbreviated as C). The light chain constant region consists of one constant domain, C L Includes. VH and V L The region is further subdivided into hypervariable regions called complementary determination regions (CDRs), which may incorporate more conserved regions called framework regions (FRs). H and V L The antibody comprises three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The heavy chain may or may not have a C-terminal lysine. In some embodiments, the antibody is a full-length antibody.

[0057] Immunoglobulins may originate from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, IgD, IgE, and IgM. The IgG subclass is well known to those skilled in the art and includes, but is not limited to, human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term “antibody” includes, by example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. The term “antibody” may also include multivalent antibodies (e.g., trivalent antibodies) capable of binding to three or more antigens. A trivalent antibody is an IgG-type bispecific antibody consisting of two symmetrical Fab arms fused via a mobile linker peptide to a third asymmetric Fab-size binding molecule. This third molecule consists of a variable region of the heavy chain that fuses to a CH3 with a “knob” mutation instead of the IgG Fc region, and a variable region of the light chain that fuses to a CH3 with a matching “hole.” The hinge region does not contain a disulfide bond that facilitates contact of the antigen to the third binding site. Unless otherwise explicitly stated or indicated by the context, the term “antibody” includes monospecific, bispecific, or multispecific antibodies as well as single-chain antibodies.

[0058] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It is known that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) Fab fragments (fragments obtained by papain cleavage) or similar monovalent fragments consisting of VL, VH, LC, and CH1 domains; (ii) F(ab')2 fragments (fragments obtained by pepsin cleavage) or similar bivalent fragments containing two Fab fragments linked by disulfide crosslinking in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolation complementarity-determining regions (CDRs); and (vii) combinations of two or more isolation CDRs that are appropriately linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using a synthetic linker that allows the VL and VH regions to pair up and form a single protein chain that forms a monovalent molecule (known as single-stranded Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-stranded antibodies are also intended to be included in the term "antigen-binding portion" of an antibody. Such antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for usefulness as intact antibodies in the same manner. Antigen-binding portions can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0059] A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody that has two different heavy / light chain pairs, resulting in two antigen-binding sites with specificity to various antigens. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment conjugation. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0060] A “fusion” or “chimeric” protein contains a first amino acid sequence that binds to a second amino acid sequence that does not naturally bind. Amino acid sequences normally present in separate proteins can be combined into a fusion polypeptide, or amino acid sequences normally present in the same protein can be placed into a new sequence of the fusion polypeptide, for example, the fusion of the factor VIII domain of this disclosure having an IgFc domain. Fusion proteins are produced, for example, by chemosynthesis or by the generation and translation of polynucleotides in which the peptide regions encode in a desired relationship. Chimeric proteins may further contain a second amino acid sequence associated with the first amino acid sequence by covalent, non-peptide, or non-covalent bonds.

[0061] "Administration" refers to the physical introduction of a composition containing a therapeutic agent to a subject using any of the various methods and delivery systems known to those skilled in the art. The routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration," as used herein, means a method of administration other than enteral and topical administration, usually by injection, and typically includes, but is not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a route other than a parenteral route, and in some embodiments, it is administered via an oral route. Other routes of administration besides parenteral administration include local, epithelial, or mucosal routes, such as intranasal, intravaginal, intrarectal, sublingual, or local administration. Furthermore, administration can be carried out, for example, once, multiple times, and / or over a single period or multiple periods.

[0062] The term "treatment" or "therapy" for a subject refers to any type of intervention, process, or administration of an activator performed on the subject with the aim of restoring, alleviating, remitting, inhibiting, or delaying the progression, onset, severity, or recurrence of symptoms, complications, or biochemical indicators associated with the disease. The Response Evaluation Criteria (RECIST) for Solid Tumors is an indicator of treatment effectiveness, an established set of rules defining when a tumor responds, stabilizes, or progresses during treatment. RECIST 1.1 is the current guideline for the measurement and definition of solid tumors for objective assessment of changes in tumor size, for use in adult and pediatric cancer clinical trials. The East Coast Cancer Group (ECOG) Performance Status is a numbered scale used to define the patient population being investigated in a trial, so that physicians enrolling patients can consistently reproduce the results. In pediatric patients, the Lansky Performance Scale is a method for describing functional status in children. It was developed and internally validated to assess response to therapy and overall condition in pediatric cancer patients.

[0063] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions of these values ​​(e.g., one-tenth and one-hundredth of an integer).

[0064] When used herein, the term “desired final protein concentration” refers to the protein concentration of the target protein concentrated using the tangential flow filtration method described herein. For example, the desired final protein concentration of the target protein is achieved by subjecting a mixture containing the target protein to the UF1, DF, and UF2 steps described herein. In some embodiments, the desired final protein concentration is up to 300 mg / mL.

[0065] As used herein, “pharmaceutically acceptable carrier” refers to a medium for a pharmacologically active drug. The carrier facilitates the delivery of the activator to the target site without interfering with the drug’s function. Non-limiting examples of suitable forms of carriers include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, salves, sprays, ointments, powders, solid mixtures, aerosols, emulsions (e.g., water in oil or oil in water), gel aqueous solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.

[0066] As used herein, the phrase “pharmaceutically acceptable composition” (or “pharmaceutical composition”) means, for example, a composition that is pharmaceutically acceptable for human administration. Such a composition may contain substances with impurities at levels not exceeding pharmaceutically acceptable levels (such levels include the absence of such impurities), and may include, for example, pharmaceutically acceptable excipients, media, carriers and other inert components in addition to any activator(s) to formulate such a composition for ease of administration. For example, a pharmaceutically acceptable anti-PD1 antibody composition may contain DNA, insofar as it is at levels acceptable for human administration.

[0067] II. Ultrafiltration Method This disclosure provides a protein purification method that enables highly concentrated final protein collection. In some embodiments, the method of this disclosure reduces the process time required for a first ultrafiltration step (for example, in a process including or consisting of a first ultrafiltration step, diafiltration and a second ultrafiltration step) in a protein purification process where there is a large reduction in feedstock and the initial volume is sufficiently large to require initial operation in a fed-batch manner.

[0068] In some embodiments, the method of this disclosure mitigates indirect challenges associated with the generation of high concentrations of active pharmaceutical ingredients (e.g., the protein of interest) by ultrafiltration. For example, the generation of high concentrations of material (e.g., approximately 150–300 g / L) significantly reduces the volume of the packing material, which not only raises challenges of equipment suitability and capacity but can also amplify challenges of aggregation due to prolonged exposure to shear and interfacial stress. During ultrafiltration, the sample volume decreases significantly (e.g., typically less than one-tenth), while the holding liquid container and system storage capacity remain fixed, resulting in a point in the process where there is a significant mismatch between the system size (e.g., holding liquid container and system storage capacity) and the sample volume. Since the system flow path, including the bottom of the container, must remain filled with liquid throughout the filtration process, the volume of the final concentration of the active pharmaceutical ingredient (e.g., the protein of interest), in conjunction with the shape of the holding liquid container, determines the minimum movable capacity of the system. As the minimum movable capacity increases with container size, the final active pharmaceutical ingredient sets an upper limit on the size of the holding liquid container. This upper limit is less than the packing capacity. Therefore, a portion of the filling material must be supplied to the holding liquid container using Fed-batch filling. The percentage of the initial filling volume that must be supplied by Fed-batch filling is a function of equipment suitability, such as the available container size and system storage capacity.

[0069] While Fed-batch filling allows for large-scale filling, this filling strategy imposes a process time penalty compared to batch operations. In a batch process, the sample liquid (buffer) volume is uniformly distributed across the total protein volume (e.g., all in the retaining solution container), whereas in a Fed-batch process, the diluent in the filling container (e.g., the supply tank) is divided between the concentrated protein in the retaining solution container. Therefore, for a given amount of remaining sample volume, the retaining solution protein concentration is higher in a Fed-batch process than in a batch process. Consequently, Fed-batch permeation flow is lower due to the higher retaining solution concentration. Therefore, in a system, the Fed-batch method operates at higher concentrations and lower flow rates for much of the first ultrafiltration step compared to a batch method, resulting in longer process times, which in turn increases the risk of aggregation induced by shear and interfacial stress.

[0070] In setting up the manufacturing scale, the operation method of the first ultrafiltration step for producing high-concentration active pharmaceutical ingredient is typically neither a full batch nor a fed-batch. Instead, the ultrafiltration step operates using a fed-batch method for supplying the packing material to the holding liquid container, and then switches to a batch method once the holding liquid container is sufficiently filled with the packing material. This mixed ultrafiltration method will hereafter be referred to as the "hybrid" method. In the hybrid method, the process time of the first ultrafiltration step is within the process time expected when operating in a full batch or fed-batch method, and this depends on the intersection where the system operation switches from fed-batch to batch. The equations describing the intersection and the hybrid process time are described in Example 1. The concentration corresponding to the intersection is then a function of the relative packing amount and system capacity, so that the process time becomes a strong function of equipment suitability.

[0071] If differences in relative packing volume and system capacity are not considered during technology transition, the specifications of process parameters developed at the laboratory scale can lead to unexpectedly long process times at the manufacturing scale, creating challenges in process development and technology transition. In contrast, since the process time of a batch process depends solely on membrane packing, the process is highly scalable.

[0072] No systematic approach has been developed to address the process time penalty or equipment compatibility dependency arising from the hybrid operating scheme in the first ultrafiltration step. To mitigate these challenges, we have developed the method disclosed herein, which enables batch-like operation of the ultrafiltration step using a fed-batch-like setting, referred herein as the pseudo-batch operating scheme.

[0073] This disclosure provides an optimized method for concentrating large quantities of protein feedstock to produce concentrated active pharmaceutical ingredients by ultrafiltration in a batch-like manner using a fed-batch setting. The disclosure also relates to a method for producing a solution containing highly concentrated protein by tangential flow filtration (TFF). The methods disclosed herein reduce the process time required for a first ultrafiltration step (e.g., in a process including or consisting of a first ultrafiltration step, diafiltration, and a second ultrafiltration step) in processes where the feedstock volume is significantly reduced and the initial volume is sufficiently large to require initial operation in a fed-batch manner. In some embodiments, the method improves product quality when quantified by particle and impurity loading generated in the ultrafiltration step due to the shorter process time. In some embodiments, the method eliminates the process time variability observed in instrumentation settings between laboratory scales, e.g., between development and manufacturing scales, when operated in a fed-batch manner. In some embodiments, improved consistency in process time across scales may enhance the accuracy of scaled-down ultrafiltration / diafiltration (UF / DF) models, leading to more efficient scaling and technology transition campaigns.

[0074] This disclosure relates to pseudobatch configurations for UF / DF in which, in some embodiments, the time penalty associated with fed-batch filling can be reduced by concentrating the target protein by converting a fed-batch setting (e.g., using a feed tank and a retaining fluid container) to a batch-like operation (e.g., connecting the feed tank and the retaining fluid container as described herein to function as a single container in a tangential flow filtration (TFF) recirculation loop).

[0075] Tangential flow filtration is an ultrafiltration procedure that relies on the use of flow pressure to drive the migration of low molecular weight molecules through an ultrafiltration membrane while simultaneously retaining high molecular weight molecules (e.g., a "retaining solution"). Generally, a membrane with a molecular weight cutoff (MWCO) of 1 / 3 to 1 / 6 of the molecular weight of the protein to be retained is selected. Other factors known to those skilled in the art, such as flow rate, processing time, intermembrane pressure, molecular shape or structure, solute concentration, presence of other solutes, and ionic conditions, may also influence the selection of an appropriate MWCO.

[0076] A traditional fed-batch TFF configuration is shown in Figure 1A. Here, a supply tank holding the packing material is connected to a retaining liquid container (e.g., a reservoir) by a supply pump, and the retaining liquid container is separately incorporated into the recirculation loop along with the TFF membrane device and recirculation pump.

[0077] In some embodiments, a method for concentrating a target protein by TFF and / or reducing filtration time comprises a first ultrafiltration step (UFI), diafiltration (DF), and a second ultrafiltration step (UF2).

[0078] In some embodiments, the flow path in the pseudobatch configuration described herein is modified to incorporate a supply tank into the recirculation loop (Figure 1B). A three-way valve is positioned after the retaining fluid port of the TFF filter module to direct the retaining fluid flow to either the supply tank or the retaining fluid container, depending on the conditions of the first ultrafiltration step. In some embodiments, a stirrer is used for both the supply tank and the reservoir (e.g., retaining fluid) tank.

[0079] In some embodiments, during the initial filling stage of UF1, a three-way valve is configured to direct the retaining fluid flow towards the supply tank and block the flow to the retaining fluid container. The packing material is supplied from the supply tank to the retaining fluid container by a supply pump, while the retaining fluid from the TFF filter module is returned to the supply tank and mixed with the remaining packing material. This new packing material mixture is then slightly further concentrated and supplied to the retaining fluid container, and the cycle is repeated. In some embodiments, the protein solutions in both the supply tank and the retaining fluid container are concentrated to the same ratio, unlike fed-batch filling where the retaining fluid is progressively further concentrated but the packing material remains fixed at its initial dilution concentration. In this configuration, similar to a batch setting, the supply tank efficiently acts as an extension of the retaining fluid container, and the two act as a single reservoir in the recirculation loop (Figure 1C). The pseudo-batch configuration described herein reduces the time penalty of the fed-batch filling section of the first ultrafiltration step and the equipment suitability dependency of the process time by converting another hybrid process into a batch-like process. In some embodiments, during the filling process, the liquid volume of the holding liquid tank is kept constant at a low volume relative to the total filling volume. In some embodiments, the liquid volume is kept constant at approximately 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the total initial filling volume during the filling process. In some embodiments, the liquid volume is kept constant at approximately 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 15% to 20%, 15% to 25%, 15% to 30%, 20% to 30%, or 25% to 30% of the total initial filling volume during the filling process. In some embodiments, the liquid volume is maintained at a constant level of approximately 10% of the total initial filling volume during the filling process. In some embodiments, the liquid volume is maintained at a constant level of approximately 20% of the total initial filling volume during the filling process.

[0080] In some embodiments, when the filling process is complete (e.g., when the total protein solution volume has decreased to less than the holding liquid container volume), a three-way valve is activated to redirect the holding liquid flow back to the holding liquid container, shutting off the flow to the supply tank, efficiently removing the supply tank from the recirculation loop, and converting the TFF setting to a substantial batch configuration. In some embodiments, the supply pump may be operated for an additional time to perform aerial expulsion of residual material in the connecting tube between the two containers (e.g., into the holding liquid container) to maximize the recovery of the filling material.

[0081] TFF membranes for concentration can be selected based on their rejection characteristics for the sample to be concentrated. As a general rule, to ensure complete retention, the molecular weight cutoff (MWCO) of the membrane should be 1 / 3 to 1 / 6 of the molecular weight of the molecule being retained (e.g., the target protein). The closer the MWCO is to that of the sample, the greater the risk of some product loss during concentration. The risk increases if diafiltration is also used, as the relative loss depends on the total volume of the filtrate produced. Membrane flow rate (filtration flow rate per unit area of ​​the membrane) is related to pore size. Smaller pores result in lower flow rates at the same pressure. Therefore, when selecting a membrane for concentration / diafiltration, the recovery rate of the product relative to the time factor must be considered. Process time can be reduced by increasing the amount of membrane area used.

[0082] Dialysis filtration (DF) is a technique that uses a filtration membrane (e.g., an ultrafiltration membrane) to completely remove, replace, or reduce the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. DF selectively utilizes permeable (e.g., porous) membrane filters to separate components of solutions and suspensions based on their molecular size. Ultrafiltration membranes retain molecules larger than the membrane pores, while allowing smaller molecules, such as 100% permeable salts, solvents, and water, to pass freely through the membrane. DF is a fractionation process that washes away smaller molecules in the retained solution through the membrane while retaining larger molecules (e.g., the protein of interest) without ultimately changing the concentration.

[0083] Diafiltration can be continuous or non-continuous. In continuous diafiltration, the diafiltration solution (e.g., buffer) is added to the sample feed reservoir at the same rate as the filtrate was produced. In this method, the volume of the sample reservoir remains constant, but small molecules that can freely permeate the membrane (e.g., salts) are washed away. Using desalination as an example, the salt concentration is further reduced by each additional diafiltration volume (DV). (DV is a measure of the degree of rinsing performed in the DF process. This is based on the volume of the introduced diafiltration buffer compared to the retained fluid volume. In constant volume DF, the retained fluid volume is kept constant, and the DF buffer is added at the same rate as the permeate is discharged. For example, a certain diafiltration volume is equivalent to adding a buffer volume equal to the volume of the product in the system to the supply reservoir, and then concentrating it back to the starting volume. For example, the first diafiltration volume (DV1) for a 200 mL starting sample is equal to 200 mL.) In the second diafiltration volume (DV2), continuous diafiltration reduces the ionic strength by approximately 99%. In non-continuous diafiltration, the solution is first diluted, then concentrated, and returned to the starting volume. The process is then repeated until the low molecular weight (e.g., salt) remaining in the reservoir reaches the desired concentration. Each further DV reduces the salt concentration further. In continuous diafiltration, less filtrate volume is required to achieve the same level of salt reduction as in non-continuous diafiltration. By initially concentrating the sample, the amount of diafiltration solution required to achieve a specific ionic strength is substantially reduced.

[0084] In some embodiments, the DF supply pump operates only in DF or recovery mode when the retained fluid container mass is below the retained fluid container mass setpoint. In some embodiments, this mass is checked every 2 seconds by the control system. In some embodiments, the DF mass is set and the DF pump is maintained until the DF endpoint. In some embodiments, the DF pump operates when the container mass falls below the input setpoint. After reaching the DF endpoint, the system proceeds to the concentration process. In some embodiments, the DF endpoint is selected from the control system's graphical user interface. In some embodiments, the default endpoint is air in the wiring. In some embodiments, the DF mass setpoint is the total retained fluid container mass.

[0085] This disclosure provides a method for reducing the filtration process time of a protein of interest, comprising continuously filling a supply tank with a protein mixture (e.g., a retaining solution) containing the protein of interest that has been filtered at least once, wherein the supply tank is separated from a main reservoir (e.g., a retaining solution) tank. In some embodiments, the method for reducing the filtration process time of a protein of interest comprises filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve connecting the filter to the supply tank and a reservoir tank valve connecting the filter to the reservoir tank, and a reservoir input connecting the supply tank and the reservoir tank, with a protein mixture containing the protein of interest.

[0086] Furthermore, the Disclosure provides a method for concentrating a target protein, comprising continuously filling a supply tank with a protein mixture (e.g., a retaining solution) containing the target protein that has been filtered at least once, wherein the supply tank is separated from a main reservoir (e.g., a retaining solution) tank. In some embodiments, the method for concentrating a target protein comprises filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve connecting the filter to the supply tank and a reservoir tank valve connecting the filter to the reservoir tank, and a reservoir input connecting the supply tank and the reservoir tank, with a protein mixture containing the target protein.

[0087] In some embodiments, the supply tank further contains an initial protein mixture containing the target protein that has not been filtered at least once, and the initial protein mixture and the retention solution are mixed therein. In some embodiments, the protein mixture and retention solution are filtered by a filter (e.g., an ultrafiltration filter). In some embodiments, the filtered protein mixture and retention solution are filled into the supply tank. In some embodiments, the protein mixture and retention solution are continuously filled into the supply tank until the target protein is concentrated to at least about 1 mg / mL, at least about 10 mg / mL, at least about 20 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, or at least about 80 mg / mL. In some embodiments, the protein mixture and retention solution are continuously filled into the supply tank until the target protein is concentrated to at least about 1 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, at least about 11 mg / mL, at least about 12 mg / mL, at least about 13 mg / mL, at least about 14 mg / mL, at least about 15 mg / mL, at least about 16 mg / mL, at least about 17 mg / mL, at least about 18 mg / mL, at least about 19 mg / mL, or at least about 20 mg / mL. In some embodiments, the protein mixture and retention solution are continuously filled into the supply tank until the target protein is concentrated to at least about 21 mg / mL, at least about 22 mg / mL, at least about 23 mg / mL, at least about 24 mg / mL, at least about 25 mg / mL, at least about 26 mg / mL, at least about 27 mg / mL, at least about 28 mg / mL, at least about 29 mg / mL, or at least about 30 mg / mL.In some embodiments, the protein mixture and retention solution are continuously filled into a supply tank until the protein of interest is concentrated to at least about 31 mg / mL, at least about 32 mg / mL, at least about 33 mg / mL, at least about 34 mg / mL, at least about 35 mg / mL, at least about 36 mg / mL, at least about 37 mg / mL, at least about 38 mg / mL, at least about 39 mg / mL, at least about 40 mg / mL, at least about 45 mg / mL, at least about 50 mg / mL, at least about 55 mg / mL, at least about 60 mg / mL, at least about 65 mg / mL, at least about 70 mg / mL, at least about 75 mg / mL, or at least about 80 mg / mL, at least about 85 mg / mL, or at least about 90 mg / mL. In some embodiments, the protein mixture and retention solution are continuously filled into a supply tank until the target protein is concentrated to approximately 1 mg / mL to 80 mg / mL, approximately 5 mg / mL to 70 mg / mL, approximately 10 mg / mL to 60 mg / mL, approximately 10 mg / mL to 50 mg / mL, approximately 10 mg / mL to 40 mg / mL, approximately 10 mg / mL to 30 mg / mL, approximately 10 mg / mL to 20 mg / mL, approximately 20 mg / mL to 70 mg / mL, approximately 20 mg / mL to 60 mg / mL, approximately 20 mg / mL to 50 mg / mL, approximately 20 mg / mL to 40 mg / mL, or approximately 20 mg / mL to 30 mg / mL. In some embodiments, the protein mixture and retention solution are continuously filled into a supply tank until the target protein is concentrated to approximately 1 mg / mL to 10 mg / mL, approximately 10 mg / mL to 20 mg / mL, approximately 20 mg / mL to 30 mg / mL, approximately 30 mg / mL to 40 mg / mL, approximately 40 mg / mL to 50 mg / mL, approximately 50 mg / mL to 60 mg / mL, approximately 60 mg / mL to 70 mg / mL, or approximately 70 mg / mL to 80 mg / mL.

[0088] In some embodiments, the filling of the retaining fluid is recirculated by a pseudobatch flow path described herein. In some embodiments, the filling of the retaining fluid is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 times.

[0089] In some embodiments, the method further includes stopping the filling of the retaining fluid into the supply tank. In some embodiments, the method further includes directing the retaining fluid into the reservoir tank. In some embodiments, the reservoir tank valve is kept closed until the protein of interest is sufficiently concentrated. In some embodiments, the method further includes frequently adding the protein mixture to the supply tank. In some embodiments, the protein mixture is directed from the supply tank to the reservoir tank.

[0090] In some embodiments, a reservoir tank is connected to a filter. In some embodiments, the filter includes an inline filtration membrane. In some embodiments, the inline filtration membrane is an ultrafiltration membrane. In some embodiments, the inline filtration membrane is polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultra-nanocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrenedivinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose, or a combination thereof. In some embodiments, the inline filtration membrane is polyethersulfone. In some embodiments, the inline filtration membrane is cellulose. In some embodiments, the inline filtration membrane is a combination of polyethersulfone and cellulose. In some embodiments, the filtration membrane has a molecular weight cutoff (MWCO) of about 50 kD to less than about 5 kD. In some embodiments, the filtration membrane has an MWCO of less than about 5 kD.

[0091] In some embodiments, the mixture is passed through (e.g., recirculated) until a desired filtration protein concentration is reached. In some embodiments, the desired filtration protein concentration is approximately 10 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 20 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 30 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 40 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 50 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 60 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 70 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 80 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 90 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 100 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 110 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 120 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 130 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 140 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 150 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 160 to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 170 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 180 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 190 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 200 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 210 mg / mL to approximately 300 mg / mL.In some embodiments, the desired filtration protein concentration is approximately 220 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 230 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 240 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 250 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 260 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 270 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 280 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is approximately 290 mg / mL to approximately 300 mg / mL. In some embodiments, the desired filtration protein concentration is 150 mg / mL.

[0092] In some embodiments, the protein viscosity is approximately 0 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 20 cP to approximately 60 cP. In some embodiments, the protein viscosity is approximately 10 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 20 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 30 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 40 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 50 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 60 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 70 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 80 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 90 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 100 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 100 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 120 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 130 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 140 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 150 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 160 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 170 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 180 cP to approximately 200 cP. In some embodiments, the protein viscosity is approximately 190 cP to approximately 200 cP.

[0093] In some embodiments, the capacity ratio between the supply tank capacity and the reservoir capacity is approximately 1:2 to 10:1, approximately 1:2 to 1:1, approximately 1:1 to 1:2, approximately 1:1 to 1:3, approximately 1:1 to 1:4, approximately 1:1 to 1:5, approximately 1:1 to 1:6, approximately 1:1 to 1:7, approximately 1:1 to 1:8, approximately 1:1 to 1:9, or approximately 1:1 to 1:10. In some embodiments, the capacity ratio between the supply tank capacity and the reservoir tank capacity is approximately 1:1, approximately 2:1, or approximately 5:1.

[0094] In some embodiments, the protein mixture is directed to a reservoir tank and / or filter using a diaphragm pump, rotary lobe pump, or peristaltic pump. In some embodiments, the protein mixture is directed to a reservoir tank and / or filter using a diaphragm pump. In some embodiments, the protein mixture is directed to a reservoir tank and / or filter using a peristaltic pump.

[0095] In some embodiments, a method for concentrating a target protein and / or reducing the filtration process time of a target protein includes filling a feed tank with an initial protein mixture containing a target protein that has not been filtered at least once, prior to the continuous filling of the feed tank with a protein mixture containing a target protein that has been filtered at least once ("retaining solution"). In some embodiments, the initial protein mixture is added to the feed tank at a concentration of about 1 mg / mL to about 30 mg / mL. In some embodiments, the initial protein mixture is added to the feed tank at a concentration of about 5 mg / mL.

[0096] In some embodiments, the process time is reduced by approximately 1%, 5%, 10%, 20%, 30%, 40%, or 50% compared to the process time of the Fed batch enrichment process. In some embodiments, the process time is reduced by approximately 40% compared to the process time of the Fed batch enrichment process. In some embodiments, the process time is reduced by approximately 0.2 hours, 0.4 hours, 0.5 hours, 0.6 hours, 0.8 hours, or 1.0 hour compared to the process time of the Fed batch enrichment process. In some embodiments, the process time is reduced by approximately 0.5 hours compared to the process time of the Fed batch enrichment process.

[0097] In some embodiments, the number of 1-2 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process. In some embodiments, the number of 5-10 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process. In some embodiments, the number of 10-25 μm particles is reduced by approximately 10%, 20%, 30%, 40%, or 50% compared to the number of particles in the Fed batch enrichment process.

[0098] In some embodiments, the protein mixture is added to the feed tank along with the loading buffer. In some embodiments, the loading buffer contains amino acids, a weak acid, a weak base, and / or sugars.

[0099] III. Target Protein In some embodiments, the methods disclosed herein can be applied to any protein product (e.g., the protein of interest). In some embodiments, the protein product is a therapeutic protein. In some embodiments, the therapeutic protein is selected from antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood coagulation factors, bone morphogenetic proteins, modified protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. In some embodiments, the protein product is an antibody or an antigen-binding fragment thereof. In some embodiments, the protein is a recombinant protein.

[0100] In some embodiments, the protein product is an antibody or an antigen-binding fragment thereof. In some embodiments, the protein product is a chimeric polypeptide containing an antigen-binding fragment of an antibody. In some embodiments, the protein product is a monoclonal antibody or an antigen-binding fragment thereof ("mAb"). The antibody may be a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the protein product is a bispecific antibody.

[0101] In some embodiments, the mixture containing protein products and impurities includes the product from the previous purification step. In some embodiments, the mixture is the original product from the previous purification step. In some embodiments, the mixture is a solution containing the original product from the previous purification step and a buffer, such as a start buffer. In some embodiments, the mixture includes the original product from the previous purification step reconstituted in the start buffer.

[0102] In some embodiments, the raw material for the protein product is bulk protein. In some embodiments, the source of the protein product is a composition comprising the protein product and non-protein components. The non-protein components may include DNA and other impurities.

[0103] In some embodiments, the source of the protein product is of animal origin. In some embodiments, the animal is a mammal, e.g., a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or human). In some embodiments, the source is human-derived tissue or cells. In certain embodiments, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, cows, cats, or dogs). In some embodiments, such terms refer to pets or livestock. In some embodiments, such terms refer to humans.

[0104] In some embodiments, the protein product purified by the method described herein is a fusion protein. A “fusion” or “fusion protein” comprises a first amino acid sequence that is in-frame bound to a second amino acid sequence that is not naturally bound. Amino acid sequences normally present in separate proteins can be combined into a fusion polypeptide, or amino acid sequences normally present in the same protein can be placed into a new sequence in the fusion polypeptide. Fusion proteins are produced, for example, by chemosynthesis, or by the production and translation of a peptide region into a polynucleotide that encodes a desired association. A fusion protein may further comprise a second amino acid sequence that is bound to the first amino acid sequence by a covalent, non-peptide bond, or non-covalent bond. A single protein is produced during transcription / translation. In this method, multiple proteins or fragments thereof can be incorporated into a single polypeptide. “Operatively bound” is intended to mean a functional bond between two or more elements. For example, an operational bond between two polypeptides causes both polypeptides to fuse in-frame to produce a single polypeptide fusion protein. In certain embodiments, the fusion protein further comprises a third polypeptide, which may include a linker sequence, as will be discussed in more detail below.

[0105] In some embodiments, the proteins purified by the methods described herein are antibodies. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies) and any of the antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. The antibody may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibody described herein is an IgG antibody, or an IgG antibody of a class (e.g., human IgG1 or IgG4) or a subclass. In some embodiments, the antibody is a humanized monoclonal antibody. In some embodiments, the antibody is a human monoclonal antibody, which is preferably an immunoglobulin. In some embodiments, the antibody described herein is an IgG1 or IgG4 antibody.

[0106] In some aspects, the protein is an anti-LAG3 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IL8 antibody, or any combination thereof. In some aspects, the protein is abatacept NGP. In other aspects, the protein is belatacept NGP.

[0107] In some aspects, the protein is an anti-PD-1 antibody.

[0108] Anti-PD-1 antibodies known in the art can be used in the compositions and methods described herein. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity are described in U.S. Pat. No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Pat. No. 8,008,449 bind to human PD-1 with (a) a K -7 D of 1×10 D M or less, (b) do not substantially bind to human CD28, CTLA-4 or ICOS, (c) increase T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (d) increase interferon-γ production in an MLR assay, (e) increase IL-2 secretion in an MLR assay, (f) bind to human PD-1 and cynomolgus monkey PD-1, (g) inhibit the binding of PD-L1 and / or PD-L2 to PD-1, (h) stimulate antigen-specific memory responses, (i) stimulate antibody responses, and (j) inhibit tumor cell proliferation in vivo, and have been demonstrated to exhibit one or more of these properties. Anti-PD-1 antibodies that can be used in the present disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some aspects at least five, of the foregoing properties.

[0109] Other anti-PD-1 monoclonal antibodies include, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Patent Application Publication No. 2016 / 0272708, and PCT International Publications WO2012 / 145493, WO2008 / 156 712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 3 5606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO20 14 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO These are described in WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825 and WO2017 / 133540, each of which is incorporated herein by reference in its entirety.

[0110] In some embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), semiprimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA (also known as toripalimab; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; also known as tislerizumab; see WO2015 / 35606 and U.S. Patent Application Publication 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136) (See 2017), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / HarbinGloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), BCD-100 (Biocad; Kaplon et al.The selection is made from the group consisting of mAbs 10(2):183-203 (2018) and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825 and WO2017 / 133540).

[0111] In some embodiments, the protein is an anti-PD-L1 antibody. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of this disclosure. An example of an anti-PD-L1 antibody useful in the compositions and methods of this disclosure is the antibody disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has a density of 1 × 10⁻¹⁶ when determined by surface plasmon resonance using a Biacore biosensor system. -7 K below M D It has been demonstrated that these antibodies bind to human PD-L1 and exhibit one or more of the following properties: (b) increased T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (c) increased interferon-γ production in an MLR assay, (d) increased IL-2 secretion in an MLR assay, (e) stimulate an antibody response, and (f) restore the effect of regulatory T cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies available in this disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments, at least five, of the aforementioned properties.

[0112] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent No. 7,943,743 and WO2013 / 173223), and atezolizumab (Roche; TECENTRIQ®; also known as MPDL3280A, RG7446; see U.S. Patent No. 8,217,149; Herbst et al. (2013) J Clin Oncol). See also 31(suppl):3000), durvalumab (AstraZeneca; IMFINZI®, also known as MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; BAVENCIO®, also known as MSB-0010718C; see WO2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; Zhang et al., Cell Discov. 7:3 (March The following are selected from the group consisting of (see 2017), LY3300054 (EliLilly Co.; see, for example, WO2017 / 034916), BGB-A333 (BeiGene; see Desai et al., JCO 36 (15suppl):TPS3113 (2018)), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0113] In some embodiments, the protein is an anti-GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) antibody. In some embodiments, the anti-GITR antibody has a 6C8 CDR sequence and includes, for example, the humanized antibody having a 6C8 CDR described in WO2006 / 105021, the antibody containing the CDR of the anti-GITR antibody described in WO2011 / 028683, the antibody containing the CDR of the anti-GITR antibody described in JP 2008278814, the anti-GITR antibody described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, WO2018 / 013818, or other antibodies containing the CDR of anti-GITR antibodies described or referred to herein, all of which are incorporated herein in their entirety.

[0114] In other embodiments, the protein is an anti-LAG3 antibody. Lymphocyte-activating gene 3, also known as LAG-3, is a protein encoded by the LAG3 gene in humans. Discovered in 1990, LAG3 is a cell surface molecule with diverse biological effects on T cell function. It is an immune checkpoint receptor and is therefore a target of various drug development programs by pharmaceutical companies striving to develop new treatments for cancer and autoimmune disorders. In its soluble form, it has also been developed as an oncological drug. Examples of anti-LAG3 antibodies include, but are not limited to, those described in U.S. Patent Application Publication No. 2017 / 087901A2, WO2016 / 028672A1, WO2017 / 106129A1, WO2017 / 198741A1, U.S. Patent Application Publication No. 2017 / 0097333A1, U.S. Patent Application Publication No. 2017 / 0290914A1, and U.S. Patent Application Publication No. 2017 / 0267759A1. All of these are incorporated herein in their entirety.

[0115] In some embodiments, the protein is an anti-CXCR4 antibody. CXCR4 is a G1-bound, seven-transmembrane protein. CXCR4 is widely expressed on hematopoietic cells and is the primary co-receptor for human immunodeficiency virus 1 (HIV-1) with CD4+. See Feng, Y., Broeder, CC, Kennedy, PE, and Berger, EA (1996) Science 272, 872-877. Examples of anti-CXCR4 antibodies include, but are not limited to, those described in WO2009 / 140124A1, U.S. Patent Application Publication 2014 / 0286936A1, WO2010 / 125162A1, WO2012 / 047339A2, WO2013 / 013025A2, WO2015 / 069874A1, WO2008 / 142303A2, WO2011 / 121040A1, WO2011 / 154580A1, WO2013 / 071068A2, and WO2012 / 175576A1. All of these are incorporated herein in their entirety.

[0116] In some embodiments, the protein is an anti-CD73 (ecto-5'-nucleotidase) antibody. In some embodiments, the anti-CD73 antibody inhibits adenosine formation. The breakdown of AMP to adenosine creates an immunosuppressive and angiogenic microenvironment within the tumor microenvironment, thereby promoting cancer development and progression. Examples of anti-CD73 antibodies include, but are not limited to, the antibodies in WO2017 / 100670A1, WO2018 / 013611A1, WO2017 / 152085A1, and WO2016 / 075176A1. All of these are incorporated herein in their entirety.

[0117] In some embodiments, the protein is an anti-TIGIT (T cell immune receptor having Ig and ITIM domains) antibody. TIGIT is a member of the PVR (poliovirus receptor) family of immunoglobulin proteins. TIGIT is expressed on several classes of T cells, including follicular B helper T cells (TFHs). This protein is known to bind to PVRs with high affinity, and this binding is thought to facilitate the interaction between TFHs and dendritic cells and regulate the T cell-dependent B cell response. Examples of anti-TIGIT antibodies include, but are not limited to, the antibodies in WO2016 / 028656A1, WO2017 / 030823A2, WO2017 / 053748A2, WO2018 / 033798A1, WO2017 / 059095A1, and WO2016 / 011264A1. All of these are incorporated herein in their entirety.

[0118] In some embodiments, the protein is an anti-OX40 (i.e., CD134) antibody. OX40 is a cytokine of the tumor necrosis factor (TNF) ligand family. OX40 functions in T cell antigen-presenting cell (APC) interactions, mediating the adhesion of activated T cells to endothelial cells. Examples of anti-OX40 antibodies include WO2018 / 031490A2, WO2015 / 153513A1, WO2017 / 021912A1, WO2017 / 050729A1, WO2017 / 096182A1, WO2017 / 134292A1, WO2013 / 038191A2, WO2017 / 096281A1, WO2 Examples include, but are not limited to, 013 / 028231A1, WO2016 / 057667A1, WO2014 / 148895A1, WO2016 / 200836A1, WO2016 / 100929A1, WO2015 / 153514A1, WO2016 / 002820A1, and WO2016 / 200835A1. All of these are incorporated herein in their entirety.

[0119] In some embodiments, the protein is an anti-IL-8 antibody. IL-8 is a chemotactic that attracts neutrophils, basophils, and T cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to inflammatory stimuli.

[0120] In some embodiments, the protein is abatacept (marketed as ORENCIA®). Abatacept (also abbreviated herein as Aba) is a drug used to treat autoimmune diseases such as rheumatoid arthritis by interfering with the immune activity of T cells. Abatacept is a fusion protein consisting of the Fc region of immunoglobulin IgG1 fused to the extracellular domain of CTLA-4. To activate T cells and elicit an immune response, antigen-presenting cells must present two signals to the T cells. One such signal is the major histocompatibility complex (MHC) complexed with the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7-2).

[0121] In some embodiments, the protein is belatacept (trade name NULOJIX®). Belatacept is a fusion protein consisting of an Fc fragment of human IgG1 immunoglobulin bound to the extracellular domain of CTLA-4, which is a key molecule in the regulation of T cell costimulation and selectively blocks the process of T cell activation. It is intended to extend graft survival while limiting toxicity caused by standard immunosuppressive therapy regimens, such as calcineurin inhibitors. It differs from abatacept (ORENCIA®) ​​by just two amino acids.

[0122] In some embodiments, the protein mixture comprises antibodies, antibody fragments, antigen-binding fragments, fusion proteins, naturally occurring proteins, chimeric proteins, or any combination thereof. In some embodiments, the protein mixture comprises antibodies selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the protein mixture comprises an antibody, which is an IgG antibody selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody comprises a bivariate domain immunoglobulin. In some embodiments, the antibody comprises a trivalent antibody. In some embodiments, the antibody or antibody fragment comprises anti-PD-1, anti-PD-L, anti-CTLA4, anti-TIM3, anti-LAG3, anti-NKG2a, anti-ICOS, anti-CD137, anti-KIR, anti-TGFβ, anti-IL-10, anti-B7-H4, anti-GITR, anti-CXCR4, anti-CD73, anti-TIGIT, anti-OX40, anti-IL-8 antibodies, or fragments of these antibodies.

[0123] In some embodiments, the protein mixture containing the target protein is derived from a bacterial, yeast, insect, or mammalian cell culture. In some embodiments, the mammalian cell culture is a Chinese hamster ovary (CHO) cell culture.

[0124] In some embodiments, the protein mixture containing the target protein is obtained from a batch cell culture. In some embodiments, the protein mixture containing the target protein is obtained from a fed batch cell culture. In some embodiments, the protein mixture is produced in a bioreactor. In some embodiments, the protein mixture is produced in a disposable bioreactor. In some embodiments, the protein mixture is obtained from a perfused cell culture. In some embodiments, the protein mixture is produced by perfusion in a TFF perfused bioreactor. In some embodiments, the protein mixture is produced using cell cultures that persist for approximately 1 to 60 days. In some embodiments, the protein mixture is produced using cell cultures that persist for approximately 25 days.

[0125] IV. Pharmaceutical Compositions The proteins produced by the methods of this disclosure can be further formulated, for example, into pharmaceutical compositions, to be suitable for human administration. The compositions are pharmaceutically acceptable and may contain substances that are impurities at levels not exceeding pharmaceutically acceptable levels (such levels include the absence of such impurities). For example, pharmaceutically acceptable excipients, media, carriers, and other inactive components may be added to any activator(s) to formulate such compositions for easier administration. Compositions prepared by the methods of this disclosure are useful for treating a variety of diseases.

[0126] V. Ultrafiltration Systems This disclosure provides a system for reducing the filtration process time of a target protein. Furthermore, this disclosure provides a system for concentrating a target protein.

[0127] In some embodiments, a system for concentrating a target protein includes a supply tank, a reservoir tank connected to the supply tank by a first fluid path, a filtration membrane connected to the reservoir tank by a second fluid path, and a three-way valve connected to the filtration membrane by a third fluid path, to the reservoir tank by a fourth fluid path, and to the supply tank by a fifth fluid path, wherein the reservoir tank receives a protein mixture containing the target protein from the supply tank via the first fluid path, the filtration membrane receives the protein mixture containing the target protein from the reservoir tank via the second fluid path and filters the protein mixture, and the three-way valve receives a retaining liquid from the filter via the third fluid path and directs the retaining liquid to either the reservoir tank via a fourth fluid path or the supply tank via a fifth fluid path.

[0128] In some embodiments, the three-way valve directs the retaining fluid to the reservoir tank when the total volume of the protein mixture in the system is less than the capacity of the reservoir tank, and directs the retaining fluid to the supply tank when the total volume of the protein mixture in the system is greater than the capacity of the reservoir tank.

[0129] In some embodiments, the system further includes sensors configured to determine the total volume, mass, and / or concentration of the protein mixture within the system, where a three-way valve automatically directs the holding fluid to either a reservoir tank or a supply tank based on feedback from the sensors. In some embodiments, an in-line UV-Vis spectrophotometer is used to monitor the protein concentration of the protein mixture in real time. In some embodiments, a level sensor is used to monitor the volume of the protein solution in either or both of the supply tank and the reservoir tank in real time. In some embodiments, such a level sensor includes, but is not limited to, a waveguide radar or a membrane-based pressure level sensor. In some embodiments, the volume of the protein solution in either or both of the supply tank and the reservoir tank is monitored by gravimetric measurement, where the mass of the protein solution in the supply tank and / or reservoir tank is measured using a balance, and the mass is converted to volume using solution density. In some embodiments, the system further includes one or more diaphragm pumps, rotary lobe pumps, or peristaltic pumps.

[0130] In some embodiments, the filter includes an in-line filtration membrane. In some embodiments, the in-line filtration membrane is an ultrafiltration membrane. In some embodiments, the in-line filtration membrane is polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultrananocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrenedivinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose, or a combination thereof. In some embodiments, the inline filtration membrane is polyethersulfone. In some embodiments, the inline filtration membrane is cellulose. In some embodiments, the inline filtration membrane is a combination of polyethersulfone and cellulose. In some embodiments, the filtration membrane has a molecular weight cutoff (MWCO) of approximately 50 kD to less than approximately 5 kD. In some embodiments, the filtration membrane has an MWCO of less than approximately 5 kD. [Examples]

[0131] Example 1 Fed batch process time is a function of several equipment suitability parameters, namely, the holding fluid container capacity, system storage capacity, fill protein concentration, and fill volume.

[0132] If the volume of the packing material is too large to be filled and fitted into the retaining liquid container, a portion of the packing material is maintained in a supply tank and slowly supplied to the retaining liquid container by fed-batch ultrafiltration. The point at which the retaining liquid volume has decreased sufficiently to contain the entire protein packing material (several grams of protein) in the retaining liquid container is called the cross-concentration (Equation 1).

number

[0133] In the formula, C0 and V0 are the protein packing concentration and volume, respectively, and V res V is the volume of the liquid container, holdup This represents the system storage capacity.

[0134] The process time required to concentrate proteins in a fed-batch manner can be numerically calculated using Equation 2c, which is obtained by integrating the flow equation (Equation 2a, defined with respect to the cumulative permeate volume V') and assuming a concentration polarization model with respect to flow (Equation 2b). The integral V in Equation 2c perm,final The limit is the expected permeate volume at cross-concentration. The protein concentration in a fed-batch operation can be defined as a function of the cumulative permeate volume V' by Equation 3, where V res V is the full capacity of the holding fluid container, which is to be kept constant during Fed batch filling. Therefore, V perm,final This can be calculated by solving Equation 3 for V' using the cross-protein concentration (Equation 1).

[0135]

number

[0136]

number

[0137]

number

[0138]

number

[0139] This equation reveals that the fed batch process time is a function of several equipment suitability parameters, namely, the holding fluid container capacity, system storage capacity, fill protein concentration, and fill volume.

[0140] Example 2 A traditional fed-batch TFF configuration is illustrated in Figure 1A. In this configuration, a supply tank holding the packing material is connected to a retaining liquid container (e.g., a reservoir) by a supply pump, and the retaining liquid container is separately incorporated into the recirculation loop along with the TFF membrane device and recirculation pump.

[0141] In the pseudo-batch configuration described herein, the flow path is modified to incorporate a supply tank into the recirculation loop, as illustrated in Figure 1B. A three-way valve is positioned after the retaining fluid port of the TFF filter module, as described below, to direct the retaining fluid flow to either the supply tank or the retaining fluid container, depending on the conditions of the first ultrafiltration step. In some embodiments, a stirrer (not shown) is used for both the tank and the container.

[0142] In the initial loading phase of the first ultrafiltration step, a three-way valve is set to direct the retaining fluid flow towards the load tank and block the flow to the retaining fluid container. The packing material is supplied from the load tank to the retaining fluid container by a supply pump, while the retaining fluid from the TFF filter module is instead returned to the load tank and mixed with the remaining packing material. This new packing material is then slightly further concentrated and supplied to the retaining fluid container, and the cycle is repeated. Thus, unlike fed-batch loading, where the retaining fluid is progressively further concentrated while the packing material remains fixed at its initial dilution concentration, the protein solutions in both the load tank and the retaining fluid container are concentrated at the same rate. In this configuration, similar to a batch setting, the load tank efficiently acts as an extension of the retaining fluid container, and the two act as a single reservoir in the recirculation loop (Figure 1C). Therefore, the pseudo-batch configuration reduces the time penalty of the fed-batch loading section of the first ultrafiltration step and the equipment compatibility dependency of the process time by converting another hybrid process into a batch-like process.

[0143] When the filling process is complete (for example, when the total protein solution volume has decreased to less than the holding liquid container volume), the three-way valve is activated to redirect the holding liquid flow to the holding liquid container, shut off the flow to the supply tank, efficiently remove the supply tank from the recirculation loop, and convert the TFF setting to a substantial batch configuration. The supply pump can be operated for a further time to perform aerial expulsion of residual material in the connecting tube between the two containers (for example, into the holding liquid container) to maximize the recovery of the filling material.

[0144] As proof of concept to demonstrate the advantages of the pseudobatch configuration described above, high-concentration solutions of mAb A (approximately 180 g / L) were produced by TFF performed at a laboratory scale using batch, hybrid, and pseudobatch configurations. Process times for each filtration step (first ultrafiltration (UF1), diafiltration (DF), and second ultrafiltration (UF2)) and quality characteristics of the recovered purified active pharmaceutical ingredient (PDS) (e.g., high molecular weight (HMW) species, particle count) were compared among the three configurations. HMW levels were measured by high-performance liquid-size exclusion chromatography using an Alliance2695 HPLC system equipped with a Model 2487 dual-wavelength detector (Waters Corporation, Milford MA USA) and TSKgel SuperSW3000 main and guard columns (Tosoh Bioscience, King of Prussia PA USA). Meanwhile, the number of particles between 1 and 100 μm was quantified by microflow imaging using an MFI5200 (ProteinSimple, San Jose CA USA). PDS turbidity was measured using a Hach 2100Q turbidimeter in triplicate, which was calibrated daily before use.

[0145] The TFF experiment used a Quattroflow 150 pump (High Purity New England, Smithfield RI USA) and an 88cm² pump. 2 The experiment was conducted using a PendoTECH control and data acquisition system (PendoTECH, Princeton, NJ, USA) equipped with a 30 kDa UltracelPellicon 3D-screen membrane (MilliporeSigma, Burlington, MA, USA). A single batch of purified 10 g / L mAb solution was divided into separate fixed volumes, and the same packing material was produced in three runs. Here, the fixed volume was approximately 600 g / m². 2The goal was to achieve membrane packing. In both pseudobatch and hybrid post-compounding, the volume ratio of the initial packing volume to the retaining fluid container, plus the system storage volume, was set to 5. In each run, the mAb was concentrated to 50 g / L in a first ultrafiltration step, and then the buffer was replaced with a dialysis filtration buffer of 5 dialysis filtration volume. The dialysis filtration protein solution was then further concentrated to 180 g / L in a second ultrafiltration step (determined by gravimetric analysis from the masses of the retaining fluid and permeate to account for the change in solution density at high concentrations). The active pharmaceutical ingredient was then recovered by expelling the residual protein solution out of storage volume with buffer. Here, the volume of expelling buffer used was 1.2 times the system storage volume.

[0146] Example 3 A. Process time The approximate retention solution mAb concentration as a function of process time (calculated from the retention solution volume and total protein mass in the system) is shown in Figure 2A for the three configurations. The corresponding process times at each process stage (UF1, DF, and UF2) are shown in Figure 2B. The corresponding permeate flow observed in the three runs is shown in Figure 3.

[0147] The process time for the UF1 step in the hybrid (fed-batch filling) run was 60% longer than in the batch run, as shown in Figure 2B. Lower permeate flow (initial part of UF1) was observed in the filling step using the fed-batch strategy (Figure 3), which may also contribute to the longer UF1 process time in the hybrid run. In contrast, both the UF1 process time (Figure 2B) and permeate flow (Figure 3) in the pseudo-batch run were nearly identical to those in the batch run. Since the DF and UF2 steps are always performed in the batch configuration, regardless of the system configuration in the filling (UF1) step, the diafiltration and UF2 process times were naturally nearly identical across all three runs (Figure 2B). The slight difference in diafiltration process time across the three runs was due to the low variability of protein concentration in the diafiltration step (49–51 g / L). These results illustrate the impact of the system configuration on the total process time in the initial sample filling (UF1) step, demonstrating the ability of the pseudobatch configuration to not only mitigate the time penalty associated with fed-batch filling in the UF1 step, but also to eliminate the process time scalability challenges associated with the hybrid (fed-batch filling) process.

[0148] B. Quality characteristics The effect of pseudobatch configuration on the quality characteristics of the active pharmaceutical ingredient was evaluated, and it was determined whether adverse effects would occur by incorporating a second pump (e.g., a supply pump) into the recirculation loop, where the protein solution passes through two pumps instead of one per recirculation cycle.

[0149] mAb A was found to be insensitive to exposure to pump shear for the formation of soluble high molecular weight (HMW) species in UF / DF. HMW levels remained essentially constant throughout the entire process for all three composition strategies (Figure 4). Therefore, increased exposure to pump shear associated with pseudobatch composition did not appear to result in adverse effects on HMW formation.

[0150] The effect of pseudobatch configuration on large insoluble aggregates was quantified by microfluidic imaging (MFI). The number of microscopically visible (1–100 μm) particles in the process pools generated using three configurations is shown in Figures 5A–D. Unlike what was observed in HMW formation, mAb A did not show a significant increase in microscopically visible particle formation over time, and both the total number of particles and the relative particle size distribution differed significantly between configurations.

[0151] In hybrid runs, particle counts were consistently higher than in batch runs across all particle size ranges, consistent with longer process times and corresponding increased protein exposure to shear and interfacial stresses. In contrast, pseudobatch runs produced particle counts equivalent to or lower than batch runs in the 1–25 μm size range (Figures 5A–C), except for the 50–100 μm size range (Figure 5D). The higher particle counts in the 50–100 μm size range may be potentially due to increased levels of (undetectable) aggregate precursors generated in the UF1 step as a result of passing through a dual pump compared to the other two packing strategies. However, it is important to note that the total particle count in the 50–100 μm size range is orders of magnitude lower than the particle count in the smaller particle size ranges. From an overall particle generation perspective, the pseudobatch configuration represents a significant improvement over the fedbatch configuration and appears to produce APIs of comparable quality to the batch configuration.

[0152] Interestingly, the largest difference in particle count among the three configurations occurs between DV5 and post-UF2, despite the fact that the UF2 step is the shortest part of the overall process (Figure 2B). Considering that all three configurations have identical process times and settings in both the DF and UF2 steps, such results may not be detectable by SEC / MFI, but demonstrate the importance of the UF1 step in the generation of aggregate precursors that affect the quality of the final drug substance.

[0153] Example 4 To determine whether the type of supply pump affects the pseudobatch process, a diaphragm supply pump was replaced with a peristaltic supply pump. A solution of approximately 180 g / L of mAb A was produced using a pseudobatch filling configuration with the same process parameters (e.g., membrane packing, packing concentration, diafiltration concentration, exchange diafiltration volume, pump supply flow rate, and TMP) as the pseudobatch run in Example 3. The volume ratio of the initial filling volume to the holding fluid tank volume was maintained at 5, as in Example 3 (i.e., the liquid volume of the holding fluid tank during the filling process was maintained at a constant value equal to 20% of the initial total filling volume). The process performance of this run was compared to that of the pseudobatch run using a diaphragm supply pump.

[0154] The approximate retaining solution mAb concentration as a function of process time (calculated from the retaining solution volume and total protein mass in the system) is shown in Figure 6A for two pseudobatch runs. The corresponding process times at each process stage (UF1, DF, and UF2) are shown in Figure 6B. The corresponding permeate flow as a function of the calculated retaining solution concentration is shown in Figure 7. The flow profiles and process times for the two runs are nearly identical at each stage of the UF / DF process, indicating that the choice of peristaltic or diaphragm pump for the supply pump does not affect the performance of the pseudobatch filling method in terms of process volume.

[0155] Furthermore, we evaluated the quality characteristics of the active pharmaceutical ingredient to determine whether the type of supply pump in the pseudobatch configuration significantly affects protein stability in the UF / DF process, which could be a determinant of the general practicality of the pseudobatch method. As can be seen from Figure 8, the use of a peristaltic supply pump did not increase HMW formation in UF / DF beyond that of a diaphragm supply pump (within the assay variability range). Therefore, it was not thought that the difference in shear exposure associated with the use of a peristaltic supply pump beyond that of a diaphragm supply pump would result in adverse effects on HMW formation.

[0156] The effect of the feed pump type on large insoluble aggregates was quantified using microfluidic imaging (MFI). The number of microscopically visible (1–100 μm) particles in the process pool generated using multiple feed pump types is shown in Figures 9A–9D. Unlike what was observed in HMW formation, both the total number of particles and the relative particle size distribution differed significantly between pump types. Peristaltic feed pumps generated significantly more small particles (<25 μm) compared to diaphragm pumps, but fewer large particles (>50 μm). This profile suggests that peristaltic pumps circulate the protein solution through the feed pump, resulting in higher shear or more turbulent flow patterns, which destabilize large particles and lead to a relative increase in the population of small particles. However, as mentioned above, the difference in particle size distribution does not appear to affect membrane flow or process rate. Since these particles are removed in the final formulation and filtration steps after the UF / DF process, differences in particle generation between the two pump types are unlikely to pose a concern regarding the quality of the final product.

[0157] Example 5 To determine whether the ratio of retained liquid to total filling volume in the filling process affects pseudobatch performance, the liquid volume of the retained liquid tank in the filling process was kept constant at a low volume relative to the total filling volume. A mAb A solution of approximately 180 g / L was produced using the pseudobatch filling method with the UF / DF process parameters and configuration (peristaltic supply pump) described in Example 4. However, the liquid volume of the retained liquid tank was maintained at 10% of the total initial filling volume in the filling portion of the UF1 process, rather than 20% in Example 4. The process performance of this run was compared to that of a pseudobatch run in which the retained liquid tank volume was maintained at 20% of the total initial filling volume in the filling process.

[0158] The approximate retaining solution mAb concentration as a function of process time (calculated from the retaining solution volume and total protein mass in the system) is shown in Figure 10A for two pseudobatch runs. The corresponding process times at each process stage (UF1, DF, and UF2) are shown in Figure 10B. The corresponding permeate flow as a function of the calculated retaining solution concentration is shown in Figure 11. The flow profiles and UF1 and UF2 process times for the two runs are nearly identical, indicating that the retaining solution tank to total filled volume ratio in the filling step does not affect the process time required to concentrate the protein. The small difference in dialysfiltration time may be due to slight variations in the actual dialysfiltration concentration around the target value of 50 g / L. This result differs from the fed-batch operation, where the UF1 process time depends on the relative volume ratio of the retaining solution tank and total filled volume, as previously described in Example 1. The independence of the UF1 process time from the relative retaining fluid and filling volume ratio in the filling process of Examples 4 and 5 is consistent with the main principle of the pseudo-batch method, where connecting the supply tank and retaining fluid tank in the UF / DF recirculation loop allows them to function efficiently as a single reservoir, thereby converting the filling process into a batch process.

[0159] Furthermore, the quality characteristics of the active pharmaceutical ingredient were evaluated to determine whether the ratio of retained liquid to total filling volume in the filling process affected protein stability in the UF / DF process. As can be seen in Figure 12, in the run where the retained liquid volume was maintained at 10% of the initial filling volume, the HMW level was the same as in the run performed with a 20% volume ratio. Similarly, the particle size distribution (1-100 μm) of the solution after UF2, quantified by MFI, was almost identical between the two runs, as seen in Figures 13A-13D. Since the protein is consequently affected by the passage of the same number of pumps and other stressors between the two runs, the similarity of the HMW and large particle profiles between the two runs may be partly attributable to the identical UF1 process time (Figure 10B) and the concentration profile as a function of process time (Figure 10A).

[0160] Example 6 The pseudo-batch method was performed on a non-mAb protein formulation (MW = approximately 20 Da). This non-mAb protein formulation was used as the packing material. The protein was concentrated from 0.7 to 15.5 g / L using both the traditional fed-batch and pseudo-batch methods. No buffer exchange was performed in these two runs. All other process parameters (membrane packing, pump feed flow, TMP) were kept constant between the two runs. The liquid volume of the holding fluid tank was kept constant at 30% of the total initial packing volume throughout the packing process in both runs, and a peristaltic pump was used as the feed pump.

[0161] The approximate retention solution protein concentration as a function of process time (calculated from the retention solution volume and total protein mass in the system) is shown in Figure 14 for two runs. The corresponding process times were 3.0 hours for the fed-batch run and 2.9 hours for the pseudo-batch run. The permeate flow as a function of the calculated retention solution concentration is shown in Figure 15. The identical flow-to-concentration profiles are consistent with findings from previous examples, indicating that differences in the packing method do not affect the intrinsic membrane performance. In this example, the process packing step was performed over a very narrow and dilute concentration range (0.7-2 g / L) where the attenuating flow over this concentration range was essentially negligible, resulting in nearly identical average permeate flow during the packing step and thus similar process times between the two runs. However, the pseudo-batch packing strategy offers further advantages in reducing process time compared to the fed-batch packing method, even though the difference in this case is very small (approximately 0.1 hours) due to the very low concentration range over which the packing step is performed.

[0162] Furthermore, the quality characteristics of the active pharmaceutical ingredient were evaluated to determine whether the pseudobatch filling strategy had a detrimental effect on the stability of non-mAb proteins in UF / DF as a result of increasing the number of pump passes the protein undergoes. As can be seen in Figure 16, the pseudobatch and fedbatch methods produced equivalent amounts of HMW species. However, the two methods differed in the amount and relative size distribution of large particles formed in UF / DF when characterized by MFI. As seen in Figures 17A to 17D, the pseudobatch filling method produced smaller particles (<25 μm) than the fedbatch method, but an equivalent amount of large particles (>50 μm). This result is consistent with what was observed for mAb A in Examples 3 to 5. The additional number of pump passes inherent in the pseudobatch filling method increased the formation of small particles, but not large particles. Since the manufacturing bioprocess typically includes final formulation and filtration steps after the UF / DF process, such particles are expected to be removed from the final active pharmaceutical ingredient. Therefore, the increase in small particle formation resulting from the pseudo-batch method is not expected to have any adverse effects on the quality of the final active pharmaceutical ingredient.

[0163] The pseudo-batch configuration for UF / DF described herein allows for the mitigation of the time penalty associated with fed-batch filling by converting the fed-batch setup (using a supply tank and a holding liquid container) to a batch-like operation (connecting two containers to act as a single container in the TFF recirculation loop). This conversion also eliminates the scale dependence of process time associated with hybrid processes (e.g., fed-batch filling + batch concentration), making the process fully scalable. In addition, the incorporation of a supply pump into the recirculation loop, resulting in twice the pump pass-through compared to the batch configuration, did not cause any significant adverse effects on product quality, as quantified by HMW formation and visible particle count under a microscope.

[0164] Hybrid UF / DF processes for generating high-concentration active pharmaceutical ingredients will inevitably become more widespread as the biopharmaceutical industry increasingly shifts to subcutaneous forms for drug delivery. By using the pseudo-batch configuration described herein instead, it is possible to reduce the time penalty associated with fed-batch filling, eliminate the scale dependence of process time arising from hybrid processes, and potentially improve product quality compared to hybrid processes. In summary, these advantages can not only improve process volume and yield but also enhance the scalability of UF / DF processes for a more streamlined technology transition from laboratory / development scale to production scale.

[0165] The descriptions of the particular embodiments described herein make it fairly clear to those skilled in the art that the general properties of the invention can be readily modified and / or adapted to various applications, for example, to specific embodiments, without the need for excessive experimentation and without departing from the general concept of the invention, by applying knowledge within the art. Therefore, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the embodiments disclosed, based on the teachings and guidance provided herein. The expressions and terms herein are for illustrative purposes only and not restrictive purposes, and it should be understood that the terms and expressions herein are to be interpreted by those skilled in the art in consideration of the teachings and guidance provided herein.

[0166] All published documents, patents, and patent applications referenced herein are incorporated by reference in the same manner as if they were specifically and individually indicated as such.

[0167] Other aspects of the present invention will become apparent to those skilled in the art from the specification and practical considerations of the present invention disclosed herein. The specification and examples are intended to be considered merely illustrative, and the substantial scope and spirit of the present invention are set forth by the following claims. This disclosure may provide the following: [Section 1] A method for reducing the filtration process time of a target protein, comprising continuously filling a supply tank with a protein mixture containing the target protein that has been filtered at least once ("retention liquid"), wherein the supply tank is separated from a main reservoir ("retention liquid") tank. [Section 2] A method for concentrating a target protein, comprising continuously filling a supply tank with a protein mixture containing the target protein that has been filtered at least once ("retention solution"), wherein the supply tank is separated from a main reservoir ("retention solution") tank. [Section 3] The method according to claim 1 or 2, wherein the supply tank further comprises an initial protein mixture containing the protein of interest that has not been filtered at least once. [Section 4] The method according to item 3, wherein the initial protein mixture and the retention solution are mixed. [Section 5] The method according to item 3 or 4, wherein the protein mixture and / or retention solution are filtered by a filter. [Section 6] The method according to item 5, wherein the filtered protein mixture and retention solution ("retention solution") are filled into a supply tank. [Section 7] The method according to any one of claims 1 to 6, wherein the filling is continued until the protein of interest is concentrated to at least about 1 mg / mL, at least about 10 mg / mL, at least about 20 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, or at least about 80 mg / mL. [Section 8] The method according to any one of claims 1 to 7, wherein the filling is continued until the protein of interest is concentrated to approximately 1 mg / mL to 80 mg / mL, approximately 5 mg / mL to 70 mg / mL, approximately 10 mg / mL to 60 mg / mL, approximately 10 mg / mL to 50 mg / mL, approximately 10 mg / mL to 40 mg / mL, approximately 10 mg / mL to 30 mg / mL, approximately 10 mg / mL to 20 mg / mL, approximately 20 mg / mL to 70 mg / mL, approximately 20 mg / mL to 60 mg / mL, approximately 20 mg / mL to 50 mg / mL, approximately 20 mg / mL to 40 mg / mL, or approximately 20 mg / mL to 30 mg / mL. [Section 9] The method according to any one of claims 1 to 8, wherein the filling of the retaining fluid is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 times. [Section 10] The method according to any one of claims 1 to 9, further comprising stopping the filling of the retaining fluid into the supply tank. [Section 11] The method according to claim 10, further comprising directing the retaining fluid to a reservoir tank. [Section 12] A method for reducing the filtration process time of a target protein, comprising filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve for connecting the filter to the supply tank and a reservoir tank valve for connecting the filter to the reservoir tank, and a reservoir input for connecting the supply tank and the reservoir tank, with a protein mixture containing the target protein. [Section 13] A method for concentrating a target protein, comprising filling a filtration system, which includes a supply tank, a reservoir tank, a filter, a three-way valve including a supply tank valve for connecting the filter to the supply tank and a reservoir tank valve for connecting the filter to the reservoir tank, and a reservoir input for connecting the supply tank and the reservoir tank, with a protein mixture containing the target protein. [Section 14] The method according to item 12 or 13, wherein the reservoir tank valve is closed until the protein of interest is sufficiently concentrated. [Section 15] The method according to any one of claims 12 to 14, further comprising frequently adding the protein mixture to the supply tank. [Section 16] The method according to any one of claims 12 to 15, for directing a protein mixture from a supply tank to a reservoir tank. [Section 17] The method according to any one of items 11 to 16, wherein the reservoir tank is connected to the filter. [Section 18] The method according to item 17, wherein the filter includes an in-line filtration membrane. [Section 19] The method according to item 18, wherein the inline filtration membrane is an ultrafiltration membrane. [Section 20] The method according to claim 18 or 19, wherein the inline filtration membrane is polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultrananocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrenedivinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose, or a combination thereof. [Section 21] The method according to any one of claims 18 to 20, wherein the filtration membrane has a molecular weight cutoff (MWCO) of approximately 50 kD to approximately 5 kD, approximately 50 kD, approximately 40 kD, approximately 30 kD, approximately 20 kD, approximately 10 kD, or less than approximately 5 kD. [Section 22] The method described in paragraph 21, wherein the MWCO is less than approximately 5kD. [Section 23] The method according to any one of claims 1 to 22, wherein the mixture is allowed to flow until a desired filtration protein concentration is reached. [Section 24] The method according to item 23, wherein the desired filtration protein concentration is about 10 mg / mL to about 300 mg / mL, for example, about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 250 mg / mL, or about 300 mg / mL. [Section 25] The method according to item 24, wherein the desired filtration protein concentration is approximately 150 mg / mL. [Section 26] The method according to any one of items 23 to 25, wherein the protein viscosity is approximately 0 cP to approximately 200 cP. [Section 27] The method according to item 26, wherein the protein viscosity is approximately 20 cP to approximately 60 cP. [Section 28] The method according to any one of items 1 to 27, wherein the volume ratio between the supply tank volume and the reservoir volume is approximately 1:2 to approximately 10:1, approximately 1:2 to approximately 1:1, approximately 1:1 to approximately 1:2, approximately 1:1 to approximately 1:3, approximately 1:1 to approximately 1:4, approximately 1:1 to approximately 1:5, approximately 1:1 to approximately 1:6, approximately 1:1 to approximately 1:7, approximately 1:1 to approximately 1:8, approximately 1:1 to approximately 1:9, or approximately 1:1 to approximately 1:10. [Section 29] The method according to any one of claims 1 to 27, wherein the capacity ratio between the supply tank capacity and the reservoir capacity is approximately 1:1, approximately 2:1, or approximately 5:1. [Section 30] The method according to any one of claims 16 to 29, wherein the protein mixture is directed to a reservoir tank and / or filter using a diaphragm pump, rotary lobe pump, or peristaltic pump. [Section 31] The method according to any one of claims 1 to 30, further comprising filling the feed tank with an initial protein mixture containing the protein of interest that has not been filtered at least once, prior to the continuous filling of the feed tank with a protein mixture containing the protein of interest that has been filtered at least once ("retaining solution"). [Section 32] The method according to item 31, wherein the initial protein mixture is added to the supply tank at a concentration of approximately 1 mg / mL to approximately 30 mg / mL. [Section 33] The method according to item 32, wherein the initial protein mixture is added to the supply tank at a concentration of approximately 5 mg / mL. [Section 34] The method according to any one of claims 1 to 33, wherein the process time is reduced by approximately 1%, approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, or approximately 50% compared to the process time of the Fed batch enrichment process. [Section 35] The method according to item 34, wherein the process time is reduced by approximately 40% compared to the process time of the Fed batch enrichment process. [Section 36] The method according to any one of claims 1 to 33, wherein the process time is reduced by approximately 0.2 hours, approximately 0.4 hours, approximately 0.5 hours, approximately 0.6 hours, approximately 0.8 hours, or approximately 1.0 hour compared to the process time of the Fed batch enrichment process. [Section 37] The method according to item 36, wherein the process time is reduced by approximately 0.5 hours compared to the process time of the Fed batch enrichment process. [Section 38] The method according to any one of claims 1 to 37, wherein the number of 1-2 μm particles is reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, or approximately 50% compared to the number of particles in the fed batch enrichment process. [Section 39] The method according to any one of claims 1 to 38, wherein the number of particles between 5 and 10 μm is reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, or approximately 50% compared to the number of particles in the fed batch enrichment process. [Section 40] The method according to any one of claims 1 to 39, wherein the number of particles between 10 and 25 μm is reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, or approximately 50% compared to the number of particles in the fed batch enrichment process. [Section 41] The method according to any one of claims 1 to 40, wherein the protein mixture comprises an antibody, an antibody fragment, an antigen-binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof. [Section 42] The method according to item 41, wherein the protein mixture comprises an antibody selected from IgM, IgA, IgE, IgD, and IgG. [Section 43] The method according to claim 42, wherein the protein mixture comprises an antibody, the antibody being an IgG antibody selected from IgG1, IgG2, IgG3, and IgG4. [Section 44] The method according to any one of claims 41 to 43, wherein the antibody comprises a bivariate domain immunoglobulin. [Section 45] The method according to any one of items 41 to 43, wherein the antibody comprises a trivalent antibody. [Section 46] The method according to item 41, wherein the antibody or antibody fragment comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-IL-8 antibody, or a fragment thereof. [Section 47] The method according to any one of claims 41 to 46, wherein the protein mixture is derived from a bacterial, yeast, insect or mammalian cell culture. [Section 48] The method according to item 47, wherein the mammalian cell culture is a Chinese hamster ovary (CHO) cell culture. [Section 49] The method according to any one of claims 1 to 48, wherein the protein mixture is obtained from a batch of cell cultures. [Section 50] The method according to any one of claims 1 to 49, wherein the protein mixture is obtained from a fed batch cell culture. [Section 51] The method according to any one of claims 1 to 50, wherein a protein mixture is produced in a bioreactor. [Section 52] The method according to item 51, wherein a protein mixture is produced in a disposable bioreactor. [Section 53] The method according to any one of claims 1 to 48, wherein the protein mixture is obtained from a perfused cell culture. [Section 54] The method according to item 53, wherein a protein mixture is produced in a perfusion or TFF perfusion bioreactor. [Section 55] The method according to any one of claims 47 to 54, wherein the protein mixture is produced by a cell culture that persists for approximately 1 to 60 days. [Section 56] The method according to item 55, wherein the protein mixture is produced by a cell culture that persists for approximately 25 days. [Section 57] The method according to any one of claims 1 to 54, wherein the protein mixture is added to a feed tank together with the loading buffer. [Section 58] The method according to item 57, wherein the loading buffer comprises an amino acid, a weak acid, a weak base and / or a sugar. [Section 59] The method according to any one of claims 1 to 58, further comprising formulating a protein into a pharmaceutical composition. [Section 60] Protein prepared by the method described in any one of items 1 to 59. [Section 61] A pharmaceutical composition containing the proteins described in items 1 to 60. [Section 62] A method of administering the pharmaceutical composition described in item 61 to a subject requiring it. [Section 63] A method for treating a disease or symptom in a subject requiring the use of the pharmaceutical composition described in item 61. [Section 64] supply tank, A reservoir tank connected to the supply tank by a first fluid path, A filtration membrane connected to the reservoir tank by a second fluid path, and A three-way valve connected to the filter membrane via a third fluid path, to the reservoir tank via a fourth fluid path, and to the supply tank via a fifth fluid path. A system for concentrating a target protein, including, The reservoir tank receives a protein mixture containing the target protein from the supply tank via the first fluid path. The filtration membrane receives the protein mixture containing the target protein from the reservoir tank via a second fluid path and filters the protein mixture. A system in which the three-way valve receives the retaining fluid from the filter via the third fluid path and directs the retaining fluid to either the reservoir tank via the fourth fluid path or the supply tank via the fifth fluid path. [Section 65] The system according to paragraph 64, wherein a three-way valve directs the retaining fluid to the reservoir tank if the total volume of the protein mixture in the system is less than the capacity of the reservoir tank, and directs the retaining fluid to the supply tank if the total volume of the protein mixture in the system is greater than the capacity of the reservoir tank. [Section 66] The system according to any one of claims 64 to 65, further comprising a sensor configured to determine the total volume and / or concentration of a protein mixture in the system, wherein a three-way valve automatically directs the retaining fluid to either a reservoir tank or a supply tank based on feedback from the sensor. [Section 67] The system according to any one of claims 64 to 66, further comprising one or more diaphragm pumps, rotary lobe pumps, or peristaltic pumps. [Section 68] The system according to any one of claims 64 to 67, wherein the filter includes an inline filtration membrane. [Section 69] The system according to item 68, wherein the inline filtration membrane is an ultrafiltration membrane. [Section 70] The system according to item 69, wherein the inline filtration membrane is polyvinyl ether, polyvinyl alcohol, nylon, silicon, polysilicon, ultra-nanocrystalline diamond, diamond-like carbon, silicon dioxide, titanium, silica, silicon nitride, polytetrafluoroethylene, silicone, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polycarbonate, graphene, graphene oxide, polysaccharide, ceramic particles, poly(styrenedivinyl)benzene, polysulfone, polyethersulfone, modified polyethersulfone, polyarylsulfone, polyphenylsulfone, polyvinyl chloride, polypropylene, cellulose acetate, cellulose nitrate, polylactic acid, polyacrylonitrile, polyvinylidene fluoride, polypiperazine, polyamide-polyether block polymer, polyimide, polyetherimide, polyamide, regenerated cellulose, composite regenerated cellulose, or a combination thereof.

Claims

1. supply tank, A reservoir tank connected to the supply tank by a first fluid path, A filtration membrane connected to the reservoir tank by a second fluid path, and A three-way valve connected to the filter membrane via a third fluid path, to the reservoir tank via a fourth fluid path, and to the supply tank via a fifth fluid path. A system for concentrating a target protein, including, The reservoir tank receives a protein mixture containing the target protein from the supply tank via a first fluid path. The filtration membrane receives the protein mixture containing the target protein from the reservoir tank via a second fluid path and filters the protein mixture. A system in which the three-way valve receives the retaining liquid from the filtration membrane via a third fluid path and directs the retaining liquid to either the reservoir tank via a fourth fluid path or the supply tank via a fifth fluid path.

2. The system according to claim 1, wherein a three-way valve directs the retaining liquid to the reservoir tank when the total volume of the protein mixture in the system is less than the capacity of the reservoir tank, and directs the retaining liquid to the supply tank when the total volume of the protein mixture in the system is greater than the capacity of the reservoir tank.

3. A method for concentrating a target protein, comprising loading the target protein into a filtration system, wherein the filtration system is supply tank, A reservoir tank connected to the supply tank by a first fluid path, A filtration membrane connected to the reservoir tank by a second fluid path, and A three-way valve connected to the filter membrane via a third fluid path, to the reservoir tank via a fourth fluid path, and to the supply tank via a fifth fluid path. Includes, The reservoir tank receives a protein mixture containing the target protein from the supply tank via a first fluid path. The filtration membrane receives the protein mixture containing the target protein from the reservoir tank via a second fluid path and filters the protein mixture. A method wherein the three-way valve receives the retaining liquid from the filtration membrane via a third fluid path and directs the retaining liquid to either the reservoir tank via a fourth fluid path or the supply tank via a fifth fluid path.

4. A method for shortening the filtration process time of a target protein, comprising loading the target protein into a filtration system, The aforementioned filtration system supply tank, A reservoir tank connected to the supply tank by a first fluid path, A filtration membrane connected to the reservoir tank by a second fluid path, and A three-way valve connected to the filter membrane via a third fluid path, to the reservoir tank via a fourth fluid path, and to the supply tank via a fifth fluid path. Includes, The reservoir tank receives a protein mixture containing the target protein from the supply tank via a first fluid path. The filtration membrane receives the protein mixture containing the target protein from the reservoir tank via a second fluid path and filters the protein mixture. A method wherein the three-way valve receives the retaining liquid from the filtration membrane via a third fluid path and directs the retaining liquid to either the reservoir tank via a fourth fluid path or the supply tank via a fifth fluid path.

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