System and method for end-to-end continuous downstream processing

Abstract

Methods for purifying a target protein using continuous countercurrent downstream processing are provided herein. Methods for purifying a target protein using continuous countercurrent affinity nanoparticle dialysis are also provided herein.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 366,159, filed on June 10, 2022, the entire content of which is incorporated herein by reference in its entirety for all purposes as part of this specification.

[0002] Incorporation by Reference of Electronically Submitted Materials A computer - readable nucleotide / amino acid sequence list identified as a 50,990 - byte xml file named "CMTFF - 100 - WO - PCT.xml", created on June 7, 2023 and submitted simultaneously with this specification, is incorporated herein by reference in its entirety for all purposes as part of this specification.

[0003] As biology moves to the forefront of drug development, the need for improved manufacturing processes has been increasing. As the predicted demand for recombinant protein therapeutics grows, a more cost - effective and flexible manufacturing process is required. In fact, various economic analyses assume that process development and clinical manufacturing costs can constitute 40 - 60 percent of drug development costs. In addition to commercial manufacturing, which is mainly driven by downstream processing of consumable material costs, this can reach up to 25 percent of the sales revenue of biological formulations. Therefore, more efficient downstream processing is required.

Summary of the Invention

[0004] The present disclosure relates to a method for purifying a product of interest using countercurrent filtration, comprising: (a) contacting a first solution containing the product of interest and impurities with a binding molecule to form a complex, the complex containing the product of interest bound to the binding molecule; (b) contacting a first flowing solution containing the complex with a first side of a semipermeable membrane, the complex having a molecular weight greater than the molecular weight cut-off of the membrane such that the complex is retained on the first side of the semipermeable membrane; (c) passing the impurities through the semipermeable membrane, the impurities having a molecular weight less than the molecular weight cut-off of the semipermeable membrane and being retained on a second side of the semipermeable membrane in a second flowing solution that is countercurrent to the first flowing solution; and (d) dissociating the complex to form the free product of interest and free binding molecule.

[0005] In one aspect, the method further comprises: (e) regenerating the binding molecule, the regenerated binding molecule being capable of binding a complex upon contact with the product of interest in the first solution or the second solution.

[0006] The present disclosure also provides a method for purifying a product of interest using countercurrent filtration, comprising: (a) contacting a first flowing solution containing the product of interest and impurities with a first side of a semipermeable membrane, such that the product of interest passes through the semipermeable membrane to form a complex containing a binding molecule on a second side of the semipermeable membrane, and the complex has a molecular weight exceeding the molecular weight cut-off of the semipermeable membrane so that the complex is retained on the second side of the semipermeable membrane; (b) optionally, retaining the impurities on the first side of the semipermeable membrane, or the impurities flow through the semipermeable membrane, wherein the impurities have a molecular weight less than the molecular weight cut-off of the semipermeable membrane and are retained on the first side of the semipermeable membrane in the first flowing solution that is countercurrent to the second flowing solution, or pass through the semipermeable membrane towards the second side of the semipermeable membrane, and the flow rate of the second flowing solution is lower than the flow rate of the first flowing solution; (c) dissociating the complex to form the free product of interest and free binding molecules; and the method further comprises: (e) regenerating the binding molecule, wherein the regenerated binding molecule is capable of binding the complex upon contact with the product of interest in the first flowing solution or the second flowing solution. In some embodiments, the regenerated binding molecule passes through the second side of the semipermeable membrane again in the second flowing solution.

[0007] In one embodiment, the second flowing solution contains a second binding molecule capable of binding impurities in the first solution and / or the second flowing solution. In another embodiment, the unbound binding molecule diffuses through the semipermeable membrane into the second flowing solution.

[0008] In some embodiments, the product of interest is a protein. In one embodiment, the binding molecule comprises Protein A, Protein G, a cation exchange resin, or an anion exchange resin. In another embodiment, the binding molecule comprises Protein A. In one embodiment, the first solution containing the product of interest is obtained from a bioreactor. In one embodiment, the second flowing solution contains a positively charged polymer. In another embodiment, the positively charged polymer is DEAE dextran.

[0009] In one aspect, the impurities include low molecular weight species. In another aspect, the positively charged polymer binds to the low molecular weight species that have diffused through the semipermeable membrane.

[0010] In one aspect, steps (a) to (c) are repeated and the binding molecule includes Protein A. In another aspect, steps (a) to (c) are repeated and the binding molecule includes a cation exchange resin. In another aspect, steps (a) to (c) are repeated and the binding molecule includes an anion exchange resin.

[0011] In some embodiments, the binding molecule comprises assembled nanoparticles. In some embodiments, the assembled nanoparticles are assembled ferritin nanoparticles comprising 24 fusion protein monomers. In some embodiments, each fusion protein monomer comprises: i) a self-assembling nanoparticle monomer; ii) a linker; and iii) an immunoglobulin binding domain. In some embodiments, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 1-3. In some embodiments, the immunoglobulin binding domain is the Z domain of Protein A. In some embodiments, the Z domain of Protein A comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 4-7. In some embodiments, the linker comprises an amino acid sequence selected from one of SEQ ID NOs: 8-27. In some embodiments, the unfused and / or unassembled fusion protein monomers diffuse through a semipermeable membrane into a second flowing solution. In some embodiments, the fusion protein monomer further comprises a purification tag at one end of the fusion protein monomer. In some embodiments, the purification tag comprises 6, 8 or 10 repeating histidines. In some embodiments, the fusion protein monomer further comprises a protease site between the purification tag and the remainder of the fusion protein monomer. In some embodiments, the fusion protein monomer comprises a protease site that is the HRV-3C protease site. In some embodiments, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 28-36. In some embodiments, the fusion protein monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 32, or is encoded by a nucleic acid comprising SEQ ID NO: 52. In some embodiments, the fusion protein monomer comprises 2-5 immunoglobulin binding domains. In some embodiments, the 2-5 immunoglobulin binding domains are the Z domains of Protein A. In some embodiments, the 2-5 immunoglobulin binding domains are separated from each other by a linker.In some embodiments, the fusion protein monomers can assemble into aggregate nanoparticles containing 24 fusion protein monomers.

[0012] In some embodiments, steps (b) and (c) are repeated. In some embodiments, steps (b) and (c) are repeated 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times.

[0013] In some embodiments, the permeate from step (b) and / or step (c) is recycled to the first flowing solution and / or the upstream flowing solution. In some embodiments, the permeate from step (b) and / or step (c) is recycled to the first flowing solution and / or the second flowing solution and / or the upstream solution. In one embodiment, the dissociated protein is diafiltered. In some embodiments, the semipermeable membrane is a dialysis membrane.

[0014] In one embodiment, the flow rate of the first flowing solution and / or the second flowing solution is about 30 to about 60 mL / min. In another embodiment, the flow rates of the first flowing solution and the second flowing solution are the same. In another embodiment, the flow rates of the first flowing solution and the second flowing solution are different. In a further embodiment, the first flowing solution and the second solution are pulsed. In another embodiment, the pulsing improves mass transfer across the semipermeable membrane. In another embodiment, the pulse volume is smaller than the volume of the pores of the semipermeable membrane. In another embodiment, the pulse volume is about half of the volume of the pores of the semipermeable membrane. In another embodiment, the pulse volume is larger than the volume of the pores of the semipermeable membrane.

[0015] In one aspect, a target product of about 0.1 kg / day, about 0.5 kg / day, about 1 kg / day, about 2 kg / day, about 3 kg / day, about 4 kg / day, about 5 kg / day, about 6 kg / day, about 7 kg / day, about 8 kg / day, about 9 kg / day or about 10 kg / day is purified. In another aspect, the target product includes an antibody, an antigen-binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein or any combination thereof. In another aspect, the protein includes an antibody selected from IgM, IgA, IgE, IgD and IgG, and optionally about 10 mM to about 1 M NaCl, or more preferably about 150 mM NaCl is added to the first flowing solution. In another aspect, the protein includes an antibody, and the antibody is an IgG antibody selected from IgG1, IgG2, IgG3 and IgG4. In another aspect, the antibody is a monoclonal antibody.

[0016] In one aspect, the first flowing solution is concentrated to the target product of about 50 g / L to about 100 g / L. In one aspect, the target product is a monoclonal antibody. In one aspect, the free target product is concentrated to about 50 g / L to about 100 g / L.

[0017] In one aspect, the complex has a molecular weight that is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times the size of the molecular weight cut-off of the semipermeable membrane. In some aspects, steps (a) - (c) are performed a second time, the second flowing solution of the second time is added to the first flowing solution of the first time, and the MWCO of the filter used the second time is the same size as or has a larger MWCO than the steps (a) - (c) performed the first time. In some aspects, steps (a) - (c) are performed a second time, the first flowing solution of the second time is added to the first flowing solution of the first time, and the MWCO of the filter used the second time is the same size as or has a larger MWCO than the steps (a) - (c) performed the first time. In some aspects, steps (a) - (c) are performed a third time, the second flowing solution of the third time is added to the first flowing solution of the second time, and the MWCO of the filter used the third time is the same size as or has a larger MWCO than the steps (a) - (c) performed the first or second time. In some aspects, steps (a) - (c) are performed a third time, the first flowing solution of the third time is added to the first flowing solution of the second time, and the MWCO of the filter used the third time is the same size as or has a larger MWCO than the steps (a) - (c) performed the first or second time. In some aspects, the second and third times of steps (a) - (c) are performed continuously. In some aspects, the first flowing solution or the second flowing solution of the third time flows into the first flowing solution of the second time, and / or the first flowing solution or the second flowing solution of the second time flows into the first flowing solution of the first time.

[0018] In one aspect, the present disclosure provides a method of using a solution that flows out from the filtrate or dialysate of a continuous downstream process as a wash for an upstream process, where the upstream process has a filter with an MWCO that is the same size as or larger than the downstream process that produced the effluent.

[0019] In one aspect, the present disclosure provides a method of using a solution that has flowed out of a retention liquid from a continuous downstream process as a wash for an upstream process, where the upstream process has a filter with a MWCO that is the same size or smaller than the downstream process that produced the effluent, and the filtrate is directed downstream. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Mode for Carrying Out the Invention

[0021] The present disclosure provides a highly effective approach for removing contaminants during protein purification using countercurrent filtration without the need for chromatography. Accordingly, the present disclosure provides a method for purifying a target product that uses approximately 1 / 10 the amount of water and solution of a chromatography process.

[0022] I. Definitions To more readily understand the present disclosure, certain terms are first defined. As used herein, unless explicitly provided otherwise herein, each of the following terms shall have the meaning set forth below. Throughout this specification, further definitions are provided.

[0023] Note that terms such as "a" or "an" refer to one or more of that entity. For example, "feed medium" is understood to represent one or more feed media. Accordingly, terms such as "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0024] As used herein, the term "and / or" shall be construed as a specific disclosure where each of the two specified features or components is either with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" shall include each of the following aspects: 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).

[0025] Whenever an aspect is described in this specification using the word "comprising", it is understood that other similar aspects regarding "consisting of" and / or "consisting essentially of" are also provided.

[0026] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in this disclosure to those of ordinary skill in the art.

[0027] Units, prefixes, and symbols are shown in the form recognized by the International System of Units (SI). Numerical ranges include the numbers defining the range. The headings provided in this specification do not limit the various aspects of the disclosure and can be obtained by referring to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by referring to the entire specification.

[0028] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of their alternatives. As used in this specification, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any listed or enumerated components.

[0029] The terms "about" or "essentially comprises" refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measuring system. For example, "about" or "essentially comprises" can mean within one or more standard deviations in each practice in the art. Alternatively, "about" or "essentially comprises" can mean a range up to 20%. Further, especially with respect to biological systems or processes, these terms can mean up to one order of magnitude or up to five times the value, whichever is greater. When a particular value or composition is provided in the present application and the claims, unless otherwise stated, the meaning of "about" or "essentially comprises" should be assumed to be within the acceptable error range for that particular value or composition.

[0030] As described herein, any concentration range, range in percentages, ratio range or integer range should be understood to include any integer value within the recited range and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0031] The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally or otherwise modified by intervening modifications such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation to a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included in this definition. The terms "polypeptide" and "protein" as used herein specifically include antibodies and Fc domain-containing polypeptides (e.g., immunoadhesins).

[0032] "Anion exchange resin" or "anion exchange polymer" refers to a positively charged solid or liquid phase, thereby having one or more positively charged ligands bound to the solid phase. Any positively charged ligand (such as a quaternary amino group) bound to a solid phase suitable for forming an anion exchange resin can be used.

[0033] "Cation exchange resin" or "cation exchange polymer" refers to a negatively charged solid phase having free cations for exchanging with cations in an aqueous solution that pass over or through the solid phase. Any negatively charged ligand (such as carboxylate, sulfonate and others described herein) bound to a solid phase suitable for forming a cation exchange resin can be used.

[0034] As used herein, the term "product of interest" is used in its broadest sense to include any product (either natural or recombinant) containing a protein, nucleic acid, polymer, or lipid present in a mixture for which purification is desired. Such products of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (such as antibodies), and / or any fusion protein. Such products of interest may also include DNA, RNA, cDNA and antisense oligonucleotides. In some embodiments, the product of interest refers to any protein that can be produced by the methods described herein. In some embodiments, the product of interest refers to any DNA or RNA sequence that can be produced by the methods described herein. In some embodiments, the product of interest is the protein of interest.

[0035] As used herein, terms such as "protein of interest" are used in their broadest sense to include any protein (either natural or recombinant) present in a mixture for which purification is desired. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins. In some embodiments, the protein of interest refers to any protein that can be produced by the methods described herein. In some embodiments, the protein of interest is an antibody. In some embodiments, the protein of interest is a recombinant protein.

[0036] Terms such as "purify", "separate", or "isolate", as used interchangeably herein, refer to increasing the degree of purity of a product of interest from a composition or sample that includes the product of interest and one or more impurities. Typically, the purity of the product of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0037] As used herein, the term "buffer" refers to a substance that, by being present in a solution, increases the amount of acid or base that must be added to cause a unit change in pH. A buffer solution resists changes in pH by the action of its acid-base conjugate components. A buffered solution for use with biological reagents can generally maintain a constant concentration of hydrogen ions such that the pH of the solution is within a physiological range. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0038] As used herein, terms such as "continuous countercurrent modular tangential flow filtration" or "CMTFF" refer to the general techniques described herein where tangential flow filtration is used, optionally in series or parallel, in one or more individual implementations, and the flow of the permeate of the membrane flows countercurrent to the retentate side of the membrane.

[0039] As used herein, terms such as "C3ANDo" or "Continuous Counterflow Affinity Nanoparticle Dialysis" refer to the general dialysis approaches used herein, and in one or more individual embodiments, optionally in series or parallel, the flow on the lumen side of the dialysis module flows countercurrently to the shell side of the module.

[0040] As used herein, terms such as "sProA" or "soluble protein A" refer to any protein A that is soluble in solution. Thus, typically, protein A is not bound to a chromatography resin or membrane. In some embodiments, protein A is derived from Staphylococcus. Soluble protein A retains the ability to bind to the Fc region of IgG immunoglobulins.

[0041] As used herein, the term "contaminant" is used in its broadest sense to include any unwanted component or compound within a mixture. In cell cultures, cell lysates, or clarified bulk (e.g., clarified cell culture supernatant), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in the cell culture medium. Host cell contaminant proteins include those produced naturally or recombinantly by the host cell, as well as proteins (e.g., proteolytic fragments) related to or derived from the product of interest and other process-related contaminants, but are not limited thereto. In certain embodiments, precipitation of contaminants is separated from the cell culture using other means such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration.

[0042] The term "HMW species" refers to any one or more unwanted proteins present in a mixture. High molecular weight species can include dimers, trimers, tetramers, or other multimers. These species are often considered impurities related to the product, which can be bound by either covalent or non-covalent bonds, and can consist of, for example, misfolded monomers where hydrophobic amino acid residues are exposed to a polar solvent, which can cause aggregation.

[0043] The term "LMW species" refers to any one or more unwanted species present in a mixture. Low molecular weight species are often considered product-related impurities and may include clipped species, charge variants, or half-molecules for compounds (such as monoclonal antibodies) that are intended to be dimers.

[0044] The term "host cell protein" or HCP refers to unwanted proteins produced by host cells that are not related to the production of the intended product of interest. Unwanted host cell proteins can be secreted into the supernatant of the upstream cell culture. Unwanted host cell proteins can also be released during cell lysis. The cells used in upstream cell culture require proteins for growth, transcription, and protein synthesis, and these irrelevant proteins are unwanted in the final drug product.

[0045] As used herein, the terms "fed-batch culture" or "fed-batch culture process" 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. A fed-batch culture can be started using a basal medium. The medium to which additional components are provided to the culture at some point after the start of the culture process is the feed medium. A fed-batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0046] The term "chromatography" refers to any kind of technique for separating a target protein (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Usually, the target protein is separated from other molecules (e.g., contaminants) as a result of differences in the rates at which the individual molecules of the mixture move through a stationary medium under the influence of a mobile phase or in a binding and elution process. The term "matrix" or "chromatography matrix" refers to any kind of adsorbent, resin or solid phase for separating a target protein (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in a mixture in a separation process. Non-limiting examples include particulate, monolithic or fibrous resins, and membranes that can be placed in a column or cartridge. Examples of materials for forming a matrix include polysaccharides (e.g., agarose and cellulose); silica (e.g., controlled pore glass), poly(styrene divinyl) benzene, polyacrylamide, ceramic particles divinyl) benzene, polyacrylamide, ceramic particles and other mechanically stable matrices such as derivatives of any of the above. Examples of typical matrix types suitable for the methods of the present disclosure are cation exchange resins, affinity resins, anion exchange resins or mixed mode resins.

[0047] "Ligand" is a functional group that binds to a chromatography matrix and determines the binding characteristics of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). Some preferred ligands that can be used herein include strong cation exchange groups such as sulfopropyl and sulfonic acid; strong anion exchange groups such as trimethylammonium chloride; weak cation exchange groups such as carboxylic acid; weak anion exchange groups such as N5N diethylamino or DEAE; hydrophobic interaction groups such as phenyl, butyl, propyl, hexyl; and affinity groups such as protein A, protein G, and protein L, but are not limited thereto. For the present disclosure to be more readily understood, certain terms are first defined. When used in this application, each of the following terms shall have the meaning described below, unless explicitly provided otherwise herein. Further definitions are set forth throughout this application.

[0048] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., a protein of interest containing an Fc region or an antibody) specifically binds to a ligand specific for the protein of interest. Such ligands are generally referred to as biospecific ligands. In some embodiments, a biospecific ligand (e.g., Protein A or a functional variant thereof) is covalently bound to a chromatography matrix material and is accessible to the protein of interest in solution when the solution contacts the chromatography matrix. The protein of interest generally retains its specific binding affinity for the biospecific ligand during the chromatography process, while other solutes and / or proteins in the mixture do not bind detectably or specifically to the ligand. The binding of the protein of interest to the immobilized ligand allows contaminants or protein impurities to pass through the chromatography matrix, while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed in active form from the immobilized ligand under suitable conditions (e.g., low pH, high pH, high salt, competing ligand, etc.) and passed through the chromatography column with the elution buffer, free of contaminating proteins or protein impurities that previously passed through the column. Any component can be used as a ligand for purifying its respective specific binding protein, e.g., an antibody. However, in various methods according to the present disclosure, Protein A is used as a ligand for the Fc region containing the target protein. The conditions for elution of the target protein (e.g., a protein containing an Fc region) from a biospecific ligand (e.g., Protein A) can be readily determined by one of ordinary skill in the art. In some embodiments, Protein G or Protein L or functional variants thereof can be used as biospecific ligands. In some embodiments, a biospecific ligand such as Protein A is used in the pH range of 5-9 for binding to the Fc region containing protein, washing or re-equilibrating the biospecific ligand / target protein conjugate, and then eluted with a buffer having a pH greater than 4 or less than 4 containing at least one salt.In some embodiments, Protein A is not bound to the chromatography column resin and is thus soluble Protein A or "sProA".

[0049] As used herein, terms such as "buffer" refer to substances that, by virtue of their presence in solution, increase the amount of acid or base that must be added to cause a unit change in pH. A buffer solution resists changes in pH by the action of its acid-base conjugate components. Buffered solutions for use with biological reagents can generally maintain a certain concentration of hydrogen ions so that the pH of the solution is within the physiological range. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0050] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Thus, as the amount of ions present in an aqueous solution increases, the solution has a higher conductivity. The unit of measurement of conductivity is millisiemens per centimeter (mS / cm) and can be measured using a conductivity meter.

[0051] As used herein, the term "mobile phase" refers to a liquid or gas that flows through a chromatography system and moves materials to be separated on a stationary phase at different rates. The mobile phase can be polar or nonpolar. Polar mobile phases are generally used in conjunction with nonpolar stationary phases, and these chromatographic separations are known as reverse-phase chromatography. Conversely, nonpolar mobile phases are often used in conjunction with polar stationary phases and are generally known as normal-phase chromatography.

[0052] As used herein in the context of chromatography, the term "stationary phase" refers to the solid or liquid phase of a chromatography system to which the substances to be separated are selectively adsorbed. Usually, silica is used as the stationary phase.

[0053] As used herein, terms related to chromatography such as "chromatography column" or "column" refer to a container in the form of a cylinder or hollow column filled with a chromatography matrix or resin. The chromatography matrix or resin is a material that provides the physical and / or chemical properties used for purification.

[0054] Terms such as "ion exchange" and "ion exchange chromatography" refer to a chromatography process in which a target ionizable solute (e.g., a target protein in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent bonding) to a solid-phase ion exchange material under appropriate conditions of pH and conductivity, such that the target solute interacts non-specifically with charged compounds more than solute impurities or contaminants in the mixture. Contaminant solutes in the mixture can be washed from the column of ion exchange material or bind to or are excluded from the resin faster or slower than the target solute. "Ion exchange chromatography" includes, in particular, cation exchange (CEX), anion exchange (AEX) and mixed-mode chromatography.

[0055] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution at a constant rate through or across a population of cells. Since perfusion systems generally involve retaining cells within a culture unit, perfusion cultures are characteristically of relatively high cell density, but the culture conditions are difficult to maintain and control. In addition, since cells are grown at high density and then retained within the culture unit, the growth rate typically decreases continuously over time, even reaching the late exponential or stationary phase of cell growth. This continuous culture strategy generally involves culturing mammalian cells (e.g., non-adherent-dependent cells) that express a polypeptide and / or virus of interest during the production phase in a continuous cell culture system.

[0056] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL includes three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain the 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 can be derived from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, IgD, IgE, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are 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 way of 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" can include multivalent antibodies (e.g., trivalent antibodies) capable of binding to three or more antigens. A trivalent antibody is an IgG-shaped bispecific antibody composed of two normal Fab arms fused via a flexible linker peptide to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain fused to a CH3 with a "knob" mutation and the variable region of the light chain fused to a CH3 with a matching "hole". The hinge region does not contain disulfide bonds that would facilitate antigen access to the third binding site. Unless explicitly stated otherwise and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific, or multispecific antibodies, as well as single-chain antibodies.

[0058] As used herein, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term “antigen-binding fragment” of an antibody include: (i) Fab fragments (fragments resulting from papain cleavage) or similar monovalent fragments consisting of the VL, VH, LC, and CH1 domains; (ii) F(ab’)2 fragments (fragments resulting from pepsin cleavage) or similar divalent fragments comprising two Fab fragments joined by disulfide bridges in the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs), and (vii) combinations of two or more isolated CDRs that may be joined by a synthetic linker at will. Further, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be joined by a synthetic linker that enables them to be made as a single protein chain (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact immunoglobulin.

[0059] "Isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to PD-L1 substantially does not contain an antibody that specifically binds to an antigen other than PD-L1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity to other antigens such as PD-L1 molecules from different species. Further, the isolated antibody may substantially not contain other cellular and / or chemical substances.

[0060] "Bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs and gives rise to two antigen-binding sites with specificities for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or conjugation of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0061] Terms such as "monoclonal antibody" (mAb) refer to a non-naturally occurring preparation of antibody molecules of a single molecular composition, i.e., antibody molecules having essentially the same primary sequence and showing a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are an example of isolated antibodies. Monoclonal antibodies can be produced by hybridomas, recombinant, transgenic or other techniques known to those of skill in the art.

[0062] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences that are normally present in individual proteins can be brought together in a fusion polypeptide, and amino acid sequences that are normally present in the same protein can be arranged in a fusion polypeptide in a new arrangement, such as, for example, the fusion of the Factor VIII domain of the present disclosure having an Ig Fc domain. Fusion proteins can be made, for example, by chemical synthesis or by creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship. A chimeric protein can further comprise a second amino acid sequence linked to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0063] In the monomeric form of the fusion protein, the self-assembling nanoparticle monomer is a ferritin monomer. Ferritin is an intracellular protein that stores iron and releases iron as needed. Ferritin is widely conserved and is found in almost all organisms. In its native state, ferritin is a spherical protein of 24 subunits that self-assemble to form hollow nanoparticles. Vertebrates have two types of ferritin, a light subunit (L) with a molecular weight of approximately 19 kDa and a heavy subunit (H) with a molecular weight of approximately 21 kDa. Amphibians also express an additional type of ferritin called M ferritin. Most organisms express a ferritin similar to the vertebrate H-type ferritin.

[0064] Ferritin is a hollow spherical protein with a mass of 474 kDa and contains 24 ferritin monomer subunits. Thus, as used herein, "aggregated ferritin nanoparticles" refers to an aggregated protein containing 24 ferritin monomer subunits. In some embodiments, the ferritin monomer subunits are "fusion protein monomers" containing at least one self-assembling (sa) ferritin monomer sequence and one or more affinity ligands separated by any number of homologous or heterologous linker sequences. A histidine tag (e.g., 8 histidines) may also be included to facilitate purification by immobilized metal affinity chromatography (IMAC). When a histidine tag is included, a cleavable sequence can be included to enzymatically and optionally cleave the histidine tag using a protease after expression.

[0065] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0066] As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culturing conditions that can be defined include temperature, gas mixture, time and media formulation.

[0067] As used herein, terms such as "inoculating" refer to the addition of cells to a medium to initiate a culture.

[0068] As used herein, terms such as "induction" or "induction phase" or "growth phase" of cell culture refer to the initial seeding of a bioreactor at the start of upstream cell culture, e.g., a seed bioreactor, and include the period of exponential cell growth (e.g., logarithmic phase) during which cells are mainly rapidly dividing. During this stage, the rate of increase in the density of viable cells is higher than at any other time point.

[0069] As used herein, terms such as "production phase" of cell culture refer to the period during which cell growth is stationary or maintained at a substantially constant level. The density of viable cells remains substantially constant over a given period. Logarithmic cell growth has ended, and protein production is the major activity during the production phase. The medium at this point is generally supplemented to support continuous protein production and achieve the desired glycoprotein product.

[0070] As used herein, terms such as "expression" or "expressing" are used to refer to transcription and translation occurring within a cell. The expression level of a product gene in a host cell can be determined based on either the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the product gene produced by the cell, or both.

[0071] As used herein, the terms "medium", "cell culture medium", "feed medium", and "fermentation medium" refer to a nutrient solution used to grow and / or maintain cells, particularly mammalian cells. Without limitation, these solutions typically include the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, usually a basic set of 20 amino acids + cysteine; (3) vitamins and / or other organic compounds required at low concentrations; (4) free fatty acids or lipids, such as linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range, and provide at least one component from one or more of the trace elements. The nutrient solution may optionally be supplemented with one or more components from the following categories: (1) hormones and other growth factors (e.g., serum, insulin, transferrin, and epidermal growth factor); (2) salts, such as magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) proteins and tissue hydrolysates, such as peptone or a peptone mixture obtainable from purified gelatin, plant material, or animal by-products; (6) antibiotics, such as gentamicin; (7) cytoprotective agents, such as pluronic polyol; and (8) galactose. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium ((DMEM), (Sigma)) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980) can be used as a medium for host cells. Any other necessary supplements may also be included at appropriate concentrations.

[0072] Various aspects of the present disclosure are described in further detail in the following subsections.

[0073] II. Countercurrent Filtration The present disclosure provides a highly effective approach for removing contaminants during protein purification using countercurrent filtration without the need for chromatography. Accordingly, the present disclosure provides a method for purifying a target product that uses approximately 1 / 10 the amount of water and solution of a chromatography process.

[0074] The present disclosure is a method for purifying a target product using countercurrent filtration, comprising: (a) contacting a first solution containing the target product and impurities with a binding molecule to form a complex, the complex containing the target product bound to the binding molecule; (b) contacting the first flowing solution containing the complex with a first side of a semipermeable membrane, the complex having a molecular weight greater than the molecular weight cut-off of the membrane such that the complex is retained on the first side of the semipermeable membrane; (c) passing the impurities through the semipermeable membrane, the impurities having a molecular weight less than the molecular weight cut-off of the semipermeable membrane and being retained on a second side of the semipermeable membrane in a second flowing solution that is countercurrent to the first flowing solution; (d) dissociating the complex to form the free target product and free binding molecule; and (e) passing either the target product or the binding molecule through the semipermeable membrane, either the smaller of the target product or the binding molecule having a molecular weight less than the molecular weight cut-off of the semipermeable membrane and being retained on the second side of the semipermeable membrane in the second flowing solution countercurrent to the first flowing solution, while the larger of the target product or the binding molecule is retained on the first side of the membrane.

[0075] The present disclosure provides a method for purifying a product of interest using molecular countercurrent filtration, comprising: (a) contacting a first solution containing the product of interest and impurities with a binding molecule to form a complex, the complex containing the product of interest bound to the binding molecule; (b) contacting the first flowing solution containing the complex with a first side of a semipermeable membrane under pressure, such that the complex is retained on the first side of the semipermeable membrane, the complex having a molecular weight exceeding the molecular weight cut-off of the membrane; (c) passing the impurities through the semipermeable membrane by convection, the impurities having a molecular weight less than the molecular weight cut-off of the semipermeable membrane and becoming the filtrate; (d) dissociating the complex to form the free product of interest and free binding molecule; and (e) passing either the product of interest or the binding molecule through the semipermeable membrane, wherein the smaller of the product of interest or the binding molecule has a molecular weight less than the molecular weight cut-off of the semipermeable membrane and is retained on a second side of the semipermeable membrane in a second flowing solution countercurrent to the first flowing solution, while the larger of the product of interest or the binding molecule is retained on the first side of the membrane.

[0076] Optionally, filtrate from one module is used to dilute another solution upstream of the semipermeable membrane such that the filtrate is used in a convective manner.

[0077] In some embodiments, the method further comprises: (e) regenerating the binding molecule by contacting a solution containing the binding molecule with the first side of the semipermeable membrane, the binding molecule having a molecular weight exceeding the molecular weight cut-off of the semipermeable membrane such that the binding molecule is retained on the first side of the membrane, and the regenerated binding molecule being capable of binding to the complex upon contact with the product of interest in the first solution or the second solution. See FIG. 9. In some embodiments, the second flowing solution contains a second binding molecule capable of binding to impurities in the first solution and / or the second flowing solution. In some embodiments, unbound binding molecules diffuse through the semipermeable membrane into the second flowing solution.

[0078] In some embodiments, the binding molecule comprises Protein A, Protein G, a cation exchange resin, or an anion exchange resin. In some embodiments, the binding molecule comprises Protein A.

[0079] A "cation exchange resin" or "cation exchange membrane" refers to a solid phase having free anions for exchanging cations in an aqueous solution that are negatively charged and pass over or through the solid phase. Any negatively charged ligand (e.g., carboxylate, sulfonate, and others described below) bound to the solid phase that is suitable for forming a cation exchange resin can be used. Commercially available cation exchange resins include, for example, but are not limited to, those having sulfonate-based groups. In some embodiments, the cation exchange resin is one having a sulfonate-based group (e.g., MonoS, Minis, Source 15S and 30S manufactured by GE Healthcare, SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance, Capto S, Capto SP ImpRes, TOYOPEARL® SP-650S and SP-650M manufactured by Tosoh, MACROPREP® High S manufactured by BioRad, Ceramic HyperD S manufactured by Pall Technologies, TRISACRYL® M and LS SP and Spherodex LS SP); a sulfoethyl-based group (e.g., FRACTOGEL® SE manufactured by EMD, POROS® S-10 and S-20 manufactured by Applied Biosystems); a sulfopropyl-based group (e.g., TSK Gel SP 5PW and SP-5PW-HR manufactured by Tosoh, POROS® HS-20, HS 50 and POROS® XS manufactured by Life Technologies); a sulfoisobutyl-based group (e.g., FRACTOGEL® EMD SO3 manufactured by EMD -); sulfonoxyethyl groups (e.g., SE52, SE53, and Express-Ion S manufactured by Whatman), carboxymethyl groups (e.g., CM SEPHAROSE (registered trademark) Fast Flow manufactured by GE Healthcare, Hydrocell CM manufactured by Biochrom Labs Inc., MACRO-PREP (registered trademark) CM manufactured by BioRad, Ceramic HyperD CM manufactured by Pall Technologies, TRISACRYL (registered trademark) M CM, TRISACRYL (registered trademark) LS CM, Matrx CELLUFINE (registered trademark) C500 and C200 manufactured by Millipore, CM52, CM32, CM23, and Express-Ion C manufactured by Whatman, TOYOPEARL (registered trademark) CM-650S, CM-650M, and CM-650C manufactured by Tosoh); sulfonic acid and carboxylic acid groups (e.g., BAKERBOND (registered trademark) Carboxy-Sulfon manufactured by J.T. Baker); carboxylic acid groups (e.g., WP CBX manufactured by J.T Baker, DOWEX (registered trademark) MAC-3 manufactured by Dow Liquid Separations, AMBERLITE (registered trademark) Weak Cation Exchangers, DOWEX (registered trademark) Weak Cation Exchanger, and DIAION (registered trademark) Weak Cation Exchangers manufactured by Sigma-Aldrich, FRACTOGEL (registered trademark) EMD COO manufactured by EMD); sulfonic acid groups (e.g., Hydrocell SP manufactured by Biochrom Labs Inc., DOWEX (registered trademark) Fine Mesh Strong Acid Cation Resin manufactured by Dow Liquid Separations, UNOsphere S, WP Sulfonic manufactured by J.T. Baker, SARTOBIND (registered trademark) S membrane manufactured by Sartorius, AMBERLITE (registered trademark) Strong Cation Exchangers, DOWEX (registered trademark) Strong Cation, and DIAION (registered trademark) Strong Cation Exchanger manufactured by Sigma-Aldrich); or orthophosphate groups (e.g., P11 manufactured by Whatman).Examples of other cation exchange resins include carboxymethyl - cellulose, BAKERBOND ABXTM, Ceramic HyperD Z, Matrex Cellufine C500, Matrex Cellufine C200, or any combination thereof.

[0080] "Anion exchange resin" or "anion exchange membrane" refers to a positively charged solid phase, which thereby has one or more positively charged ligands bound to the solid phase. Any positively charged ligand (such as a quaternary amino group) bound to a solid phase suitable for forming an anion exchange resin may be used. Examples of commercially available anion exchange resins include DEAE cellulose. In some embodiments, the anion exchange resin is DEAE cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystem, SARTOBIND® Q, MonoQ, MiniQ, Source 15Q and 30Q from Sartorius, Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT from J.T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, MACRO-PREP® DEAE and MACRO-PREP® High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q from Pall Technologies, DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHERE 77, weak base anion from Dow Liquid Separations, INTERCEPT® Q membrane from Millipore, Matrex CELLUFINE® A200, A500, Q500 and Q800, FRACTOGEL® EMD from EMDSelected from TMAE, FRACTOGEL® EMD DEAE and FRACTOGEL® EMD DMAE, AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE®, TSK gel Q and DEAE 5PW and 5PW-HR manufactured by Tosoh, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q manufactured by Whatman, and SARTOBIND® Q (Sartorius Corporation, New York, USA). Other anion exchange resins include POROS XQ, Sartobind® Q, Q Sepharose™ XL, Q Sepharose™ big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q Sepharose™ high performance, Q Sepharose™ XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMD TMAE HiCap, Nuvia Q, or PORGS PI or any combination thereof.

[0081] In some embodiments, the first solution containing the product of interest can be obtained from the bioreactor. In some embodiments, the first solution can be obtained from the bioreactor without any other downstream processing steps prior to performing the methods described herein.

[0082] In some embodiments, the second flowing solution contains a positively charged polymer. In some embodiments, the positively charged polymer can include DEAE dextran. In some embodiments, the positively charged polymer can bind to low molecular weight species diffused through the semipermeable membrane.

[0083] In some embodiments, steps (a) to (c) of the method described herein are repeated, and the binding molecule includes protein A. In some embodiments, steps (a) to (c) of the method described herein are repeated, and the binding molecule includes a cation exchange resin. In some embodiments, steps (a) to (c) of the method described herein are repeated, and the binding molecule includes an anion exchange resin.

[0084] In some embodiments, the dissociated protein is diafiltered.

[0085] In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be at least about 10 mL / min to about 1,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 9,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 8,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 7,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 6,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 5,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 4,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 3,000 mL / min. In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be about 10 mL / min to about 2,000 mL / min.In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can be from about 30 mL / min to about 60 mL / min, from about 31 mL / min to about 60 mL / min, from about 32 mL / min to about 60 mL / min, from about 33 mL / min to about 60 mL / min, from about 34 mL / min to about 60 mL / min, from about 35 mL / min to about 60 mL / min, from about 36 mL / min to about 60 mL / min, from about 37 mL / min to about 60 mL / min, from about 38 mL / min to about 60 mL / min, from about 39 mL / min to about 60 mL / min, from about 40 mL / min to about 60 mL / min, from about 41 mL / min to about 60 mL / min, from about 42 mL / min to about 60 mL / min, from about 43 mL / min to about 60 mL / min, from about 44 mL / min to about 60 mL / min, from about 45 mL / min to about 60 mL / min, from about 46 mL / min to about 60 mL / min, from about 47 mL / min to about 60 mL / min, from about 48 mL / min to about 60 mL / min, from about 49 mL / min to about 60 mL / min, from about 50 mL / min to about 60 mL / min, from about 51 mL / min to about 60 mL / min, from about 52 mL / min to about 60 mL / min, from about 53 mL / min to about 60 mL / min, from about 54 mL / min to about 60 mL / min, from about 55 mL / min to about 60 mL / min, from about 56 mL / min to about 60 mL / min, from about 57 mL / min to about 60 mL / min, from about 58 mL / min to about 60 mL / min, or from about 59 mL / min to about 60 mL / min. In some embodiments, the flow rates of the first flowing solution and the second flowing solution are the same. In some embodiments, the flow rates of the first flowing solution and the second flowing solution are different.

[0086] In some embodiments, the flow rate of the first flowing solution and / or the second flowing solution can include about 30 mL / min, about 31 mL / min, about 32 mL / min, about 33 mL / min, about 34 mL / min, about 35 mL / min, about 36 mL / min, about 37 mL / min, about 38 mL / min, about 39 mL / min, about 40 mL / min, about 41 mL / min, about 42 mL / min, about 43 mL / min, about 44 mL / min, about 45 mL / min, about 46 mL / min, about 47 mL / min, about 48 mL / min, about .......

[0087] It seems there is an ellipsis in the middle of line ID=4 in the original text which is not fully translated in the given content. If you want a more precise translation, please provide the complete text.In some embodiments, the first flow rate is higher than the second flow rate. In some embodiments, the second flow rate is higher than the first flow rate.

[0088] In some embodiments, the first flowing solution and / or the second flowing solution is / are pulsed. In some embodiments, pulsing improves mass transfer across the semipermeable membrane. In some embodiments, the pulse volume is less than the total volume of the pores of the semipermeable membrane. In some embodiments, the pulse volume is about half of the total volume of the pores of the semipermeable membrane. In some embodiments, the pulse volume is greater than the total volume of the pores of the semipermeable membrane. In some embodiments, a peristaltic pump can be used to generate pulses in the first flowing solution and / or the second flowing solution. In some embodiments, a piston pump can be used to generate pulses in the first flowing solution and / or the second flowing solution. In some embodiments, a piezoelectric actuator can be used to generate pulses in the first flowing solution and / or the second flowing solution. In some embodiments, the pulsed flow is not completely reversible and creates diffusible mass transfer.

[0089] In some embodiments, the semipermeable membrane has a molecular weight cut-off of about 1,000 kDa. In some embodiments, Protein A has a molecular weight greater than 1,000 kDa and Protein A cannot diffuse through the semipermeable membrane. In some embodiments, the product of interest is a monoclonal antibody having a molecular weight of about 150 kDa. In some embodiments, the complex containing the monoclonal antibody and the protein has a molecular weight greater than 1,000 kDa. In some embodiments, the semipermeable membrane is a ceramic membrane.

[0090] In some embodiments, the semipermeable membrane has a molecular weight cut-off of about 500 kDa to about 1,000 kDa. In some embodiments, the complex containing the monoclonal antibody and Protein A has a molecular weight greater than 1,000 kDa. In some embodiments, the host cell protein (HCP) has a molecular weight less than 150 kDa.

[0091] In some embodiments, a complex comprising a product of interest (e.g., a monoclonal antibody) and a binding molecule (e.g., Protein A) dissociates to form the free product of interest (e.g., a monoclonal antibody) and a free binding molecule (e.g., Protein A). In some embodiments, the product of interest (e.g., a monoclonal antibody) has a molecular weight greater than the molecular weight cut-off of the semipermeable membrane, the free binding molecule (e.g., Protein A) has a molecular weight less than the molecular weight cut-off of the semipermeable membrane, and the free binding molecule (e.g., Protein A) is removed by diffusing through the semipermeable membrane. In some embodiments, the product of interest (e.g., a monoclonal antibody) has a molecular weight less than the molecular weight cut-off of the semipermeable membrane, the free binding molecule (e.g., Protein A) has a molecular weight greater than the molecular weight cut-off of the semipermeable membrane, and the product of interest (e.g., a monoclonal antibody) is removed by diffusing through the semipermeable membrane.

[0092] In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 1 kDa to about 10 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 100 kDa to about 200 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 300 kDa to about 400 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 400 kDa to about 500 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 500 kDa to about 600 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 600 kDa to about 700 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 700 kDa to about 800 kDa. In some embodiments, the molecular weight cut-off of the semipermeable membrane is from about 900 kDa to about 1,000 kDa.

[0093] In some embodiments, the binding molecule can include a Protein A mimetic peptide. In some embodiments, the Protein A mimetic peptide can include amino acids that make up a protein, and / or non-natural amino acids, and / or synthetic amino acids, arranged in an acyclic form without disulfide bonds, or in a branched or cyclic form having one or more covalent bonds such as disulfide bonds.

[0094] In some embodiments, the methods described herein can purify a target product at about 0.1 kg / day, about 0.5 kg / day, about 1 kg / day, about 2 kg / day, about 3 kg / day, about 4 kg / day, about 5 kg / day, about 6 kg / day, about 7 kg / day, about 8 kg / day, about 9 kg / day, or about 10 kg / day.

[0095] In some embodiments, the purified target product is free of residual Protein A.

[0096] In some embodiments, the methods described herein can include a first cross-flow filtration, and the binding molecule includes Protein A. In some embodiments, the method further includes a second cross-flow filtration, and the binding molecule includes a cation exchange resin or an anion exchange resin. In some embodiments, the method further includes a third cross-flow filtration, and the binding molecule includes a cation exchange resin and an anion exchange resin. In some embodiments, the ion exchange resins are different between the second cross-flow filtration step and the third cross-flow filtration step. In some embodiments, the method further includes a diafiltration step.

[0097] In some embodiments, the methods described herein can purify from about 0.1 kg / day to about 0.5 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 0.5 kg / day to about 1 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 1 kg / day to about 2 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 2 kg / day to about 3 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 3 kg / day to about 4 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 4 kg / day to about 5 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 5 kg / day to about 6 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 6 kg / day to about 7 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 7 kg / day to about 8 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 8 kg / day to about 9 kg / day of the product of interest. In some embodiments, the methods described herein can purify from about 9 kg / day to about 10 kg / day of the product of interest.

[0098] While not wishing to be bound by theory, in some embodiments, the methods described herein can use affinity colloid separation. In some embodiments, the methods described herein can use anion exchange polymer affinity colloid separation. In some embodiments, the methods described herein can use Protein A to capture a product of interest (e.g., a monoclonal antibody) by diffusion through a semipermeable membrane. In some embodiments, the methods described herein can use Protein A to capture a product of interest (e.g., a monoclonal antibody) and impurities (e.g., HCP) by diffusion through a semipermeable membrane. In some embodiments, a product of interest complexed with a binding molecule (e.g., a monoclonal antibody bound to Protein A) can be dissociated at low pH. In some embodiments, Protein A can be diffused through a semipermeable membrane.

[0099] In some embodiments, the methods for purifying a product of interest can be combined modularly. In some embodiments, a product of interest obtained from a bioreactor (e.g., a monoclonal antibody) can first be subjected to the Protein A affinity colloid separation described herein. The product of interest (e.g., a monoclonal antibody) can then be further subjected to cation exchange polymer affinity colloid separation, followed by anion exchange polymer affinity colloid separation, followed by a diafiltration step.

[0100] In some embodiments, a contaminated product (e.g., the product of interest) is obtained from a bioreactor and concentrated and purified using one of the separation methods described herein (e.g., affinity colloid separation, anion exchange polymer affinity colloid separation, etc.). A second separation method is then performed on the concentrated product. The contaminated permeate from the second separation is returned to the contaminated product stream. The concentrated product (e.g., the product of interest) from the second separation method is then diafiltered. The slightly contaminated permeate from the diafiltration step is returned to the product (e.g., the product of interest) concentrated by the first separation method.

[0101] In some embodiments, the methods disclosed herein can be performed at elevated temperatures since they increase diffusivity and decrease viscosity. In some embodiments, the methods described herein can be performed at a temperature of about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the semipermeable ceramic membrane is used in the methods described herein when performed at a temperature of about 37°C, about 38°C, about 39°C, or about 40°C.

[0102] In some embodiments, the semipermeable ceramic membrane can be used in dialysis or diafiltration / ultrafiltration. These have several advantages for cleaning and use compared to polymeric semipermeable membranes.

[0103] In some embodiments, fouling of the semipermeable membrane can be reduced by various methods including, but not limited to, backflushing, forward flushing, pulsating operation, ultrasonic fouling reduction, gas entrainment, and / or electrophoretic fouling reduction.

[0104] In some embodiments, high performance tangential flow filtration (HPTFF) can also be used in diafiltration / ultrafiltration. HPTFF is known to those skilled in the art as an ultrafiltration operation in which the permeate is recycled simultaneously with the retentate and a more consistent transmembrane pressure is maintained.

[0105] In some embodiments, the method herein can be performed using a first solution comprising a product of interest having a concentration of the product of interest of about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, about 200 g / L, or greater than 200 g / L.

[0106] Dialysis eluate In some embodiments, the dialysis solution is allowed to be used upstream in a prior step. In some embodiments, the dialysis solution is titrated to a new pH and / or mixed with a high-concentration salt solution to achieve a desired ionic strength for use in an upstream process. In some embodiments, other solutions including, but not limited to, arginine, urea, guanidine, ethanol, isopropyl alcohol, other alcohols, solutions of caprylic acid, and other solutions suitable for washing the product of interest can be added to the dialysis solution. In some embodiments, the system operates continuously. In some embodiments, the dialysis solution is stored in a small surge tank. In some embodiments, the surge tank is smaller than the solution volume for one day. In some embodiments, recycling of the solution from a downstream process to an upstream process requires the use of a surge tank.

[0107] In some embodiments, the permeate from the ultrafiltration step can also be used in a countercurrent manner (e.g., the manner described herein) for each step. In some embodiments, the permeate from the single-pass tangential flow filtration step can also be used in a countercurrent manner (e.g., the manner described herein) for each step. In some embodiments, the dialysis eluate from the dialysis step can be used in a countercurrent manner for each step. In some embodiments, the retentate from the dialysis step can also be used in a countercurrent manner for each step.

[0108] In some embodiments, the pre-upstream step of receiving the countercurrent solution, or the step after the receiving step, must have an effluent to be discarded that facilitates the removal of any impurities that may be present in the recycled solution. In some embodiments, if the recycled solution is derived from the permeate of a step having a semipermeable membrane with a molecular weight cut-off (MWCO), the receiving step of the subsequent step must have a membrane with a MWCO greater than that directed for discard. If not, even a small amount of material that is larger than the MWCO of the upstream step can accumulate on the system over time. In some embodiments, the upstream step can have a semipermeable membrane with a MWCO greater than that of the downstream step such that any large material passing through the downstream filter can easily pass from the upstream filter into the waste stream.

[0109] In some embodiments, if the recycled solution is derived from the retained portion of the semipermeable membrane, the receiving step must have a semipermeable membrane of the same size or smaller than the MWCO of the downstream step, and the retained solution of the upstream step must be discarded.

[0110] In some embodiments, all combinations of recycling that are retained or derived from the permeate portion from the downstream step to the upstream step are contemplated. Here, the upstream step (or a step subsequent to the upstream step even if before the downstream step) has a waste stream in the permeate retention portion of the filter.

[0111] In some embodiments, the accumulation of trace materials in the reuse loop can be addressed by diverting a portion of the recycle line to be discarded or by periodically emptying the recycle loop.

[0112] In some embodiments, when the step is an anion exchange step, the receiving step or a subsequent step must have an effluent that removes anionic compounds. In some embodiments, when the step is a cation exchange step, the receiving step must have an effluent that removes cationic compounds. For example, when the downstream step involves an anion exchange nanoparticle or polymer, the cationic component passes through the permeate or dialysate of the membrane. If this material is used upstream, the upstream process must be able to flow in such a way as to discharge the fraction of the cationic component. This can be done using cation exchange nanoparticles or polymers that bind and retain the cationic compound on the retentate side, while the effluent flowing through the downstream process is from the permeate. Alternatively, the upstream process can be an affinity process. This is orthogonal to ion exchange, and the cationic component passes through the affinity process and flows to the waste line.

[0113] In some embodiments, if such a removal step does not exist upstream, impurities can accumulate in the recycle loop, albeit at trace levels. In some embodiments, this accumulation can be managed by a split flow where a portion of the recycle loop is directed towards waste. This split flow is a somewhat inefficient method since the product is also being directed towards waste.

[0114] In some embodiments, the present disclosure relates to a method of using the solution flowing out from a continuous downstream process as a wash for an upstream process. In some embodiments, the flowing out solution is mixed with the solution stream to adjust the pH, or salt concentration, or other excipient concentration to a desired level.

[0115] In some embodiments, the present disclosure relates to a method of using the solution flowing out from the filtrate or dialysate of a continuous downstream process as a wash for an upstream process, where the upstream process has a filter with an MWCO of the same size or larger than the downstream process that produced the effluent.

[0116] In some embodiments, the present disclosure relates to a method of using a solution that has flowed out of a holding solution of a continuous downstream process as a wash for an upstream process, where the upstream process has a filter with a MWCO that is the same size as or smaller than the downstream process that produced the effluent, and the filtrate is directed downstream.

[0117] In some embodiments, the present disclosure relates to a method of using a solution that has flowed out of a continuous downstream process as a wash for an upstream process, where the downstream process retains anions and the upstream process can discard cations.

[0118] In some embodiments, the present disclosure relates to a method of using a solution that has flowed out of a continuous downstream process as a wash for an upstream process, where the downstream process retains cations and the upstream process can discard anions.

[0119] In some embodiments, the present disclosure relates to a method of using a solution that has flowed out of a continuous downstream process as a wash for an upstream process, where the downstream process retains a particular class of impurities and the upstream process can discard these impurities.

[0120] In some embodiments, the upstream process is a concentration process for an Fc-containing protein, and the upstream process is a continuous Protein A wash process.

[0121] In some embodiments, the present disclosure provides an effluent stream from a continuous concentration process for recycling a binding molecule to wash a complex of the binding molecule and a product of interest. In some embodiments, a titrant, salt, or other excipient is added to the effluent stream. In some embodiments, a dialysis or ultrafiltration process is continuous. In some embodiments, the effluent enters a small tank, and the tank size is smaller than the daily effluent storage volume. In some embodiments, the effluent enters a small tank, and the tank size is smaller than the 3-hour effluent storage volume.

[0122] In some embodiments, the recycling of the binding molecule is a Protein A affinity ligand.

[0123] In some embodiments, the present disclosure relates to a method of using an effluent from a dialysis or ultrafiltration concentration step upstream of a dialysis or ultrafiltration step. In some embodiments, a titrant, salt, or other excipient is added to the effluent. In some embodiments, the dialysis or ultrafiltration step is continuous. In some embodiments, the effluent enters a small tank, the size of which is smaller than the daily effluent storage volume. In some embodiments, the effluent enters a small tank, the size of which is smaller than the 3-hour effluent storage volume. In some embodiments, the product of interest is part of a protein, DNA, RNA, virus or virus-like particle, synthetic molecule, or affinity complex.

[0124] ProA recycle SPTFF effluent Typically, the affinity substream collected in the step of separating the affinity ligand from the product of interest is diluted. A semipermeable membrane with an MWCO lower than the MW of the affinity ligand can be used to concentrate the affinity ligand before recycling. The permeate or dialysate from this concentration step can be used in countercurrent mode as part of the wash for the affinity step. This solution can be mixed with a titrant or other solution having related other compounds useful for removing impurities in high-concentration salts or washes.

[0125] In some embodiments, the present disclosure relates to a method of using an effluent stream from a continuous concentration step in the recycling of an affinity moiety to wash a complex of a binding molecule and a product of interest. In some embodiments, the method further comprises adding a titrant, salt, or other excipient to the effluent stream. In some embodiments, the dialysis or ultrafiltration step is continuous. In some embodiments, the effluent enters a small tank, the size of which is less than the daily effluent storage volume. In some embodiments, the effluent enters a small tank, the size of which is less than the 3-hour effluent storage volume. In some embodiments, the binding molecule is Protein A. In some embodiments, the product of interest has a HIS tag. In some embodiments, Protein A is greater than 500 kDa. In some embodiments, Protein A is less than 50 kDa.

[0126] Binding molecule In some embodiments, the binding molecule (also referred to as an affinity moiety, for example) is a recombinant protein, the product of interest is a recombinant protein, and the binding molecule can be produced in the same cell expression system as the product of interest. In some embodiments, the growth medium used to produce the binding molecule is the same as the growth medium used to produce the product of interest. This method of producing the binding molecule reduces the overall cost of the binding molecule because the impurities in the binding molecule are the same as the impurities in the product of interest. This similarity of impurities means that the binding molecule (for example, the affinity moiety) can be purified such that the binding molecule (for example, the affinity moiety) is cleaner than the step of mixing the binding molecule (for example, the affinity moiety) and the product of interest. When this step is a capture step, the product of interest has a large amount of impurities, and since this step purifies the binding molecule / product of interest complex, the binding molecule can be more contaminated than expected.

[0127] For example, the product of interest has, at capture, host cell proteins (HCPs) far exceeding 100,000 ppm. The binding molecule can also have 1,000 ppm or 10,000 ppm of HCP and still be able to purify the product of interest with minimal impact. Typically, the binding molecule is expected to have an HCP purity of less than 100 ppm, or even less than 10 ppm. The higher the acceptable impurity level for the binding molecule, the less expensive it will be to manufacture.

[0128] The initial purification of the binding molecule in such a system is relatively easy. If the binding molecule is larger than the product of interest, the binding molecule can pass through the same series of filters used with the product of interest. The binding molecule is first concentrated by an asymmetric dialysis or single pass TFF process and then washed in a dialysis or CM-TFF process. Finally, the material is filtered by a CM-TFF process or single pass TFF process with an MWCO or pore size larger than the affinity moiety.

[0129] If the binding molecule is smaller than the product of interest, this can be purified by utilizing a system similar to the "elution" step of the affinity / target cycle.

[0130] Washing solution In some embodiments, solutions suitable for washing the product of interest during one or more washing steps include solutions made from arginine, urea, guanidine, ethanol, isopropyl alcohol, other alcohols, caprylic acid, and other suitable solutions.

[0131] Design of the binding molecule (e.g., affinity moiety) In some embodiments, the binding molecule has two distinct properties. First, it is designed to be readily separable from the product of interest under non-binding conditions. Second, the complex formed between the binding molecule and the product of interest contains two or more products of interest (e.g., monoclonal antibodies) under binding conditions. In some embodiments, the number of products of interest per complex is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or a number greater than 20. In some embodiments, the molar ratio of the binding molecule to the product of interest is about 1:2 or about 1:3 or greater. In some embodiments, the binding molecule is capable of binding at twice its weight or more of its weight in the product of interest. In some embodiments, the mass ratio of the binding molecule to the product of interest is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10.

[0132] In some embodiments, the binding molecule must be readily separable from the product of interest under "elution" or non-binding conditions. For example, the Fc-containing portion elutes or ceases to bind to Protein A when the pH drops to approximately 3.5.

[0133] In some embodiments, Protein A (e.g., the binding molecule) can be configured to be much smaller than the Fc-containing compound (e.g., the product of interest). In some embodiments, for ultrafiltration separation, this means that Protein A (e.g., the binding molecule) must be 1 / 5 to 1 / 10 the size of the Fc-containing compound (e.g., the product of interest). In some embodiments, for dialysis separation, when Protein A is less than 1 / 3 the size of the Fc-containing compound, acceptable purification can be achieved. In some embodiments, when the product of interest is a typical antibody (150 kDa MW), the Protein A moiety must be less than 30 kDa. A 100 kDa MWCO membrane is sufficient to retain the antibody and allow a significant portion of the affinity ligand to pass through. Native Protein A has an MW of 40 - 60 kDa, which is about 1 / 3 the MW of the antibody. However, native Protein A cannot be separated in an ultrafiltration or dialysis device. Protein A will either be retained with the antibody or both will pass through the membrane.

[0134] For example, a single domain of Protein A can bind to an antibody in solution but does not form a complex of two or more antibodies, and ultrafiltration or diafiltration of the crude feed stream does not remove significantly more impurities than the same system and only removes unbound antibody.

[0135] In some embodiments, a construct of Protein A having two binding domains can bind to two antibodies (e.g., the product of interest), thereby enabling the formation of a complex that is filterable. Under elution conditions, some constructs of Protein A can pass through an ultrafiltration membrane or dialysis membrane having an MWCO of 100 kDa or 75 kDa while retaining the antibody.

[0136] In some embodiments, Protein A can be configured to be much larger than the Fc-containing compound (e.g., the product of interest). For example, a Protein A moiety greater than 500 kDa can be produced. A 500 kDa membrane is sufficient to allow antibodies to pass through the membrane by convection while retaining the affinity ligand. A 300 kDa membrane also allows the passage of antibodies, but may be less efficient. In dialysis, a 300 kDa membrane is sufficient to diffuse antibodies. Membranes with larger MWCOs are also efficient.

[0137] In some embodiments, a binding molecule made from a peptide or protein can have a his-tag (typically, a six-histidine tag on the amino or carboxy terminus of the protein). When the elution conditions exceed pH 7, divalent cations such as copper or cobalt can be added to aggregate the binding molecule to enable filtration. At pH 4 or 5, the his-tag is positively charged, and cation exchange methods can be used to separate the binding molecule from the product of interest.

[0138] In some embodiments, the product of interest containing DNA can be separated because it can have a sequence that specifically binds to large nanoparticles or microparticles.

[0139] In some embodiments, the binding molecule contains an elastin-like peptide sequence that allows for natural aggregation under certain conditions.

[0140] In some embodiments, the binding molecule includes multiple executable incorporation methods that enable separation under elution / unbound conditions.

[0141] In some embodiments, a binding molecule containing a small peptide can be found, for example, by screening phage display for small molecular weight synthetic or naturally occurring molecules. In some embodiments, the peptide can be separated from small molecular weight compounds when it is much larger than the compound under unbound conditions. Most peptides are larger than the small molecule targets described above.

[0142] In some embodiments, the binding molecule must have an affinity coefficient that is much smaller than the concentration of the product of interest in solution.

[0143] In some embodiments, a polymer can be used as the binding molecule. In some embodiments, the polymer is ionic and / or hydrophobic.

[0144] In some embodiments, the complex formed by the binding molecule and the product of interest contains at least two products of interest per complex, or at least three products of interest per complex. In some embodiments, the size of the complex is much larger than the product of interest itself, facilitating separation through a semipermeable membrane.

[0145] In some embodiments, a Protein A molecule (e.g., the binding molecule) can bind to three antibodies (e.g., the product of interest) and can be purified using a 300 kDa membrane or a 500 kDa membrane. There are complexes that pass through the 700 kDa membrane.

[0146] In some embodiments, a very large Protein A (e.g., the binding molecule) can bind to one antibody (e.g., the product of interest) and be retained by a semipermeable membrane, but the cost of Protein A is extremely high. Furthermore, separation of the large MW Protein A from the antibody becomes more difficult. This is due to the high concentration of Protein A being retained by the membrane. Retention of the high concentration Protein A reduces the performance of the semipermeable membrane. The flux decreases, the resistance increases, and the antibody can be retained more highly. Therefore, a lower concentration of Protein A compared to the antibody is preferred. Thus, in some embodiments, the ratio of antibody to Protein A is 2:1 per mole or more preferably 3:1 mole. In some embodiments, for very large Protein A moieties such as those higher than 500 kDa, the molar ratio is 4:1 or 8:1 or even 20:1. The 8:1 molar ratio is a 2.4:1 antibody:Protein A mass ratio. Considering the Protein A cost and the filter efficiency, a higher mass ratio is preferred.

[0147] In some embodiments, very small Protein A molecules (e.g., binding molecules) must efficiently bind to at least two antibodies (e.g., products of interest) such that a chain reaction can occur and a complex of three or more antibodies can be formed. In some embodiments, the size of the complex can be controlled by the addition of Protein A molecules that bind to only one antibody, thereby creating a "dead end" in the chain reaction. The size of the complex should not be too large as it would make filtering the complex difficult. In some embodiments, for Protein A / mAb complexes, the size of the complex can be 300 kDa, 500 kDa, 700 kDa, 1 MDa. In some embodiments, the size can be as small as about 70 nm and as large as about 1 μm, and the complex is filtered by a 0.2 μm filter. In some embodiments, the size can be 10 μm or 100 μm. Since the cost of microfilters is typically lower than that of nanofilters, the largest particles that can be filtered are preferred. Even if a 300 kDa membrane is more effective than a 0.2 μm membrane, 300 kDa particles allow for economical purification.

[0148] In some embodiments, very small Protein A molecules (e.g., binding molecules) can bind to one antibody (e.g., the product of interest), which can contain another affinity domain that binds to another Protein A molecule that itself is bound to the antibody. Thus, in some embodiments, large complexes can be formed. For example, in some embodiments, single-domain Protein A can have a his tag that can bind to the domain of Protein A in the presence of chelating metal ions such as cobalt, copper, nickel, or others so that a complex of Protein A and the antibody is formed. His-tagged proteins are known to those skilled in the art regarding protein expression and purification. Without limitation, in some embodiments, the domain of Protein A, a binding molecule, can be tagged with 3 to 6 histidines. In some embodiments, the molar ratio of Protein A to the mAb is about 2:1, but the mass ratio is very high, about 1:10 or 1:15, or even 1:30. In some embodiments, the size of the complex can be controlled by adding a domain of Protein A that does not contain a his tag. In some embodiments, the ratio of non-his-tagged Protein A to his-tagged Protein A can be 1:2 (which creates relatively small particles), 1:3, 1:5, 1:10, or higher. In some embodiments, a greater number of his-tagged Protein A relative to non-his-tagged Protein A produces increasingly larger particles. In some embodiments, larger particles make it difficult to filter the particles. In some embodiments, a preferred ratio is 1:3 to 1:5. Single-domain Protein A (with or without histidine) can be separated by ultrafiltration or dialysis using a 50 kDa membrane, preferably a 75 kDa membrane, most preferably a 100 kDa membrane. The his-tagged Protein A passes through the membrane while the antibody is retained. In some embodiments, the chelating metal is added to Protein A before the antibody, and in some embodiments, the antibody is bound to Protein A and then the chelating metal is added.

[0149] The methods of the present disclosure may also relate to the use of self-assembling nanoparticles. Such nanoparticles have the ability to assemble and simultaneously bind to multiple antibodies, resulting in the formation of large complexes. In some embodiments, the binding molecule is a nanoparticle. Self-assembling nanoparticles are formed from the self-assembly of monomers encoded by each plasmid. The number of monomers that assemble into a nanoparticle varies (e.g., about 2, about 5, about 12, about 24 to 60 or more). The MW of the fusion nanoparticle is the product of the size of the monomer in the nanoparticle and the number of monomers, whereas the MW of the mAb bound to the species is the product of the monomer MW, mAb MW, the number of monomers in the nanoparticle, and the volume % (mol mAb / mol ProA binding domain). The nanoparticles present on the retentate side of the filtration membrane after elution must have an apparent MW greater than the cut-off of the elution membrane filter (e.g., 300 or 500 kDa MWCO), whereas the affinity-bound mAb nanoparticle complex must have an MW greater than the cut-off of the washing and binding membrane filter (e.g., 500, 750, 1000 kDa MWCO or 0.22 μm). Any additional Z domain of Protein A adds mass to the nanoparticle and additional mass to the mAb nanoparticle complex. Similarly, the mass ratio of the binding mAb to the nanoparticle increases with the number of Z domains only if additional mAb can bind.

[0150] Self-assembling nanoparticles span a variety of sizes, number of homomonomers, and arrangements. Ferritin nanoparticles are composed of 24 monomers and form 12 nm pores containing hollow spheres each having 5 helical domains. This was selected for its less than 4 pH tolerance required to maintain its structure when faced with the conditions used to dissociate mAb from the affinity ligand. The Helicobacter pylori ferritin was modified from the WT form to include N-terminal amino acids that allow for favorable spatial separation of the N-terminus to be modified with the affinity ligand. Stable protein 1 (SP1) is composed of 12 subunits that exhibit stability at high temperature and pH stability and have been shown to be a reliable carrier for enzymes and carbon nanotube-binding proteins that form biomaterials. SP1 also has the feature that it can reversibly form nanorods that may be favorable for retaining the size of mAb (and nanoparticles) over a large pore size.

[0151] In an aspect of the method, each fusion protein monomer comprises: i) a self-assembling nanoparticle monomer; ii) a linker; and iii) an immunoglobulin binding domain; In an aspect, the linker connects the self-assembling nanoparticle monomer and the immunoglobulin binding domain.

[0152] In an aspect, the method further comprises regenerating the fusion protein monomer, wherein the regenerated fusion protein monomer is capable of reforming aggregates of nanoparticles and complexes upon contact with the protein of interest in a first flowing solution or a second flowing solution.

[0153] In an embodiment of the method, the self-assembling nanoparticle monomer is a ferritin monomer. In an embodiment of the fusion protein monomer, the ferritin monomer is a Helicobacter pylori ferritin monomer or a Pyrococcus furiosus ferritin monomer. In an embodiment, the ferritin monomer is a Helicobacter pylori ferritin monomer. In an embodiment, the ferritin monomer is a Pyrococcus furiosus ferritin monomer. In an embodiment, the ferritin monomer is a human ferritin monomer. In an embodiment, the ferritin monomer is a human heavy chain ferritin monomer. In an embodiment, the ferritin monomer is a human light chain ferritin monomer. In an embodiment, the ferritin monomer is the ferritin monomer disclosed in Table 1 below.

[0154] In an embodiment of the method, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 1. In an embodiment, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 1. In an embodiment, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 2. In an embodiment, the ferritin monomer comprises an amino acid sequence that is at least 90%, 95%, 98% or 100% identical to SEQ ID NO: 2.

[0155] In an embodiment, the assembled nanoparticle is an assembled ferritin nanoparticle. In an embodiment, the assembled ferritin nanoparticle comprises any one of the above-mentioned ferritin monomers. In an embodiment, the assembled ferritin nanoparticle comprises two or more of the above-mentioned ferritin monomers.

[0156] In some embodiments, the bispecific nature of the small molecule binding molecule does not require binding to the same site on the product of interest. By binding to two different sites on the product of interest, a complex of two or more products of interest can be created. In some embodiments, this heterogeneous bispecific affinity is preferred for molecules that do not have two binding sites thereon. Since antibodies are dimers, antibodies can bind to two Protein A molecules, and as a result, the complex can be large. Most molecules do not have two binding sites for affinity, so the bispecific nature of the affinity ligand requires two different affinity moieties on the same site.

[0157] In some embodiments, the bispecific peptide can be created by linking two affinity peptides together such that the combined peptides bind to two different epitopes on a small synthetic or natural target, thereby enabling the finding and purification of larger complexes in a nanofiltration system such as those described. In some embodiments, the MWCO is expected to be much lower than that of proteins, DNA, RNA, or viruses or virus-like particles. MWCOs near 500 Da, 1 kDa, 2 kDa, 4 kDa, 8 kDa, 16 kDa, 32 kDa can be useful in such systems.

[0158] In some embodiments, the affinity moieties made from small DNA polymerases or RNA strands can be found for small molecular weight synthetic molecules, for example, by common screening methods. The bispecific moiety is created from two different affinity moieties that bind to two different epitopes on a small synthetic target, thereby enabling the finding and purification of larger complexes in a nanofiltration system such as those described. In some embodiments, the MWCO is expected to be much lower than that of proteins, DNA, RNA, or viruses or virus-like particles. MWCOs near 500 Da, 1 kDa, 2 kDa, 4 kDa, 8 kDa, 16 kDa, 32 kDa can all be useful in such systems.

[0159] In some embodiments, binding molecules made from small peptides can be found, for example, by screening phage display for small molecular weight synthetic or naturally occurring molecules. Some targets may include metabolites such as aspirin and those discussed by Mulukutla et al, Biotech Bioeng, V 114, 2017, that inhibit the growth of CHO cells. These affinity ligands must have an affinity coefficient that is much smaller than the concentration of the product in solution. In some embodiments, the peptide can be separated from the small molecular weight compound when it is extremely large compared to the compound under non-binding conditions.

[0160] In some embodiments, the present disclosure provides a binding molecule having two or more binding sites, each of the binding sites binding to an epitope on a product of interest, and the complex comprising the binding molecule and the product of interest being larger than the product of interest. In some embodiments, the complex is about 25%, about 50%, about 100%, about 200%, or about 400% larger than the product of interest. In some embodiments, the complex comprises one binding molecule and about two or more products of interest. In some embodiments, the complex loses affinity for the product of interest under specific elution conditions. In some embodiments, the binding molecule has two, three, four, five, six, seven, eight, nine, or ten binding sites for the product of interest. In some embodiments, the binding molecule consists of two or more protein A binding domains having a molecular weight of about 300 kDa, about 400 kDa, about 600 kDa, or about 1,000 kDa. In some embodiments, the two or more protein A binding domains are capable of binding to the mass of a target Fc-containing molecule that is twice the mass of protein A. In some embodiments, the complex comprises three or more fab fragments having a molecular weight of about 100 kDa, about 300 kDa, about 600 kDa, or about 1,000 kDa. In some embodiments, the complex comprises DNA having two or more affinity sites having a molecular weight of about 300 kDa, about 400 kDa, about 600 kDa, or about 1,000 kDa. In some embodiments, the complex comprises a peptide having two or more binding sites having a molecular weight of about 1 kDa, about 2 kDa, about 4 kDa, about 8 kDa, or about 16 kDa. In some embodiments, the complex comprises RNA having two or more affinity sites having a molecular weight of about 300 kDa, about 400 kDa, about 600 kDa, or about 1,000 kDa.

[0161] In some embodiments, the filtration is continuous dialysis. In some embodiments, the filtration is continuous modular countercurrent tangential flow filtration. In some embodiments, the complex is separated into the product of interest and the binding molecule by filtration under specific elution conditions, and the MWCO of the membrane or the pore size of the membrane is smaller than the binding molecule and larger than the product of interest.

[0162] In some embodiments, the binding molecule has two or more binding sites for the product of interest, one of the sites binds to a first product of interest and the other site binds to a second product of interest. In some embodiments, the binding molecule has a molecular weight smaller than the product of interest. In some embodiments, the second binding molecule has only one of these binding sites. In some embodiments, the mixture of the binding molecule and the product of interest forms a complex having a weight ratio of at least one unit of the binding molecule to about two or more units of the product of interest. In some embodiments, the ratio of the binding molecule to the complex is controlled by the size of the complex. In some embodiments, the ratio of the binding molecule to the product of interest is about 1:3, about 1:5 or about 1:10 or more. In some embodiments, filtration of the complex is used to purify the product of interest. In some embodiments, the filtration is continuous dialysis. In some embodiments, the filtration is continuous modular countercurrent tangential flow filtration. In some embodiments, the binding molecule and the product of interest are separated by filtration under elution conditions, and the binding molecule passes through the filter while the product of interest is retained. In some embodiments, the filter area is larger than 1 m 2 2. In some embodiments, the filtration is larger than the molecular weight cut-off of the filter being used and requires the addition of polymers or particles that bind to the affinity moiety.

[0163] In some embodiments, the binding molecule has two or more binding sites, one of the sites binds to one of two similar epitopes on the product of interest, and the other site binds to one of two similar epitopes on an individual product of interest. In some embodiments, an individual binding molecule has only one of these binding sites. In some embodiments, the complex comprises at least one binding molecule and two or more products of interest. In some embodiments, this ratio is controlled by the size of the complex. In some embodiments, the ratio is about 1:3, about 1:5 or about 1:10. In some embodiments, the complex is filtered to purify the product of interest. In some embodiments, the filtration is continuous dialysis. In some embodiments, the filtration is modular countercurrent tangential flow filtration. In some embodiments, the binding molecule and the product of interest are separated by filtration under elution conditions. In some embodiments, the filtration is greater than the MWCO of the filter used and requires the addition of a polymer or particle that binds to the binding molecule. In some embodiments, the binding molecule has a molecular weight three times greater than that of the product of interest.

[0164] Dialysis-based affinity separation In some embodiments, when the size of the binding molecule is greater than the MWCO of the dialysis membrane, the binding molecule can be placed in the dialysis solution, and the product of interest is smaller than the MWCO of the membrane. In some embodiments, the product of interest flows into the dialysis device on the retentate side of the filter, and the larger binding molecule is suspended in the dialysis solution on the other side of the membrane in a countercurrent flow to the flow of the product of interest. Unbound large binding molecules cannot diffuse across the membrane to the retentate side because they are larger than the MWCO. The product of interest diffuses through the membrane and binds to the large binding molecule. After binding, the complex is too large to diffuse or convect back to the retentate side.

[0165] In some embodiments, the dialysis process uses a higher solution flux on the dialysis side of the membrane, which is cleaner, compared to the retentate side of the membrane that allows for diffusion. The ratio of the dialysis flux to the retentate flux, α, is typically 2, 4, 8, or 16 times greater than the retentate flux.

[0166] The addition of large binding molecules to the dialysis solution can reduce the dialysis solution flux below what it would be without them. The useful flux ratio, i.e., α, is proportional to the affinity coefficient and inversely proportional to the concentration of the affinity ligand. Some equilibrium models, such as the Langmuir isotherm, can be applied. For example, in some embodiments, in a system with an MWCO of 500 kDa, or 700 kDa, or 1000 kDa, a large Protein A moiety can be used to maintain a target concentration equivalent to that in the retentate (α = 1), and more preferably, α can be reduced to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, or 1 / 128, thereby increasing the antibody concentration from the retentate to the dialysate by 2, 4, 8, 16, 32, 64, or 128 times.

[0167] Under flowing conditions, the amount of the product of interest passing through the semipermeable membrane is related, inter alia, among other factors, to the diffusibility of the product of interest in the membrane, the affinity and concentration of the binding molecules on the dialysis side of the membrane, and inversely to the membrane area and the membrane thickness. In some embodiments, the mass transport of the product of interest in the lumen may affect the amount of the product of interest passing through the membrane, particularly when the thickness of the flow field of the retention fluid is much greater than the membrane thickness (e.g., in the case of hollow fibers, this is when the hollow fiber lumen is much larger than the membrane). Methods for introducing a lateral flow in the lumen of the membrane, or an orthogonal flow in the case of a flat sheet, are known. Similarly, mass transport can be affected by the flow distribution of the dialysate. In some embodiments, methods for providing a lateral flow are used to enable the dialysate to reach the inner portion of the bundle of hollow fiber membranes. In some embodiments, these methods include using Dean vortices, pulsating flow, bubbles, and separating the hollow fibers at the top and bottom of the cartridge to provide lateral mixing in the lateral flow. In some embodiments, a twisted or coiled membrane can also provide lateral mixing. In some embodiments, in the case of a flat sheet, many of these same methods can be used. In some embodiments, a screen can also be used to generate mixing orthogonal to the membrane.

[0168] III. Product of Interest In some embodiments, the methods disclosed herein can be applied to any protein product (e.g., the product 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, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, receptors, 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.

[0169] 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 comprising an antigen-binding fragment of an antibody. In certain embodiments, the protein product is a monoclonal antibody or an antigen-binding fragment thereof ("mAb"). The antibody can be a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the protein product is a bispecific antibody.

[0170] In some embodiments, the mixture containing the protein product and contaminants includes the product before the purification step. In some embodiments, the mixture is the untreated product before the purification step. In some embodiments, the mixture is a solution and buffer containing the untreated product of the pre-purification step, such as a solution containing the starting buffer. In some embodiments, the mixture includes the untreated product of the pre-purification step reconstituted in the starting buffer.

[0171] In some embodiments, the source of the protein product is a bulk protein. In some embodiments, the source of the protein product is a composition containing the protein product and non-protein components. The non-protein components can include DNA and other contaminants.

[0172] In some embodiments, the source of the protein product is from an animal. In some embodiments, the animal is a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). In some embodiments, the source is human tissue or cells. In certain embodiments, such terms refer to non-human animals (e.g., pig, horse, cow, cat or dog). In some embodiments, such terms refer to pets or livestock. In some embodiments, such terms refer to humans.

[0173] In some embodiments, the protein product purified by the methods described herein is a fusion protein. A "fusion" or "fusion protein" includes a first amino acid sequence joined in-frame to a second amino acid sequence that is not naturally joined in nature. Amino acid sequences that are normally present in individual proteins can be brought together in a fusion polypeptide, and amino acid sequences that are normally present in the same protein can be arranged in a new sequence in a fusion polypeptide. Fusion proteins can be made, for example, by chemical synthesis or by creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship. A fusion protein can further include a second amino acid sequence joined to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond. Upon transcription / translation, a single protein is produced. In this way, multiple proteins, or fragments thereof, can be incorporated into a single polypeptide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between two polypeptides fuses both polypeptides together in-frame to produce a single polypeptide fusion protein. In certain embodiments, the fusion protein can further include a third polypeptide that can include a linker sequence, as discussed in more detail below.

[0174] In some embodiments, the protein purified by the methods described herein is an antibody. Antibodies can be, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intracellular antibodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFv), camelized antibodies, affibodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to a polyclonal antibody population. Antibodies can be of any type of immunoglobulin molecule (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 antibodies described herein are IgG antibodies, or of the IgG class (e.g., human IgG1 or IgG4), or subclasses thereof. In one embodiment, the antibody is a humanized monoclonal antibody. In some embodiments, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In some embodiments, the antibodies described herein are IgG1 or IgG4 antibodies.

[0175] The present disclosure relates to the methods disclosed herein, wherein the product of interest is an antibody, an antigen-binding antibody fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof. In some embodiments, the product of interest is a full-length IgG antibody. In some embodiments, the antibody is IgG1, IgG2, IgG3 and / or IgG4, or hybrids thereof. In some embodiments, the antibody is a monoclonal antibody.

[0176] In some embodiments, the methods disclosed herein are achieved using bacterial cells, yeast cells, insect cells, or mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary cells. In some embodiments, the product of interest is prepared by the methods disclosed herein.

[0177]

Table 1-1

[0178]

Table 1-2

[0179]

Table 1-3

[0180]

Table 1-4

Examples

[0181] Example 1: System and Method for End-to-End Continuous Downstream Purification Capture Step Experiments were performed using a Chinese hamster ovary (CHO) cell line expressing monoclonal antibody IgG1. The studies were carried out in a 3-liter glass stirred tank reactor with a working volume (Chemglass Life Sciences, USA) of 2 liters, or in a 500-liter perfusion bioreactor. The cell culture was passed through a filter with a pore size of 0.2 μm and a surface area of 980 cm 2Using a tangential flow filtration (TFF) perfusion system (Repligen Corporation, USA) with a polyethersulfone (PES) hollow fiber membrane having a membrane area, samples were continuously collected from day 0 to day 14. In the TFF system, a low shear magnetic levitation centrifugal pump (Levitronix, Zurich, Switzerland) was used as the recirculation device.

[0182] Affinity colloid capture step The continuous countercurrent affinity colloid capture step included four steps as shown in Figure 1. First, dehydration of the conditioned medium (CM); second, addition of soluble protein A (sProA); third, dehydration of the CM & sProA complex; and fourth, two-stage countercurrent washing.

[0183] During the dehydration stage, a 30 kDa Ultracel Pellicon capsule (catalog number PCC030C05 Millipore-Sigma, USA) with an area of 0.5 m 2 was used. Two peristaltic pumps (Watson-Marlow Fluid Technology Group, USA) were used to control the flow rates of the feed and the retentate. In the feed, retentate, and permeate lines, PendoTech pressure sensors (Cole-Parmer, USA) were placed to monitor the transmembrane pressure (TMP). A critical flux study was performed using the flux stepping procedure by Li and Zydney (Li Z, Zydney AL. Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates. Biotechnology Prog. 2017;33(6):1561-1567). The TMP was evaluated as a function of time during a constant flux operation, and the filtrate flux was increased stepwise to determine the onset of fouling.

[0184] To determine the optimal conditions for mixing sProA and mAb, a series of studies were conducted in batch mode using a well-behaved mAb ("mAb1"). The mAb was aliquoted into 100 mL glass beakers and placed on a magnetic stirring plate at a constant mixing speed such as 300 RPM at room temperature. sProA (Catalog number 10-2001-1M Repligen Corporation, USA) was added to the beaker using a programmable syringe pump at a flow rate of 0.05 mL / min. The optimal binding conditions were determined by varying the molar ratio of sProA:mAb, and the data were evaluated by dynamic light scattering (DLS), size exclusion chromatography (SEC), and bindable Protein A.

[0185] To concentrate the mixture of CM and sProA, a 0.1 m Pellicon capsule with a Biomax 300 kDa membrane, C screen was used. Two peristaltic pumps (Watson-Marlow Fluid Technology Group, Wilmington, MA) were used to control the flow rates of the feed and the hold-up liquid, and the transmembrane pressure (TMP) was monitored by placing PendoTech pressure sensors on the feed, hold-up liquid, and permeate lines. To further remove impurities, the hold-up liquid at this stage was fed to a two-stage countercurrent washing process. The area of each filter during washing was 0.1 m 2 and the feed and hold-up liquid fluxes of each filter were selected based on critical flux studies performed using a flux stepping procedure. The continuous countercurrent affinity colloidal capture process is shown in Figure 1. 2

[0186] Analysis The mAb concentration was determined by analytical protein A affinity chromatography using a 20×2.1 cm I.D. POROS A / 20 protein A column (Thermo Fisher Scientific). The antibody concentration was calculated using a standard curve prepared for the molecule of interest according to Beer's law. The mAb purity was evaluated by size exclusion chromatography (SEC) using a 30×7.8 cm I.D. TSKgel G2000SWxl column (Tosoh Bioscience LLC). The product purity was calculated from the SEC chromatogram as the ratio of the monomer peak area to the sum of all peak areas. The size of the complex of mAb and sProA was evaluated by dynamic light scattering (DLS) using a Zetasizer Nano (Malvern Panalytical, UK). The HCP level was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cygnus Technologies, USA). The sample was diluted using a sample dilution buffer (Cygnus Technologies) until the HCP concentration in the sample was in the range of 1 - 100 ng / mL. The ELISA procedure followed the kit manufacturer's protocol.

[0187] CM dehydration Figure 2 shows the critical flux studies for the diafiltration stage at feed fluxes of 5, 10, and 15 LMH with a feed (CM mAb1) concentration of 1 mg / mL. As shown in Figure 2, a maximum concentration factor of 7 was achieved at a feed flux of 5 LMH, while the TMP was less than 1 psi. Using the data generated here, the diafiltration process can be designed based on the flow rate exiting the perfusion bioreactor. Considering the initial concentration of CM used, the permeate flow rate can also be adjusted based on the desired final concentration factor. Based on the generated critical flux data, a three-stage TFF for diafiltration was designed to concentrate the 670 L of mAb1 CM generated on days 11 - 13 of a 500 L perfusion bioreactor. Two peristaltic pumps were used to control the feed and retentate fluxes at each filter as shown in Figure 3. The average concentration of CM was 1 mg / mL, which was concentrated 8.3-fold during the process where the fluxes and pressures at each filter are described in Table 1. Nearly 1 log reduction in HCP was achieved during this step, and little to no measurable retention of HCP was seen on the membrane. Additionally, the final purity of the captured product can also increase by 0.5 - 1 log reduction during this step, mainly due to the interaction of HCP with the complex of sProA and mAb.

[0188]

Table 2

[0189] Mixing of mAb and sProA The optimal binding conditions for sProA and mAb were determined by varying the molar ratio of sProA:mAb. Figure 4 demonstrates the size of the complexes formed with different molar ratios of mAb of sProA:mAb1. The final purified mAb at a concentration of 4 mg / mL and pH 7.4 was used for each study, and the molar ratio was increased by adding 40 mg / mL of sProA at pH 7.2. The size of the complexes was investigated using DLS and SEC, based on the data that a soluble complex of approximately 700 kDa was formed at a molar ratio of sProA:mAb of 0.3. In addition to the size of the complexes, ProA-binding capable HPLC was used to investigate the binding yield as well. A molar ratio of 0.3 or higher with a 100% yield was achieved for mAb1, where 100% yield means that all of the mAb formed complexes and there was no free mAb present in the solution. Based on the data generated in this study, a molar ratio of sProA:mAb of 0.3 was used for all of the remaining studies reported here.

[0190] Three-stage dehydration and CC washing As shown in Figure 5, three-stage dehydration and CC washing were used, where the mAb & sProA complex was first concentrated. Then, the retentate at this stage was diluted with the permeate of the second washing stage and concentrated again in the first washing stage. Finally, the retentate of the first washing stage was diluted again using a fresh buffer of 50 mM Tris at pH 7.4 and concentrated further during the second washing stage. To determine the optimal flow rate for this process, a critical flux study was performed for the washing stage. As shown in Figure 6, when the feed flux is 5 LMH, a concentration factor of 5 can be achieved. Therefore, based on this data, the feed and retentate fluxes for each stage were controlled at 5 and 1 LMH, respectively, to achieve a concentration factor of 5 during each stage. At the end of the capture process, a concentration factor of 40 was achieved with a final HCP level of 590 ppm.

[0191] These data demonstrate a 590 ppm HCP level with 90% antibody yield, which is most effective at high protein concentrations to reduce the amount of wash solution per kg of product. The final purity of the product is comparable to Protein A chromatography columns and has the advantage of reducing the Process Mass Index (PMI) by at least a factor of 3. Additionally, the final cost of the purification process is further reduced by replacing expensive Protein A chromatography resin with solubilized Protein A in a form that can be recycled and reused at least 10 - 20 times. This process has been shown to be economically viable using commercially available sProA. Moreover, since sProA can be produced by any manufacturer, including domestic ones, constraints in the supply chain are eliminated.

[0192] The various removal degrees (R) in TFF are shown as follows: R = C0 / C, where C0 is the concentration of the species in the feed entering the filter and C is the concentration of the species in the final retentate remaining in the filter. Figure 7 shows the variable removal for mAb, sProA, and the mAb&sProA complex with a molar ratio of 3, where mAb and sProA pass through the filter while the mAb & sProA complex is 100% retained by the filter.

[0193] CEX Final Purification To separate sProA and mAb after the capture step, a CEX final purification step was developed. To determine the optimal conditions for separating mAb and sProA, a 96-well plate (catalog number 136101, Fisher Scientific) was used and the study was carried out in batch contact mode. UNOSphere cation exchange resin (catalog number 1560111, BioRad) was prepared and washed with a pH 3 buffer containing 50 mM glycine, then this was sedimented to determine the volume concentration and centrifuged at 1000×g for 10 minutes. The mAb&sProA complex from the capture step was titrated to pH 3 using glycine / HCl and mixed with 20% resin at a binding capacity of 20 mg / mL. After incubating the sample for 5 minutes, this was centrifuged at 1000×g for 10 minutes and the filtrate containing the unbound protein was collected. In the next step, to elute sProA, the first elution buffer was mixed with the sample and then centrifuged to collect the filtrate containing sProA. In the final step, the second elution buffer was mixed with the sample and centrifuged to recover mAb as the filtrate in this step. The buffer volume in both elution steps was 20 times the volume of the sedimented resin, and the incubation times were 10 minutes and 20 minutes for the first and second elution steps before centrifuging the sample, respectively.

[0194] Figure 8 shows the data generated during the first elution of the CEX final purification step measuring the partition coefficients of sProA and mAb1 at pH 3 and different concentrations of NaCl. Based on the data, 0.8 M NaCl can elute 91% of sProA at pH 3 without eluting any mAb in this step. During the second elution step, 50 mM Tris and 1 M NaCl at pH 7.8 were used, which resulted in the elution of 70% of the mAb.

[0195] AEX Final Purification Materials and Methods A synthetic feed containing the monoclonal antibody mAb1 and impurities, i.e., HCP (imitating the neutralized Protein A elution pool), was adjusted to a pH of 8.5 and a salt concentration of 10 mM NaCl (conductivity, 2.5 ± 0.3 mS / cm). The synthetic feed was prepared by spiking the mAb with HCP concentrated from the conditioned medium (mAb1). The concentrated HCP was prepared by loading the mAb1 conditioned medium (adjusted to pH 8.5, conductivity 2.5 mS / cm) onto a Capto Q column (20 cm bed height, 1.1 cm diameter) pre-equilibrated with Buffer A, 10 mM Tris (pH 8.5), 10 mM NaCl. After washing the column after loading with Buffer A, the bound HCP was recovered by eluting with 10 mM Tris (pH 8.5), 1 M NaCl. To digest the host cell DNA and RNA, the eluted HCP pool was treated with 10 μL of Benzonase endonuclease (EMD Merck, Germany) for 2 hours. The resulting HCP pool was buffer-exchanged by dialysis against Buffer A using a Slide-A-Lyzer™ Dialysis Flask (Thermo Scientific, USA) with a MWCO of 3.5K. The mAb concentration was determined using Solo-VPE (Repligen, USA). Chromatographic separation was performed using Fractogel TMAE(M) anion exchange resin (EMD Merck, Germany) with a particle size range of 40 - 90 μm as specified by the manufacturer. The resin was washed and resuspended in the binding buffer, 10 mM Tris HCl (pH 8.5), 10 mM NaCl. The adsorbent and binding buffer conditions were experimental and are described elsewhere (Kelley et al., 2008). The resin slurry concentration (v / v%) was determined from the resin volume sedimented after centrifugation at 1000 × g for 5 minutes.

[0196] Batch binding experiment Optimal binding conditions for adsorbent screening and impurity removal were performed using batch binding experiments. This approach enables easier and faster screening while maintaining functional similarity to the intended continuous operation. An anion exchange resin (e.g., Fractogel TMAE) was pre-equilibrated to make a 50% (v / v) resin slurry and resuspended in buffer A (as described above). The required amount of resin slurry was pipetted into 1.5 mL low-protein binding tubes (catalog number 90411, Thermo Scientific) containing the mAb pool with or without impurities to obtain a 1% or 5% final resin suspension. The mAb pool was pre-conditioned with buffer A. The resulting mixture was incubated on a rotary mixer at room temperature (22 ± 2 °C) for 20 minutes. After incubation, the resin containing the sample was filtered through a 0.45 μm spin filter to recover the final purified mAb. The mAb concentration was measured using Solo-VPE (Repligen, USA). HCP and host cell (CHO) DNA in the final unpurified mAb and the final purified mAb were quantified using in-house assays for HCP ELISA and real-time PCR, respectively.

[0197] Note that some of the screening studies were performed in 1 mL, 0.45 μm 96-well filter plates (catalog number 8129, Pall Corporation, USA). The operating conditions (pH and conductivity) were selected based on the weak-partitioning mode (WPC) of the operation (citation). In WPC, the differential interaction between the product of interest and impurities and the chromatography resin is represented by the partition coefficient K p For a protein (mAb or impurity), K p is the ratio of the concentration of the solute bound to the resin divided by the concentration in the solution at equilibrium (citation).

[0198]

Number

[0199] Continuous single-pass tangential flow filtration (TFF) anion exchange chromatography As shown in Figure 9, the continuous single-pass TFF anion exchange chromatography system consists of a hollow fiber module with a surface area of 980 cm 2 (catalog number S04-P20U-10-N, Repligen, USA) and a peristaltic pump. The flow rates of the feed and permeate were controlled using the peristaltic pump. This equipment enabled the adjustment of the permeate flux considering the highly normalized water permeability (400 LMH / psi) of the hollow fiber module used in this study. The resin slurry (40 v / v%) was pre-equilibrated with binding buffer, 10 mM Tris HCl (pH 8.5), and 10 mM NaCl. The slurry was manually batch-mixed with the feed (10 g / L mAb and impurities) to obtain a loading material with a desired final resin slurry concentration of 1% - 5%. This mixture was incubated for 5 minutes and applied to the hollow fiber module at a feed flux of 50 LMH using the supply pump P1. The flow rate between pumps P1 and P2 was adjusted to achieve the desired permeate flux and slurry concentration factor along the hollow fiber membrane. Here, P1 > P2, and the slurry concentration factor is the ratio between the feed flow rate and the retentate flow rate. The selection of the slurry concentration factor depended on the proportion of the slurry in the loading material during single-pass operation and the intended product recovery rate. The selected slurry concentration factors were targeted at 20 times and 5 times of the initial resin slurry of 1% - 5% respectively (Table 2), and nominally the final slurry concentrations were 20% and 25% after SPTFF. The purified mAb product was continuously recovered in the permeate, while the concentrated resin containing impurities was recovered from the retentate.

[0200] [Table 3] * Without Benzonase treatment Water regeneration and PMI

[0201] Slurry concentration factor The product recovery percentage or process yield in single-pass operation was directly proportional to the slurry concentration factor, both for the hollow fiber module. The slurry concentration factors that were successfully evaluated for the 5% resin slurry were 5-fold, 10-fold, and 15-fold. For concentration factors exceeding 15, the sharp increase at a maximum membrane transmembrane pressure of 25 psi might have been due to the blockage of the hollow fibers.

[0202] Batch binding experiment Figure 11 shows the calculated "Kp impurity" values obtained at various HCP concentrations while keeping the mAb concentration constant at 10 g / L using 5% Fractogel TMAE resin. For HCP challenges below 600 PPM, the residual HCP level in the final purified mAb material was below the detection limit of the assay. For higher (above 600 ppm) HCP challenges, a 2.3 log reduction in HCP was observed, and the Kp impurity value for HCP was above 3500. Similarly, the partition coefficient for the purified mAb product was determined and found to be less than 1.0 (data not shown) at 10 mM salt (NaCl). From the principle of WPC, a lower product Kp value is preferred, especially at higher mAb loads, as it reduces the potential for product loss, thereby enabling maximum utilization of the effective binding capacity of the adsorbent for HCP and DNA removal.

[0203] Continuous single-pass TFF anion exchange chromatography By the functional similarity of batch binding studies for the TFF system, the switch to the TFF mode of operation was simplified while leveraging the results from batch binding. Table 1 shows the HCP reduction observed during the operation of the TFF mode. For both 1% and 5% resin slurries with a 10 mg / mL mAb feed, a 1.9 log reduction in HCP comparable to the batch binding results was observed. The selection of the 1% resin slurry yielded a 91% product recovery compared to the 74% product recovery observed using the 5% slurry in the TFF mode. It should be noted that the products discussed here are the result of the slurry concentration factor and not due to product Kp (Kp < 1). Interestingly, a significant decrease in HCP log reduction was observed with an 85 mg / mL mAb feed.

[0204] In - process water regeneration from the purified product in the permeate offsets the PMI of this step. As shown in Figure 9, an mAb feed (35 g / L) was concentrated to 80 g / L using a 30 kDa single - pass TFF module, and 56% of the water feed with no detectable HCP leakage in the regenerated water was recovered (Table 3).

[0205]

Table 4

[0206] Additional regeneration strategies include, but are not limited to, that the permeate concentrated to 150 g / L containing 10 g / L of mAb recovered 93% of the water using the loading material; the permeate concentrated to 150 g / L containing 25 g / L of mAb recovered 83% of the water using the loading material; the permeate concentrated to 150 g / L containing 50 g / L of mAb recovered 66% of the water using the loading material; and the permeate concentrated to 150 g / L containing 100 g / L of mAb recovered 33% of the water using the loading material.

[0207] As outlined above, 1% and 5% resin slurries in the loading material were evaluated for slurry concentration factors of 20 and 5, respectively. The HCP loads in 1% and 5% resin were 0.6 mg / mL resin and 0.12 mg / mL resin, respectively. At a feed mAb concentration of 10 g / L, 2 log of HCP removal was observed.

[0208] Example 2: Design and Escherichia coli expression of ferritin nanoparticles Particle design Protein nanoparticles can be composed of monomers that self-assemble into 24-mer particles via a self-assembly domain derived from Helicobacter pylori ferritin (Hpftn). A version with an n-terminal modification, Bf-HpFtn, was utilized. This design is constructed at the n-terminus of Bf-HpFtn by adding a linker domain, an antibody-binding domain, and an affinity tag. Optimization of the particle design occurred mainly by changing the linker region resulting from the selection of nanoparticle 384.

[0209] The self-assembly domain (Bf-HpFtn) of nanoparticle 384 contains the peptide set forth in SEQ ID NO: 1. This is connected to the antibody-binding domain by a linker, and the embodiment of this linker contains SEQ ID NO: 26. The antibody-binding domain (Z domain) contains SEQ ID NO: 4. The affinity tag contains the polyhistidine tag and enterokinase cleavage site set forth in SEQ ID NO: 50. Thus, the entire protein monomer composed of the above parts in the nanoparticle of nanoparticle 384 is SEQ ID NO: 30.

[0210] Construct design and generation The protein sequence of the protein monomer was reverse translated into a nucleotide sequence using Geneious Prime® software, which selects for E. coli optimization using randomized codon usage frequencies with rare codons subtracted. The sequence obtained from the designed coding gene can be found in SEQ ID NO: 52. Synthetic nucleotides with vector overlaps designed for cloning into an in-house vector derived from pET28 but with the β-lactamase resistance gene replaced in the vector were ordered from a commercial vendor. The insert was cloned by overlap assembly using a commercially available Gibson reaction-based cloning kit and transformation into an E. coli cloning strain. Plasmids obtained from single colony isolates were confirmed by Sanger sequencing and then transformed into E. coli BL21(DE3) for expression, forming strain Bl_NP0384.

[0211] Expression of Nanoparticles The protein from strain B1_NP0384 was produced by fed-batch growth and IPTG induction of the expression strain. The growth medium contains 20.3 g / kg yeast extract, 10.1 g / kg sodium sulfate, and 7 g / kg dipotassium phosphate, and this is supplemented with 100 μg / mL carbenicillin after autoclaving. The strain was grown overnight at 37 °C in a shake flask and then inoculated into 30 L of the same medium at 37 °C in a bioreactor, and the feed cycle was started after the first oxygen rebound. The feed contains 550 g / kg glucose supplemented with 5.4 g / kg magnesium sulfate and 333 g / kg yeast extract. Incubation was started at an optical density of 80 at 550 nm by adding IPTG to a final concentration of 0.3 mM, taking all precautions available to maintain oxygen supply. After 3 h of incubation, the culture was cooled before harvesting. The cell paste was recovered by centrifugation and stored at -80 °C.

[0212] Purification of Nanoparticles for the Insoluble Fraction (Inclusion Bodies) For protein purification, cells were suspended in phosphate-buffered saline (pH 7.4) up to 6 volumes (mL / g cell paste). After complete resuspension by stirring, the cell suspension was continuously stirred with a magnetic stir bar, and protease inhibitors (2.3 tablets / L of Roche Complete 04693132001) and DNase (Pierce Universal Nuclease 2656968, 100 uL / L; DNase I Roche 10104159001, 33 mg / L) were added. The cell suspension was stirred and then lysozyme (Thermo 89833) was added up to 40 mg / L, and the cells were stirred at room temperature for about 40 minutes. The cell suspension was cooled on ice and then passed twice through an LM20 Microfluidizer at 12,000 psi while maintaining the cells in a cooling loop immersed in a reaction chamber and an ice water bath. The lysed cells were centrifuged at 3000×g in a 1 L centrifuge bottle, and the supernatant was decanted. The inclusion body pellet was then suspended in 0.5× B-PER II (Thermo 78260) extraction reagent in PBS at a volume of 5 mL / g pellet while adding an additional 33 mg / L of Dnase I. The suspension was made complete by stirring and then centrifuged at 10,000×g for 20 minutes in a 500 mL centrifuge bottle. The supernatant was decanted and then the pellet was washed in PBS (5 mL / g pellet). The suspension was centrifuged at 10,000×g for 20 minutes and decanted.

[0213] Urea solubilization of inclusion bodies The washed inclusion pellet was then suspended by stirring in 5 mL / g pellet in PBS until completely resuspended, and then 3 volumes of 8 M / kg urea in 50 mM Tris at pH 7.8 were added to the stirred solution of the inclusion suspension (ending up with 6 M urea / kg). The solution was maintained at 4°C overnight and then warmed to RT and centrifuged at 15,000×g for 20 minutes. The supernatant containing the solubilized protein monomer was further purified by immobilized metal affinity chromatography (IMAC).

[0214] Immobilized Metal Affinity Chromatography (IMAC) Purification (2.1×19 cm) The supernatant containing the urea-solubilized protein was filtered through a 0.45 μm filter and loaded onto a 77 mL Ni-NTA column (2.1×19 cm) pre-equilibrated with 5 mM imidazole in 50 mM Tris-HCl, 6 M urea (pH 7.8) at a flow rate of 15.2 mL / min for 5 column volumes (CV). After loading the sample, the column was washed with 5 mM imidazole in 50 mM Tris-HCl, 6 M urea (pH 7.8) for CV to remove weakly bound impurities. After column washing, the bound protein was eluted with a step gradient of 500 mM imidazole in 50 mM Tris-HCl, 6 M urea (pH 7.8). After elution, the column was washed with 0.5 M sodium hydroxide for 5 CV, followed by 5 CV of deionized (DI) water and a 20% ethanol storage solution. To avoid loss of chelated nickel on the Ni-NTA resin, 3 CV of 0.1 M NiSO4 was refilled into the column before each run, followed by washing with 5 V of DI water and the equilibration buffer. The eluted protein was collected with an AKTA Avant fraction collector and kept at 6 °C until the refolding step.

[0215] Dialysis for Urea Removal and Refolding Batch dialysis was mainly performed to remove urea. This enabled the simultaneous implementation of nanoparticle refolding and self-assembly, along with conditioning into the preferred buffer. The IMAC eluate equilibrated to ambient temperature was loaded into a Slide-A-Lyzer™ Dialysis Cassette (Thermo Fisher) with a MWCO of 10 kDa, washed and stirred once overnight in 50 mM Tris buffer at pH 7.8 with 200 dialysate volumes (DV), filtered through a 0.22 μm filter, and stored at 4 °C.

[0216] Size-Exclusion Chromatography (SEC) 3 mL of nanoparticles 384 (ferritin) filtered through a 0.22 μm syringe and dialyzed were loaded onto a HiPrep™ 16 / 60 Sephacryl® S-500 HR (120.6 mL column volume (CV); Cytiva) size exclusion chromatography column equilibrated with 50 mM Tris (pH 7.8) using AKTA™ Pure (Cytiva). The column was isocratically eluted at 0.5 mL / min (15 cm / hour) using an equilibration buffer to collect at least 2 CV of fraction A with a signal of 5 mAU or more in a 96-well deep well plate with a maximum fraction volume of 1 mL. 280 The column was washed with 0.5 CV of 0.5 M NaOH, 1.5 CV of equilibration buffer, 1 CV of water, and 1.2 CV of 20% ethanol at the same flow rate. All buffers were filtered through 0.22 μm before use. Fractions were analyzed for protein concentration (A280, EC1% = 7.7) using a Stunner (Unchained Labs, Pleasanton CA) and for purity using the TSKgel SuperSW mAb method described herein. Fractions were pooled to include only those with peaks 1 and 2 in Figures 12A - 12B. Peak 3 was excluded because it contained smaller fragments suspected to be incompletely assembled ferritin species.

[0217] Binding, Dissociation, and Characterization of Nanoparticles When a sample containing both nanoparticles and antibodies was prepared, precipitation could be visually observed immediately. To solve this, a precipitated sample containing an excessive amount of antibodies against the nanoparticles was added to multiple salt solutions, and re-solubilization was visually evaluated by appearance and absorbance readings at 600 nm as a measure of light scattering by the particles in the solution. In addition, nanoparticles alone and antibodies alone were also added as controls to these salts to determine whether the salts themselves affected the precipitation of any of these components. The sample in which the antibodies and nanoparticles had precipitated was found to have the best re-solubilization by being placed in a 15 mM NaCl solution. In addition, under this condition, precipitation of either component did not occur alone. However, adding salt to a sample that had already precipitated did not result in complete re-solubilization of the precipitated sample.

[0218] Based on the hypothesis that although it is difficult, it may be possible to prevent the precipitation interaction from completely reversing, salt was added to the antibodies before adding the nanoparticles. A method for creating a bound nanoparticle and antibody sample was devised, where a 5 M NaCl stock solution was added to a solution containing the antibodies, and then the nanoparticles were added to this solution. The amount of the NaCl stock solution added to the antibodies was calculated so that the final concentration of the solution would be equal to 150 mM NaCl. When this method was tested, as can be confirmed in Figure 13, a clear sample with no visible precipitation was produced.

[0219] IgG Sepharose 6 affinity column (“reverse-phase Protein A, mAb column”) A set of samples containing nanoparticles was loaded onto a 3.9 mL Tricorn 5 / 20 column (Cytiva, Marlborough, MA) filled to a bed height of 19.7 cm and equipped with IgG Sepharose 6 Fast Flow affinity resin (Cytiva) connected to an AKTA (registered trademark) Pure 25 (Cytiva) FPLC system. For all runs, the method followed the sequence in Table 4 at a set linear velocity of 300 cm / h.

[0220]

Table 5

[0221] As shown in Table 5, four chromatography runs were performed and the results are shown in FIGS. 14A - 14D. An engineering run (cycle 0) was performed first to confirm the binding capacity of the resin (i.e., that the resin can actually bind to Protein A). Recombinant native Protein A ligand (rSPA, Repligen, Waltham, MA) derived from S. aureus diluted to pH 7.4 with 50 mM Tris (pH 7.4) was loaded. Despite a 25% loss in the column by mass balance, all of the chromatogram area and 75% of the quantified mass were due to the eluate, suggesting that the column is functional. When less than half of the mass of the soluble Protein A load was loaded (cycle 1), approximately half of the purified nanoparticle load bound and appeared in the eluate while half appeared in the flow - through, as seen from the chromatogram peak area. Cycle 1b shows that the population in the flow - through also has the ability to bind to the column, suggesting that it was overloaded in cycle 1, resulting in a lower capacity than for soluble Protein A and loss of access to some ligands due to its much larger size. Cycle 2 shows that the eluate from cycle 1 was titrated to neutral pH with 0.5 M Tris (pH 11) and re - loaded and was able to bind again. Overall, these data demonstrate that the nanoparticles can bind to mAb, can dissociate from mAb (enable elution), and can be regenerated by pH titration and bind to mAb at least twice.

[0222]

Table 6

[0223] Combined SEC and SDS-PAGE for Complex Formation and Dissociation To demonstrate complex formation between an antibody and nanoparticles, experiments using both SEC and SDS-PAGE were performed. First, while stirring 4.2 mL of antibody at a concentration of 9.6 mg / mL in a buffer containing 50 mM Tris and 215 mM NaCl at pH 7.8, 1.8 mL of nanoparticles in 50 mM Tris at a concentration of 5.1 mg / mL and pH 7.8 were continuously added at a flow rate of 0.1 mL / min. The resulting mixture contained 6.7 mg / mL of antibody and 1.5 mg / mL of nanoparticles in a buffer containing 50 mM Tris and 150 mM NaCl at pH 7.8.

[0224] This material was then analyzed using a HiPrep 16 / 60 Sephacryl S-500 HR column (Cytiva). The column was connected to an AKTA (registered trademark) pure, and each step was performed at a flow rate of 14.9 cm / hr. Starting from the initial storage in 20% ethanol in water, the column was washed with 1 CV of water and then equilibrated with 2 CV of 50 mM Tris at pH 7.8. Using a 50 mL Superloop from Cytiva, a mixture of antibody and nanoparticles was loaded at a volume of 3 mL, or 2.5% of the column volume, and the column was run through with 2 CV of the same equilibration buffer to elute the mixture. The elution peaks measured at an absorbance of 280 nm were fractionated in 2 mL increments. The column was then sanitized with 0.75 CV of 0.5 M NaOH, washed with 1 CV of water, and then returned to the 20% ethanol in water storage buffer by applying 1.2 CV. The same method was repeated two more times with the antibody alone at a 3 mL load of 17.4 mg / mL and the nanoparticles alone at a 3 mL load of a concentration of 5.0.

[0225] Fraction runs from the mixture of antibody and nanoparticles were then analyzed using SDS-PAGE according to the protocol described below.

[0226] The SDS-PAGE results (Figure 16) show that the front of the peak contains nanoparticles and antibodies, while the back of the peak contains only antibodies. This can be confirmed by comparing fraction B2 and B8 on the gel. The line indicating the nanoparticle monomer is visible in B2 but not in B8. Additionally, the evaluation in Figure 15 shows that the antibody does not elute alone before an elution volume of approximately 100 mL corresponding to fraction B6 on the gel, but when the antibody and nanoparticles are combined, the antibody can be initially confirmed at around 80 mL. The antibody appears at an earlier elution volume than when alone, indicating a larger size, and it is concluded that the antibody is bound to the nanoparticles.

[0227] To demonstrate the low-pH dissociation of the antibody and nanoparticles, this combination of SEC and SDS-PAGE was repeated, but at low pH. As performed above, a mixture containing the antibody at 6.7 mg / mL and nanoparticles at 1.5 mg / mL in a buffer containing 50 mM Tris and 150 mM NaCl at pH 7.8 was prepared. Next, 1 M glycine at pH 3.5 was used to titrate this solution to lower the pH to 3.5. 1 mL of the low-pH buffer was added per 2 mL of the original mixture, and the samples were diluted to an antibody concentration of 4.5 mg / mL and a nanoparticle concentration of 1.0 mg / mL, all performed at pH 3.5.

[0228] For the SEC procedure, the same HiPrep 16 / 60 Sephacryl S-500 HR column (Cytiva) was connected to AKTA (registered trademark) pure and the same operating method was used with two major differences. First, the equilibration buffer for this run was 100 mM glycine (pH 3.5) so that the column environment could be maintained at low pH conditions. Second, to prevent any interaction between the acid and the base, after sterilization with 0.5 M NaOH, an additional washing step with 0.5 CV of water was added. The mixture of antibody and nanoparticles was loaded at 5.5 mL or 4.5% of the column volume, which was increased to account for dilution by the titration procedure. This same procedure was repeated for the control, injecting 5.5 mL of antibody alone at 4.5 mg / mL and 5.5 mL of nanoparticles alone at 1.0 mg / mL.

[0229] Fractions for the low pH SEC run using the mixture of antibody and nanoparticles were then run on an SDS-PAGE gel according to the method shown below. Two peaks were generated by the mixture of antibody and nanoparticles (Figures 17 and 18); the first peak was completely that of the nanoparticles and eluted where the peak of nanoparticles alone typically elutes, and the second peak was the antibody peak that eluted where the peak of antibody alone typically elutes. Different from the neutral pH experiment, where the antibody elutes earlier (due to its larger size) than when typically injected alone, here the antibody does not show an increase in size by the time it elutes from the SEC column but instead elutes at the same retention time as when no nanoparticles were mixed during loading. Thus, this demonstrates that at low pH the antibody and nanoparticles are completely dissociated.

[0230] In Figure 19, SEC traces of the mixture loading of antibody and nanoparticles at neutral pH and low pH are compared. The sterilization peaks are located at different points on the x-axis due to washing with excess water in the low pH method. However, it was immediately apparent that the peak after sterilization was much larger in the neutral pH run than in the low pH run. This indicates that more material is bound or retained on the column for the neutral pH run than for the low pH run, and it is speculated that this may be due to larger protein complexes formed under neutral pH conditions.

[0231] Nanoparticle filter performance To understand the complex filtration performance on a small scale under elution conditions, a 500 μL volume, 300 kDa MWCO PES centrifugal filter (VS0152; Satorius Stedim, PA) was used.

[0232] First, two mixtures of antibody and nanoparticle 384 in 50 mM Tris, which are pH 7.4 and pH 7.8 respectively, were mixed to concentrations of 0.86 mg / mL and 0.40 mg / mL for antibody and nanoparticle respectively after adding 5 M NaCl (in water) to a final concentration of 150 mM. The mixtures were further diluted to pH 3.5 using 100 mM glycine (pH 3.0) or 50 mM Tris (pH 7.4), 150 mM NaCl, with final concentrations of 0.20 and 0.43 for nanoparticle 384 and mAb respectively at pH 7.4 or pH 3.5. Each of the two pH mixtures was complemented by two sets of controls that contained either mAb or nanoparticle in the same protein, NaCl concentration, and pH as in the mixture, but with the absent component replaced by buffer. In total, six spin filters were first equilibrated (500 μL, 1 spin, 2000 g, 1 minute) in buffer matching the subsequent buffer, then the samples were loaded and spun at 2000 g for 15 seconds at ambient temperature (RT).

[0233] The volumes of the load, filtrate, and retentate were recorded, and the absorbance at 280 nm (A280) was measured with a Nanodrop 2000 spectrophotometer (Thermo-Fisher, Waltham MA) blanked with the corresponding sample buffer. A 0.5 mL sample was titrated to neutral pH units by adding a 2 v / v% 0.5 M Tris base solution to which a volume addition ratio was applied to reduce the required volume of load, filtrate, and retentate samples, and SDS-PAGE was performed as follows.

[0234] Figure 20 shows an SDS-PAGE gel for the mixtures separated at neutral pH and low pH. At neutral pH, when the nanoparticles were present alone or in complexes, they appeared in both the filtrate and the retentate, and fewer nanoparticles passed through the filter when present in complexes. The mAb was evenly distributed across the filter when alone at neutral pH but was more highly retained when present in complexes. At pH 3.5, under the elution conditions, the separation demonstrated that only the mAb passed through the filter when complexed, while the nanoparticles were found only in the retentate.

[0235] To avoid gel contamination, only a small amount of the load was passed through the filter, and a realistic scaled-down model was created to speculate on what was happening in TFF (Figure 10). A negligible amount of loss was observed. The pH decreased by the fractionation of both the mAb and the nanoparticles, but comparable fractionations were also observed for the mAb complexes at each pH.

[0236] Analytical methods The purity was determined using an Agilent (Santa Clara, CA) 1260 Infinity II Bio-inert LC system equipped with a TSKgel SuperSW mAb HR; 4 μm; 7.8 mm × 30 cm (Tosoh, King of Prussia, PA). Samples were injected and eluted isocratically at 0.5 mL / min for 30 min while recording the absorbance at 280 nm. An arginine-rich mobile phase (100 mM dibasic sodium phosphate anhydrous, 100 mM sodium sulfate, 1 M arginine, pH 6.8) was used and no guard column was included to avoid material loss.

[0237] SDS PAGE Sample buffer was prepared by using 5% bME and adding Novex™ 2X Tris-Glycine SDS (LC2676, Thermo-Fisher), added to the sample at a 1:1 volume ratio, heated at 95 °C for 2 min, and cooled to RT. All samples and SeeBlue™ Plus 2, 3 kDa~198 kDa ladder (LC5925; Thermo-Fisher; no heating / cooling) were electrophoresed on a 4~12% Bis-Tris PAGE using 1X MES running buffer at 200 V for 35 min or 150 V for 90 min and then stained and imaged with SimplyBlue™ Safestain (LC6060; Thermo-Fisher). An additional 2.5 v / v% pure β-mercaptoethanol was added to low pH samples after cooling and before loading.

[0238] Example 3: Materials & Methods for TFF and C3ANDO Isothermal Titration Calorimetry (ITC) The titrant material was a 28 mg / mL monoclonal antibody (mAb) purified with binding - elution Protein A and flowed through anion - exchange chromatography to remove impurities. The ITC cell material was nanoparticles at 0.5 - 1.0 g / L and was manufactured by the method described in this section. Both of the obtained materials were buffer - exchanged by dialysis using Slide - A - Lyzer™ dialysis cassettes with a 3.5K MWCO (Thermo Scientific, USA) to a final formulation of 50 mM Tris (pH 7.8), 150 mM NaCl (Tris - saline). This material was degassed using a vacuum degassing device. Solo - VPE (Repligen, USA) was used to determine the mAb and nanoparticle concentrations. MicroCai PEAQ - ITC Automated (Malvern Panalytical, USA) was used to titrate the ITC cell material (nanoparticles) with the titrant (mAb) while measuring the energy released or absorbed by the cells through titration compared to a reference cell filled with water (WFI) for injection. The titration was performed by injecting 3 μL of each titrant at 2 μL / s for 12 times and mixing the ITC sample cell of 200 μL of the ITC cell material at 1000 RPM at 20 °C. Control titrations were performed with Tris - saline, nanoparticles and Tris - saline as the ITC cell material, and mAb, Tris - saline and Tris - saline as the titrant material, respectively. The energy released or absorbed by the cells throughout the titration was converted to thermodynamic properties (KD, enthalpy, entropy, free energy) and molar ratios using the PEAQ - ITC Automated software after inputting the concentrations of the titrant and the ITC cell material. The three control titrations performed were also uploaded to the software to minimize the energy not directly related to the binding of the nanoparticles and the mAb.

[0239] Conditioned Medium (CM) - dehydrated Dehydrating the conditioned medium by perfusion cell culture can significantly reduce the membrane area and water requirements for subsequent product capture and final purification steps. Single-pass TFF operations evaluated with different membrane units / geometries having an MWCO of 30 - 50 kDa were used to demonstrate dehydration. The conditioned medium (CM) fortified with mAb or mAb-containing mAb CM (obtained from perfusion cell culture) was filtered through a 0.22 μm PES filter. The mAb-fortified conditioned medium was prepared by spiking purified mAb into CM with low mAb. As shown in Figure 23, the continuous single-pass TFF system includes either a cassette-type membrane or a hollow fiber module and a peristaltic pump.

[0240] The flow rates of the feed and retentate were controlled using peristaltic pumps. With this equipment, the permeate flux and volume concentration factor (VCF = Q フィード / Q 保持液) can be adjusted. This membrane unit was pre-equilibrated with phosphate buffered saline, 10 mM phosphate buffer, 150 mM NaCl (pH 7.4) at a feed flux of 40 LMH, which is 20 times the hold-up volume of the membrane unit. In a typical study, a supply pump P1 was used to apply 0.22 μm filtered CM to the TFF module at a feed flux of 10 LMH. The flow rate between supply pumps P1 and P2 was adjusted to achieve the desired permeate flux and VCF along the membrane module, where P1 > P2 in this case. The selection of VCF depends on the mAb concentration in the load material and the intended dehydration in single-pass mode. Some bias runs of feed flux and mAb concentration were evaluated with respect to the process performance measured by the transmembrane pressure for membranes in the MWCO range of 30 - 50 kDa. The concentrated CM was continuously recovered with the retentate while the mAb-depleted CM was recovered with the permeate. An analytical protein A column was used to determine the mAb concentration. The mAb samples were loaded onto a 0.1 mL POROSTM Prepacked protein A affinity column (ThermoFisher, Waltham, MA) filled to a bed height of 3.0 cm and connected to an Agilent Series Gradient 1200 (Agilent) HPLC system. For all runs, this method had a set linear velocity of 6000 cm / h using a binding buffer of 1× PBS (pH 7.2) and an elution buffer of 1× PBS (pH 7.2) + 0.1% H3PO4.

[0241]

Table 7

[0242] Concentration of nanoparticle size A feed containing purified nanoparticles at approximately 0.5 mg / mL (the nanoparticle purification method was as described above) was used in an ultrafiltration / diafiltration (UF / DF) process for buffer exchange to remove small-sized (less than 300 kDa) impurities, particularly monomers and partially formed nanoparticles. UF / DF was carried out using a 0.11 m2 300 kDa PES Pellicon® 2 (Millipore, USA) membrane. Before the start of UF / DF, the membrane storage solution was flushed with 0.25 M NaOH, then flushed with water (WFI), and then equilibrated with 50 mM Tris (pH 7.8), 150 mM NaCl (Tris-buffered saline). Concentration was carried out at a feed flux of 260 LMH using ultrafiltration at a concentration of approximately 2 mg / mL, followed by diafiltration with 6 DV of Tris-buffered saline buffer to chase the Tris-buffered saline membrane to a final concentrated nanoparticle of 1.0 mg / mL. Solo-VPE (Repligen, USA) with an extinction coefficient of 0.77 M-1 cm-1 was used to determine the nanoparticle concentration. The used membrane was washed with 0.25 M NaOH and stored in 0.1 M NaOH for later use.

[0243]

Table 8

[0244] Continuous countercurrent TFF - capture, HCP removal and elution The partitioning of solutes during membrane filtration is expressed as the sieving coefficient (S). S is a measure of the ability of a solute to pass through the membrane and is calculated by the following equation (where C P is the concentration of the solute in the permeate and C f is the concentration of the solute in the retentate).

[0245]

Equation

[0246] The sieving coefficient 1 indicates unhindered transport of the solute through the membrane, while a sieving coefficient of 0 indicates complete retention of the solute by the membrane. The experimental determination of S allows prediction of the ability to separate, recover, or remove the molecule of interest in the retentate or permeate using a reasonable number of stages in a multi-stage single-pass TFF (SPTFF) configuration, as shown in Figure 25.

[0247] Sieving coefficient of pure mAb The sieving coefficients for pure mAb in Tris-buffered saline were determined for membranes of various MWCOs using the experimental setup shown in Figure 23 and presented in Table 8.

[0248]

Table 9

[0249] Sieving coefficient of pure nanoparticles The sieving coefficients for pure nanoparticles at pH 7.4 and pH 3.5 were determined for a 300 kDa MWCO membrane using the experimental setup shown in Figure 23 and presented in Table9.

[0250]

Table 10

[0251] Single-pass TFF binding and retention of mAb-nanoparticle complexes In a typical conjugation process, to achieve a molar ratio of approximately 10 (mAb:nanoparticle), the final purified mAb at a concentration of approximately 1 mg / mL was immediately mixed with an equal volume of nanoparticles at 0.5 g / L. Prior to mixing, the mAb and nanoparticles were exchanged into Tris-buffered saline buffer at pH 7.4 to avoid some pH / salt drift during mixing. The resulting mAb-nanoparticle mixture was then used as feed for a one-stage SPTFF configuration using a membrane module with a 300 kDa MWCO, as shown in Figure 23. The retentate flow rate was adjusted to achieve a VCF of 3 throughout the operation while monitoring the cartridge TMP and permeate flow rate. Free or unbound mAb in the permeate was quantified using ProA-binding HPLC (Table 10).

[0252]

Table 11

[0253] As shown in Table 10, 15% of the mAb was detected in the permeate, while the remaining portion of the mAb was retained on the retentate side of the 300 kDa membrane. This observation is consistent with the results of the sieving coefficients of the mAb and nanoparticles described above. At pH 7.4, pure mAb shows S = 1, indicating unrestricted partitioning across the membrane, while the transport of pure nanoparticles is completely restricted (i.e., S = 0). Upon binding to the nanoparticles, 85% of the mAb was retained on the retentate side of the membrane by the resulting affinity complex (>900 kDa), allowing partitioning of the permeate by free mAb.

[0254] Single-pass TFF HCP removal HCP removal is another aspect of any mAb capture process. After successfully demonstrating the formation and retention of mAb-nanoparticle complexes in a pure buffer system, the study was repeated using conditioned media (CM). Typically, to achieve a molar ratio of approximately 10 (mAb:nanoparticle), perfusion CM with an mAb titer of approximately 1 mg / mL was immediately mixed with an equal volume of 0.5 g / L nanoparticles in Tris-buffered saline at pH 7.8. A one-stage SPTFF configuration with 300 kDa and 1000 kDa MWCO membrane modules was pre-filled with CM by flushing the membranes at a feed flow rate of 40 LMH corresponding to 20 times the hold-up volume of the system. After pre-filling, the mAb-nanoparticle mixture was used as feed for the SPTFF configuration as shown in Figure 23. The retentate flow rate was adjusted to achieve a VCF of 3 throughout the operation while monitoring the TMP of the cartridge and the outlet flow. Free or unbound mAb in the permeate was quantified using ProA-binding HPLC (Table 11).

[0255]

Table 12

[0256] Similar to the previous results, 11% of the mAb was detected in the permeate, while the remaining portion of the mAb was retained on the retentate side of the 300 kDa membrane. The observed HCP removal can be further improved by a multi-stage SPTFF configuration using an appropriate wash buffer. For the 300 kDa membrane, a significantly lower operating TMP of 5 psi was observed compared to 9 psi for mAb-nanoparticles in a pure buffer system (Figure 26).

[0257] Single-pass TFF elution To evaluate the mAb capture during dissolution, the configuration shown in Figure 23 was used. The procedure involved stepwise adjusting the pH of the solution containing the mAb-nanoparticle complex from 7.8 to 3.5 using 2M glycine HCl buffer (pH 3.0). The acidified solution functioned as the feed for a single-pass TFF configuration, which utilized a membrane module with a MWCO of 300 kDa. Throughout the operation, the flow rate of the retentate was adjusted to achieve a VCF of 3 while closely monitoring the TMP of the cartridge and the outlet flow rate. Samples recovered in the retentate and permeate were reduced and denatured at 70°C for 5 minutes. Subsequently, they were loaded onto an SDS-PAGE gel for electrophoresis using 1X NuPAGE™ MES SDS buffer supplemented with 1 mM sodium metabisulfite. After electrophoresis, the gel was transferred to a fixing solution, stained with Coomassie blue, and then decolorized with deionized water. The mAb recovery in the permeate relative to the feed was calculated using densitometry of the gel image using ImageJ software.

[0258] Analysis of the SDS-PAGE gel image (Figure 27) revealed that the mAb recovery in the permeate was 64%. This is comparable to the maximum theoretical recovery (66%) for molecules with S = 1 on a 300 kDa membrane where the VCF for a single-step configuration is 3. However, a multi-step configuration has the potential to increase the mAb recovery up to 90%. On the other hand, for the nanoparticles, it was found that approximately 50% of the nanoparticles were partitioned into the permeate, which is higher than the 20% partition observed for pure nanoparticles at pH 3.5.

[0259]

Table 13

[0260] Example 4: Schematic of Continuous Countercurrent Affinity Nanoparticle Dialysis Scanning (C3ANDo) Diffusion coefficient (De) of mAb Convection and diffusion are two mechanisms that drive the movement of molecules across a semipermeable membrane. Unlike convection, diffusion is driven by the difference in concentration gradients across the membrane. The effectiveness of the membrane in promoting diffusion depends on the characteristics of the membrane as well as the size and shape of the protein. Therefore, the measurement of the diffusion properties or diffusion coefficients of real-world mAbs enables the estimation of mass transfer in systems dominated by diffusion. The following text represents a typical C3ANDo configuration using a hollow fiber membrane operating in a countercurrent mode. Using the C3ANDo configuration discussed herein, the diffusivity of mAbs and other model proteins was evaluated as shown in Table 13 below using multiple membranes, followed by demonstration of mAb capture and elution.

[0261] The hollow fiber module was mounted vertically, and the feed was introduced from the port at the bottom of the lumen side through pump P1 (Figure 28). The shell side port (shell outlet) near the supply port was attached to pump P2. The distal shell side (shell inlet) port was attached to pump P3. The shell side buffer was introduced into the module using pump P3, while pump P2 adjusted the flow rate at the shell outlet. The depleted flow was collected at the lumen outlet. The peristaltic pumps P1, P2, and P3 were equipped with appropriate pump heads and tubes. The pressure was monitored using pressure sensors placed immediately before and after the input / output ports. Before the experiment, the shell and lumen compartments of the hollow fiber module were flushed with buffer. The relationship between the flow rates on the feed side and the shell side was defined using α.

[0262]

Number

[0263] In a typical experimental setup with a feed flow (P1) of 0.05 mL / min (flux 0.12 LMH), both shell-side pumps were adjusted to 0.112 mL / min at an α value of 2.25. For some experiments, values in the range of 0.2 - 2.25 were evaluated. The configuration was operated in single-pass or feed recycle mode. Small samples were periodically collected from the lumen output and shell output for offline analysis.

[0264]

Table 14

[0265] C3ANDo Capture In a typical mAb capture study, 1 g / L of the final purified mAb in Tris-buffered saline (pH 7.8) was used as the feed. On the other hand, Tris-buffered saline (pH 7.8) was simultaneously introduced into the shell-side inlet. The feed and shell-side flow rates were maintained at 0.05 mL / min and 0.01 mL / min, resulting in an α of 0.2. Similar experiments were conducted to evaluate the effect of affinity nanoparticles on mAb recovery. As shown in Figures 29A and 29B, nanoparticles in Tris-buffered saline (pH 7.8) were supplied to this shell-side inlet. The mAb recovery on the shell side was calculated by quantifying the mAb at the lumen outlet using Solo-VPE (Repligen, USA).

[0266] As shown in Figure 14 below, the presence of nanoparticles on the shell side increased the mAb recovery compared to buffer alone. The formed mAb-nanoparticle complexes were recovered at the shell outlet and then utilized to evaluate the mAb recovery during the C3ANDo elution step.

[0267]

Table 15

[0268] C3ANDo Elution The feasibility of the diffusion-driven elution approach was evaluated using the scheme shown in Figure 31. To demonstrate C3ANDo elution, the mAb-nanoparticle complex (pH 7.8) obtained from the capture step was dialyzed against 0.1 M glycine HCl (pH 3.5) buffer. As the feed solution moves along the membrane, the pH of the solution changes from 7.8 to 3.5 by buffer exchange, causing dissociation of the mAb (150 kDa) from the nanoparticles (750 kDa). Due to its relatively small size, the dissociated mAb preferentially diffuses across the membrane into the shell-side compartment.

[0269] The lumen-outlet flow was recirculated through the membrane. During feed recirculation, the eluted mAb was collected at the shell-side outlet. After 1000 minutes of runtime, the feed recirculation was stopped and the operation was continued in single-pass mode, collecting the lumen-output flow. Samples were neutralized to pH 7.4 using 0.5 M tris base whenever applicable. These samples were reduced and denatured at 70 °C for 5 minutes. They were then loaded onto an SDS-PAGE gel for electrophoresis using 1X NuPAGE™ MES SDS buffer supplemented with 1 mM sodium pyrosulfite. After electrophoresis, the gel was transferred to a fixing solution, stained with Coomassie blue, and then decolorized with deionized water. The mAb recovery in the permeate relative to the feed was estimated using image densitometry with ImageJ software.

[0270] As described above, the C3ANDO elution study was divided into two operating modes: a recirculation mode (0 - 1000 minutes; Figure 32) and a single pass mode (1000 - 1600 minutes; Figure 33). From Figure 32, it can be observed that when compared to the feed, an increasing amount of mAb diffused into the shell compartment of the hollow fiber (40 - 640 minutes) that contained some detectable nanoparticles. Due to the low pH on the shell side, mAb was dissociated / eluted from the nanoparticles and subsequently diffused across the membrane into the shell side compartment. A significant decrease in the mAb concentration can be confirmed in the recirculated feed samples when compared to the feed (Figure 32). In Figure 33, wells 4 - 9 correspond to lumen outlet samples collected at time points 1040 minutes, 1120 minutes, 1240 minutes, 1320 minutes, 1440 minutes, and 1520 minutes after switching to the single pass mode at the 1000 minute time point. These samples show a relative enrichment of nanoparticles as the system reaches a steady state. The eluted mAb from the recirculation phase at the 600 and 640 minute time points was loaded along the single pass samples in wells 10 and 11, respectively, for comparison (Figure 33).

[0271] Preferred embodiments of the present disclosure are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the present disclosure includes any combination of the above elements in all possible variations of the invention as encompassed by the present invention, unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A method for purifying a target protein product using countercurrent filtration, (a) A step of forming a complex by contacting a first solution containing the target protein product and impurities with a binding molecule, wherein the complex contains the target protein product bound to the binding molecule, and the binding molecule contains protein A, protein G, a cation exchange resin, or an anion exchange resin, (b) A step of bringing a first fluid solution containing the composite into contact with a first side of a semipermeable membrane, wherein the composite has a molecular weight exceeding the molecular weight cutoff of the membrane, such that the composite is retained on the first side of the semipermeable membrane. (c) A step of passing the impurity through the semipermeable membrane, wherein the impurity has a molecular weight less than the molecular weight cutoff of the semipermeable membrane and is retained on the second side of the semipermeable membrane in a second fluid solution that is countercurrent to the first fluid solution, (d) The process includes the step of dissociating the complex to form a free target protein product and free bound molecules, A method comprising the following steps: (a) to (c), wherein the target protein product comprises an antibody, an antigen-binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof.

2. A method for purifying a target protein product using countercurrent filtration, (a) A step of bringing a first fluid solution containing the target protein product and impurities into contact with a first side of a semipermeable membrane, wherein the target protein product passes through the semipermeable membrane to form a complex containing binding molecules on the second side of the semipermeable membrane, and the complex has a molecular weight exceeding the molecular weight cutoff of the semipermeable membrane, and the binding molecules include protein A, protein G, a cation exchange resin, or an anion exchange resin, such that the complex is retained on the second side of the semipermeable membrane. (b) Optionally, a step of retaining the impurity on a first side of a semipermeable membrane, or a step of allowing the impurity to flow through the semipermeable membrane, wherein the impurity has a molecular weight less than the molecular weight cutoff of the semipermeable membrane, and the impurity is either retained on the first side of the semipermeable membrane in a first fluid solution that is countercurrent to the second fluid solution, or passes through the semipermeable membrane toward the second side, and the flow rate of the second fluid solution is lower than the flow rate of the first fluid solution. (c) The process includes the step of dissociating the complex to form a free target protein product and free binding molecules, A method wherein unbound molecules diffuse through the semipermeable membrane into a second fluid solution, and steps (a) to (c) are repeated.

3. The method according to claim 1, further comprising the step of (e) regenerating the binding molecules, wherein the regenerated binding molecules are capable of forming a complex upon contact with the target protein product in a first or second fluid solution, the regenerated binding molecules pass again through the second side of the semipermeable membrane in the second fluid solution, and the unbound binding molecules diffuse through the semipermeable membrane into the second fluid solution.

4. The method according to claim 1, wherein the second fluid solution contains a second binding molecule that can bind to impurities in the first solution and / or the second fluid solution.

5. The method according to claim 1, wherein the second fluid solution contains a positively charged polymer, and the positively charged polymer binds to low molecular weight seeds diffused through the semipermeable membrane.

6. The method according to claim 1, wherein the binding molecule comprises assembled ferritin nanoparticles containing 24 fusion protein monomers, each fusion protein monomer comprising i) a self-assembled nanoparticle monomer, ii) a linker, and iii) an immunoglobulin-binding domain, the immunoglobulin-binding domain being the Z domain of protein A, and the Z domain of protein A comprising an amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to one of sequence numbers 4 to 7.

7. The aforementioned fusion protein monomer i) An amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to one of sequence numbers 1 to 3, ii) An amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to one of sequence numbers 28-36, iii) An amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 32, or iv) Amino acid sequence encoded by nucleic acid including SEQ ID NO: 52 The method according to claim 6, including the method described in claim 6.

8. The method according to claim 1, wherein the permeate from step (b) and / or step (c) is recycled into the first fluid solution and / or the second fluid solution and / or the upstream fluid solution.

9. The method according to claim 1, wherein the flow rate of the first fluid solution and / or the second fluid solution is about 30 to about 60 mL / min.

10. The method according to claim 9, wherein the flow rates of the first fluid solution and the second fluid solution are the same.

11. The method according to claim 9, wherein the flow rates of the first fluid solution and the second fluid solution are different.

12. The method according to claim 1, wherein the first fluid solution and / or the second fluid solution are pulsed.

13. The method according to claim 12, wherein the pulse volume is smaller than the volume of the pores in the semipermeable membrane.

14. The method according to claim 1, wherein the released target protein product is concentrated to about 50 g / L to about 100 g / L.

15. The method according to claim 1, wherein steps (a) to (c) are performed a second time, the second fluid solution from the second time is added to the first fluid solution from the first time, and the MWCO of the filter used the second time is the same size as or larger than that of steps (a) to (c) performed the first time.

16. The method according to claim 3, wherein steps (a) to (c) are performed a second time, the second first fluid solution is added to the first first fluid solution, and the MWCO of the filter used the second time is the same size as or larger than that of steps (a) to (c) performed the first time.

17. The method according to claim 15, wherein steps (a) to (c) are performed a third time, the second fluid solution of the third time is added to the first fluid solution of the second time, and the MWCO of the filter used in the third time is the same size as or larger than the MWCO of steps (a) to (c) performed in the first or second time.

18. The method according to claim 16, wherein steps (a) to (c) are performed a third time, the third first fluid solution is added to the second first fluid solution, and the MWCO of the filter used in the third time is the same size as or larger than that of steps (a) to (c) performed in the first or second time.

19. The method according to claim 15, wherein a third flow of the first or second fluid solution flows into the second flow of the first fluid solution, and / or the second flow of the first or second fluid solution flows into the first flow of the first fluid solution.

20. A method for using a solution discharged from the filtrate, dialysate, or retaining solution of a continuous downstream process as a cleaning method for an upstream process, i) If the effluent or dialysate is the wash, the upstream process has a filter having an MWCO of the same size as or larger than that of the downstream process that generated the effluent, and further ii) A method wherein, if the retaining liquid is the wash, the upstream step has a filter having an MWCO of the same size as or smaller than that of the downstream step that produced the effluent, and the filtrate is directed downstream.

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