Method for integrated concentration and buffer exchange

Abstract

Provided herein is a method for purifying a target protein using asymmetric dialysis.

Description

【Technical Field】 【0001】 (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 366,147, filed on June 10, 2022, the entire content of which is incorporated herein by reference for all purposes. 【0002】 As biological agents move 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 agents. Therefore, more efficient downstream processing is needed. 【Summary of the Invention】 【0003】 The present disclosure relates to a method for purifying a target protein using countercurrent concentration dialysis, comprising: (a) flowing a first flowing solution containing the target protein and impurities through a first hollow fiber dialysis cassette at a first flow rate, wherein the dialysis cassette includes dialysis fluid inflow at a dialysis fluid inflow rate and dialysis fluid outflow at a dialysis fluid outflow rate, and the first flowing solution is countercurrent to the dialysis fluid inflow and outflow; (b) flowing the impurities through a semipermeable membrane of the dialysis cassette, wherein the dialysis fluid inflow rate is higher than the first flow rate, a second flowing solution containing the target protein and a low amount of impurities exits the dialysis cassette at a second flow rate, and the dialysis fluid outflow rate is the sum of the dialysis fluid inflow rate and the difference between the first flow rate and the second flow rate; (c) optionally, flowing the second flowing solution directly from the first dialysis cassette to a second dialysis cassette; and (d) optionally, repeating steps (a) and (b) using the second flowing solution and the second dialysis cassette, thereby forming a third flowing solution containing a lower amount of impurities compared to the first flowing solution and the second flowing solution. 【0004】 In one aspect, the method further comprises flowing the third flowing solution directly from the second dialysis cassette to a third dialysis cassette and repeating steps (a) and (b), thereby forming a fourth flowing solution containing a lower amount of impurities compared to the first flowing solution, the second flowing solution, and the third flowing solution. In another aspect, the method further comprises flowing the fourth flowing solution directly from the third dialysis cassette to a fourth dialysis cassette and repeating steps (a) and (b), thereby forming a fifth flowing solution containing a lower amount of impurities compared to the first flowing solution, the second flowing solution, the third flowing solution, and the fourth flowing solution. 【0005】 In one aspect, the dialysate inflow rate is about 0.1 times, about 0.2 times, about 0.3 times, about 0.4 times, about 0.5 times, about 0.6 times, about 0.7 times, about 0.8 times, about 0.9 times, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.25 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 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 higher than the first flow rate. In another aspect, the dialysate inflow rate is about 2.25 times higher than the first flow rate. In another aspect, the second flow rate is about 0.1 times, about 0.15 times, about 0.2 times, about 0.25 times, about 0.3 times, about 0.35 times, about 0.4 times, about 0.45 times, about 0.5 times, about 0.55 times, about 0.6 times, about 0.65 times, about 0.7 times or about 0.75 times that of the first flow rate. In another aspect, the second flow rate is about 0.25 to about 0.5 times that of the first flow rate. In another aspect, the first flow rate is about 0.01 mL / min to about 25 mL / min. In another aspect, the first flow rate is about 0.5 mL / min, about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 11 mL / min, about 12 mL / min, about 13 mL / min, about 14 mL / min, about 15 mL / min, about 16 mL / min, about 17 mL / min, about 18 mL / min, about 19 mL / min, about 20 mL / min, about 21 mL / min, about 22 mL / min, about 23 mL / min, about 24 mL / min, or about 25 mL / min. 【0006】 In one aspect, the impurities include low molecular weight species. In another aspect, the low molecular weight species are ionic impurities such as inorganic acids / bases and salts of other species (amino acids), culture additives, metal salts, carbohydrates (less than 1000 kDa), and chelating agents such as EDTA. 【0007】 In one aspect, the target protein is diafiltered. 【0008】 In one aspect, the target protein is obtained from a bioreactor. 【0009】 In one aspect, the target protein is purified 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. 【0010】 In one aspect, the target protein 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. In another aspect, the protein includes an antibody, and the antibody is an IgG antibody selected from IgG1, IgG2, IgG3, and IgG4. In yet another aspect, the antibody is a therapeutic antibody. 【Brief Description of the Drawings】 【0011】 【Figure 1】 A schematic diagram of a single-pass asymmetric dialysis system is shown. A feed containing monoclonal antibody (mAb) is pumped into a hollow fiber module using a pump (P1). The concentration factor along the cartridge is adjusted using pumps P2 and P4. Fresh dialysis buffer is delivered to the shell side using pump P3. 【Figure 2】 Shows the relationship between a' and the buffer consumption per gram of mAb for asymmetric dialysis of a 20 g / L mAb feed at a 10-fold target concentration factor. 【Figure 3】 A schematic diagram of a fully continuous downstream process incorporating asymmetric dialysis is shown. 【Modes for Carrying Out the Invention】 【0012】 The present disclosure provides a highly effective approach for removing contaminants during protein purification using asymmetric continuous countercurrent concentration dialysis without the need for chromatography. Thus, the present disclosure provides a method for purifying a target protein using approximately 1 / 10 the amount of water and solution of a chromatography process. 【0013】 I. Definitions To make the present disclosure easier to understand, certain terms are first defined. As used herein, each of the following terms shall have the meaning set forth below, unless explicitly provided otherwise herein. Throughout this specification, further definitions are set forth. 【0014】 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. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein. 【0015】 When the term "and / or" is used herein, each of the two specified features or components should be construed as a specific disclosure 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). 【0016】 Whenever an aspect is described herein using the word "comprising", it is understood that other similar aspects with respect to "consisting of" and / or "consisting essentially of" are also provided. 【0017】 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 one of ordinary skill in the art with many common dictionaries of the terms used in this disclosure. 【0018】 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, which can be obtained by referring to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification. 【0019】 The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of these 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. 【0020】 Terms such as "about" or "essentially comprising" refer to values or compositions 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 comprising" can mean within one or more standard deviations in each practice in the art. Alternatively, "about" or "essentially comprising" 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. When a particular value or composition is provided in the present application and the claims, unless otherwise stated, the meaning of "about" or "essentially comprising" should be assumed to be within the acceptable error range for that particular value or composition. 【0021】 As described herein, any concentration range, percentage range, 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. 【0022】 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). 【0023】 As used herein, the term "protein of interest" is used in its 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. 【0024】 As used interchangeably herein, the terms "purify", "separate", or "isolate" refer to increasing the degree of purity of a protein of interest from a composition or sample containing the protein of interest and one or more impurities. Typically, the purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition. 【0025】 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 the physiological range. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases. 【0026】 As used herein, terms such as "impurity" are used in their broadest sense to encompass any unwanted component, contaminant, 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 naturally or recombinantly produced by the host cell, and proteins related to or derived from the protein of interest (e.g., proteolytic fragments), as well as other process-related contaminants, but are not limited thereto. In certain embodiments, precipitation of impurities is separated from the cell culture using other means such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration. 【0027】 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 associated with the product, which can be linked by either covalent or non-covalent bonds, and can consist, for example, of misfolded monomers where hydrophobic amino acid residues are exposed to a polar solvent, which can cause aggregation. 【0028】 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 can include truncated species or half-molecules for compounds intended to be dimers (such as monoclonal antibodies). 【0029】 The term "host cell protein" or HCP refers to unwanted proteins produced by host cells that are not related to the production of the protein 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. 【0030】 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. Fed-batch culture can be started with 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. Fed-batch culture is typically stopped at some point, and the cells and / or components in the medium are recovered and optionally purified. 【0031】 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 the culture unit, perfusion cultures typically have a relatively high cell density, but the culture conditions are difficult to maintain and control. In addition, since the 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 the polypeptide and / or virus of interest during the production phase in a continuous cell culture system. 【0032】 The term "ultrafiltration" refers to a membrane-based separation process that separates molecules in a solution, for example, based on size. This can achieve the separation of different molecules or the concentration of similar molecules. 【0033】 The term "tangential flow filtration" refers to a specific filtration method in which a solution containing a solute flows tangentially across an ultrafiltration membrane, and a low molecular weight solute passes through the membrane by applying pressure. A solution containing a higher molecular weight solute that flows tangentially across the ultrafiltration membrane is retained, and this solution is thereby referred to herein as the "retentate". A lower molecular weight solute that passes through the ultrafiltration membrane is referred to herein as the "permeate". Thereby, the retentate is concentrated, for example, tangentially, by flowing along the surface of the ultrafiltration membrane under pressure. The ultrafiltration membrane has a pore size at a certain cut-off value. In some embodiments, the cut-off value is about 50 kDa or less. In some embodiments, the cut-off value is about 30 kD or less. 【0034】 The term "diafiltration" or "DF" refers to the use of an ultrafiltration membrane, for example, to remove solvents, buffers and / or salts from a solution or mixture containing proteins, peptides, nucleic acids or other biomolecules, to exchange them, or to reduce their concentrations. 【0035】 An "antibody" (Ab), which is not limited thereto, includes a glycoprotein immunoglobulin that specifically binds to an antigen and contains 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 contains 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. 【0036】 Immunoglobulins can be derived from any of the commonly 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) that can bind 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 a variable region of the heavy chain fused to a CH3 with a "knob" mutation and a 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. 【0037】 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 optionally. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but they 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 included within the term "antigen-binding portion" of an antibody. These antibody fragments can be 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. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. 【0038】 "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. Furthermore, an isolated antibody may not substantially contain other cellular and / or chemical substances. 【0039】 "Bispecific" or "bifunctional antibody" refers to 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 various 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). 【0040】 Terms such as "monoclonal antibody" (mAb) refer to non-naturally occurring preparations 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. 【0041】 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 new sequence in a fusion polypeptide, 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. 【0042】 As described herein, any range of concentrations, range of percentages, 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. 【0043】 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. 【0044】 As used herein, the term "seeding" refers to the addition of cells to a medium to initiate a culture. 【0045】 As used herein, the terms "induction" or "induction phase" or "growth phase" of cell culture refer to the initial seeding of a bioreactor (e.g., a seed bioreactor) at the start of an upstream cell culture, and include the period of exponential cell growth (e.g., the logarithmic phase) during which cells are mainly dividing rapidly. During this stage, the rate of increase in the density of viable cells is higher than at any other time point. 【0046】 As used herein, terms such as the "production phase" of cell culture refer to the period during which cell growth is stationary or maintained at a nearly constant level. The density of viable cells remains nearly 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. 【0047】 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. 【0048】 As used herein, the terms "medium" and "cell culture medium" and "feed medium" and "fermentation medium" refer to nutrient solutions used for growing and / or maintaining 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 can be selectively 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 culture medium for host cells. Any other necessary supplements can also be included at appropriate concentrations. 【0049】 Various aspects of the present disclosure are described in further detail in the following subsections. 【0050】 II. Countercurrent dialysis The present disclosure provides a highly effective approach for removing contaminants during protein purification using sequential countercurrent dialysis without the need for chromatography. Accordingly, the present disclosure provides a method for purifying a target protein that uses approximately 1 / 10 the amount of water and solution of a chromatography process. 【0051】 In some aspects, the present disclosure is a method for purifying a target protein using countercurrent concentration dialysis, comprising: (a) flowing a first flowing solution containing the target protein and impurities through a first hollow fiber dialysis cassette at a first flow rate, the dialysis cassette comprising a dialysis fluid inflow at a dialysis fluid inflow rate and a dialysis fluid outflow at a dialysis fluid outflow rate, the first flowing solution being countercurrent to the dialysis fluid inflow and outflow; (b) flowing the impurities through a semipermeable membrane of the dialysis cassette, the dialysis fluid inflow rate being higher than the first flow rate, a second flowing solution containing the target protein and a low amount of impurities exiting the dialysis cassette at a second flow rate, the dialysis fluid outflow rate being the sum of the dialysis fluid inflow rate and the difference between the first flow rate and the second flow rate; and (c) optionally, flowing the second flowing solution directly from the first dialysis cassette to a second dialysis cassette, and optionally repeating steps (a) and (b) using the second flowing solution and the second dialysis cassette, thereby forming a third flowing solution containing a lower amount of impurities compared to the first flowing solution and the second flowing solution. 【0052】 In some aspects, the method further comprises flowing the third flowing solution directly from the second dialysis cassette to a third dialysis cassette and repeating steps (a) and (b), thereby forming a fourth flowing solution containing a lower amount of impurities compared to the first flowing solution, the second flowing solution, and the third flowing solution. 【0053】 In some embodiments, the method further includes flowing a fourth flowing solution directly from the third dialysis cassette to the fourth dialysis cassette and repeating steps (a) and (b), thereby forming a fifth flowing solution that contains a lower amount of impurities compared to the first, second, third, and fourth flowing solutions. In some embodiments, the asymmetric dialysis at various production scales can be carried out using a hollow fiber membrane having an area of 1.0 m 2 , 2.0 m 2 , 2.5 m 2 , 3.6 m 2 , 5.4 m 2 , 7 m 2 , 8 m 2 or 10 m 2 . 【0054】 In some embodiments, the dialysate inflow rate is about 0.1 times, about 0.2 times, about 0.3 times, about 0.4 times, about 0.5 times, about 0.6 times, about 0.7 times, about 0.8 times, about 0.9 times, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.25 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 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 higher than the first flow rate. In some embodiments, the dialysate inflow rate is about 2.25 times higher than the first flow rate. 【0055】 In some embodiments, the second flow rate is about 0.1 times, about 0.15 times, about 0.2 times, about 0.25 times, about 0.3 times, about 0.35 times, about 0.4 times, about 0.45 times, about 0.5 times, about 0.55 times, about 0.6 times, about 0.65 times, about 0.7 times or about 0.75 times the first flow rate. In some embodiments, the second flow rate is about 0.25 to about 0.5 times the first flow rate. 【0056】 In some embodiments, the first flow rate is from about 0.01 mL / min to about 25 mL / min. In some embodiments, the first flow rate is about 0.5 mL / min, about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 11 mL / min, about 12 mL / min, about 13 mL / min, about 14 mL / min, about 15 mL / min, about 16 mL / min, about 17 mL / min, about 18 mL / min, about 19 mL / min, about 20 mL / min, about 21 mL / min, about 22 mL / min, about 23 mL / min, about 24 mL / min, or about 25 mL / min. In some embodiments, the second flow rate is about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 11 mL / min, about 12 mL / min, about 13 mL / min, about 14 mL / min, about 15 mL / min, about 16 mL / min, about 17 mL / min, about 18 mL / min, about 19 mL / min, about 20 mL / min, about 21 mL / min, about 22 mL / min, about 23 mL / min, about 24 mL / min, about 25 mL / min, about 26 mL / min, about 27 mL / min, about 28 mL / min, about 29 mL / min, 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 49 mL / min, or about 50 mL / min. 【0057】 In some embodiments, the asymmetric continuous countercurrent diafiltration reduces the amount of impurities present in the solution containing the protein of interest. In some embodiments, the removal of molecules having a molecular weight of 100 kDa, 200 kDa, 300 kDa, 500 kDa, 700 kDa or 1000 kDa is removed from the mAb feed. 【0058】 In some embodiments, the impurities include host cell protein (HCP). In some embodiments, the asymmetric continuous countercurrent diafiltration reduces the amount of HCP by about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%. 【0059】 In some embodiments, the impurities include low molecular weight species. In some embodiments, the methods described herein can remove about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% of the low molecular weight species from a solution containing the protein of interest. 【0060】 In some embodiments, the impurities include DNA. In some embodiments, the asymmetric continuous countercurrent diafiltration reduces the amount of DNA to about 20 pg / mL or less, about 18 pg / mL or less, about 16 pg / mL or less, about 14 pg / mL or less, about 12 pg / mL or less, about 10 pg / mL or less, about 8 pg / mL or less, about 6 pg / mL or less, about 4 pg / mL or less or about 2 pg / mL or less. 【0061】 In some embodiments, the contaminants include residual Protein A. In some embodiments, the asymmetric continuous countercurrent diafiltration reduces the residual Protein A by about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%. 【0062】 In some embodiments, the protein of interest is diafiltered. In some embodiments, the protein of interest is diafiltered into a buffer having a pH of about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8. In some embodiments, the protein of interest is diafiltered into a buffer having a pH of about 6. In some embodiments, the protein of interest is diafiltered into a buffer having a conductivity of about 1.0 mS / cm. In some embodiments, the protein of interest is diafiltered into a buffer having a conductivity of at least 0.5 mS / cm, at least 1.0 mS / cm, at least 1.5 mS / cm, at least 2.0 mS / cm, at least 2.5 mS / cm, at least 3.0 mS / cm, at least 3.5 mS / cm, at least 4.0 mS / cm, at least 4.5 mS / cm or at least 5.0 mS / cm. In some embodiments, the protein of interest is diafiltered into a buffer having a pH of about 6 and a conductivity of about 1.0 mS / cm. In some embodiments, the method is used to exchange other carbohydrates such as sucrose, trehalose, lactose, maltose, or sugar alcohols such as mannitol, sorbitol, xylitol, lactitol, maltitol, etc. into the product containing the protein of interest. 【0063】 In some embodiments, the protein of interest is obtained from a bioreactor. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 10 g / L to about 100 g / L. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 15 g / L to about 95 g / L. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 20 g / L to about 40 g / L. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 25 g / L to about 35 g / L. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 30 g / L to about 35 g / L. In some embodiments, the protein of interest is obtained from a bioreactor at a concentration of about 25 g / L to about 30 g / L. 【0064】 In some embodiments, the protein of interest is obtained after an ultrafiltration step. In some embodiments, the ultrafiltration step is combined with a dialysis step and performed simultaneously (e.g., sequential asymmetric continuous countercurrent concentration dialysis, Figure 3). 【0065】 In some embodiments, the methods described herein can purify 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 of the protein of interest. 【0066】 III. Protein of Interest In some embodiments, the methods disclosed herein can be applied to any protein product (e.g., the protein of interest). In some embodiments, the protein product is a therapeutic protein. In some embodiments, the therapeutic protein is selected from antibodies or antigen-binding fragments thereof, Fc fusion proteins, anticoagulants, blood coagulation factors, 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. 【0067】 In other embodiments, the protein of interest is produced within a host cell. In some embodiments, the protein of interest is produced in a culture containing mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cell (BHK), monkey kidney fibroblast cell (COS-7), Madin-Darby bovine kidney cell (MDBK), or any combination thereof. In some embodiments, the starting mixture can be a recovered cell culture fluid, a supernatant of a cell culture, a conditioned supernatant of a cell culture, a cell lysate, and a clarified bulk. 【0068】 In some aspects, the protein product is an antibody or an antigen-binding fragment thereof. In some aspects, 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 (an "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. 【0069】 In some aspects, the mixture containing the protein product and contaminants includes the product before the purification step. In some aspects, the mixture is the untreated product before the purification step. In some aspects, the mixture is a solution containing the untreated product and buffer of the pre-purification step, such as a starting buffer. In some aspects, the mixture includes the untreated product of the pre-purification step reconstituted in a starting buffer. 【0070】 In some aspects, the source of the protein product is a bulk protein. In some aspects, 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. 【0071】 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. 【0072】 In some embodiments, the protein product purified by the methods described herein is a fusion protein. "Fusion" or "fusion protein" includes a first amino acid sequence joined in - frame 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 new sequence in the fusion polypeptide. Fusion proteins are 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. 【0073】 In some embodiments, the protein purified by the methods described herein is an antibody. Antibodies include, 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 foregoing. 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. 【0074】 The present disclosure relates to the methods disclosed herein where the protein 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 protein 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 therapeutic antibody. 【0075】 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 protein of interest is prepared by the methods disclosed herein. 【0076】 The present disclosure is further illustrated by the following examples, which should not be construed as further limitations. 【Examples】 【0077】 Example 1 Systems and Methods for Integrated Concentration and Buffer Exchange Protein and Membrane A feed containing monoclonal antibody (mAb) was adjusted to a salt content of pH 5.0, 50 mM sodium acetate buffer, and 200 mM NaCl (conductivity, 20 + 3 mS / cm) to reproduce the cation exchange elution pool. The mAh concentration in the feed was adjusted to 7 - 30 g / L. The hollow fiber modules used in this study were composed of polysulfone or polyethersulfone. The polysulfone membranes used were Optiflux 180NR (Fresenius, USA) and Renaflow HF1200 (Minntech, USA), with surface areas of 1.8 m 2 and 1.2 m 2 of membrane, respectively. The polyethersulfone membrane Minikros 0.16 m 2 (S04 - E030 - 05 - N) was purchased from Repligen (USA). Each test used a new hollow fiber module, except for the Minikros module that was washed with 0.1 N sodium hydroxide for 20 minutes prior to reuse. 【0078】 Asymmetric Dialysis Method The hollow fiber module was mounted vertically and the feed was introduced through pump P1 from the port at the bottom on the lumen side (Figure 1). The shell side port (shell outlet) close to the supply port was attached to pump P2. The distal shell side (shell inlet) and lumen side (lumen outlet) ports were attached to pumps P3 and P4 respectively. The dialysis buffer was introduced into the shell side using pump P3 while the flow rate was adjusted at the shell outlet by pump P2. The concentrated and buffer-exchanged product was collected by pump P4. The peristaltic pumps P1, P2, P3 and P4 equipped with appropriate pump heads and tubes were calibrated by periodic collection using a digital scale before the start of the process. 【0079】 Pressure was monitored using Pendotech pressure sensors placed immediately before and after the inlet / outlet ports. The shell and lumen compartments of the hollow fiber module (1.8 m 2 , OptiFlux 180NR) were flushed with dialysis buffer (20 mM histidine, pH 5.7 ± 1) before the experiment. All solutions used in the experiment were filtered through a 0.45 μm PES filter. In a typical experimental setup using a feed flow (P1) of 20 mL / min (Flux 0.7 LMH), pump P4 was adjusted to 5 mL / min to achieve a 4-fold concentration factor along the hollow fiber membrane. While on the shell side, pumps P2 and P3 were adjusted to 60 mL / min and 45 mL / min respectively. Subsequently, the flow rates of all pumps were adjusted to enable simultaneous concentration of the product and buffer exchange. The paired inlet and outlet flow rates for the feed and exchange buffer were proportional but not identical (Table 1). All experiments were carried out at room temperature (22 ± 2 °C) without planned temperature control. In some experiments, vitamin B12 was dissolved in the pre-conditioned mAb feed as a model impurity. All experiments were carried out in single-pass mode without feed or recirculation of the dialysis buffer. 【0080】 For offline pH, conductivity, mAb and histidine concentration, small samples were periodically collected from the lumen outlet. 【0081】 【Table 1】 【0082】 In a particular experiment, to achieve the desired concentration factor, pump P4 was either not used or replaced with a backpressure regulator. 【0083】 Strategies for reducing buffer consumption Buffer utilization in asymmetric dialysis depends on operation α’, 【0084】 【Number】 【0085】 Several values of α’ were evaluated to reduce buffer consumption. At α’ = 5, there was a 75% reduction in buffer consumption without affecting process performance. For an asymmetric dialysis process with a feed mAb at 20 g / L and a 10-fold concentration factor with an α’ value of 22.5, the resulting buffer consumption was over 0.1 L / g of mAb (Figure 2). 【0086】 Results Example 2 In this example, a pump P1 with a flow rate of 20 mL / min (0.7 LMH) was used to supply a 30 g / L mAb feed (pH 5, 200 mM NaCl) to the hollow fiber, and pump P4 was adjusted to 5 mL / min for a desired 4-fold concentration factor along the hollow fiber membrane. For the shell side, dialysis buffer pumps P2 and P3 were adjusted to 60 mL / min and 45 mL / min, respectively. As shown in Table 2, both the Fresenius and Repligen hollow fibers showed equivalent salt removal and buffer exchange performance. 【0087】 【Table 2】 【0088】 Example 3 In this example, a pump P1 with a flow rate of 45 mL / min (1.5 LMH) was used to supply a 20 g / L mAb feed (pH 5, 200 mM NaCl) to the hollow fiber, and the pump P4 was adjusted to 4.5 mL / min for a desired 10-fold concentration factor along the hollow fiber membrane. For the shell side, the dialysis buffer pumps P2 and P3 were adjusted to 141.75 mL / min and 101.25 mL / min (20 mM histidine, pH 5.9), respectively. 【0089】 Example 4 In this example, a lower α' of 5 was selected to reduce buffer consumption. A pump P1 with a flow rate of 45 mL / min (1.5 LMH) was used to supply a 20 g / L mAb feed (pH 5, 200 mM NaCl) to the hollow fiber device, and the pump P4 was adjusted to 4.5 mL / min for a desired 10-fold concentration factor along the hollow fiber membrane. For the shell side, the dialysis buffer pumps P2 and P3 were adjusted to 63 mL / min and 22.5 mL / min, respectively. 【0090】 Example 5 In this example, asymmetric dialysis was used with an α' of 5 to remove relatively large model impurities / tracers from the feed. For this evaluation, a pump P1 with a flow rate of 45 mL / min (1.5 LMH) was used to supply a 20 g / L mAb feed (pH 4.9, 200 mM NaCl) containing 3.2 g / L vitamin B12 (approx. 1,356 kDa) to the hollow fiber device, and the pump P4 was adjusted to 4.5 mL / min for a desired concentration factor along the hollow fiber membrane. For the shell side, the dialysis buffer pumps P2 and P3 were adjusted to 63 mL / min and 22.5 mL / min, respectively. Based on the experimental data from the experiments associated with this evaluation, the flow rate of the fresh dialysis buffer was adjusted to pH 5.6 to obtain the target product at pH 6.0. 【0091】 【Table 3】 【0092】 Example 6 In this example, glucose, a non-ionic molecule such as a carbohydrate, was exchanged into the product using asymmetric dialysis with a dialysis buffer (4.6 g / L) containing 5 α' and glucose. For this evaluation, a 20 g / L mAb feed (pH 4.9, 200 mM NaCl) was supplied to a hollow fiber device using a pump P1 with a flow rate of 45 mL / min (1.5 LMH), and pump P4 was adjusted to 4.5 mL / min for the desired concentration factor along the hollow fiber membrane. For the shell side, dialysis buffer pumps P2 and P3 were adjusted to 63 mL / min and 22.5 mL / min, respectively. 【0093】 【Table 4】 【0094】 Example 7 In this example, an ionic compound such as NaCl was exchanged into the product using asymmetric dialysis with 5 α' and a dialysis buffer, 20 mM histidine (pH 5.9), 100 mM NaCl. For this evaluation, a 20 g / L mAb feed with low conductivity (1.2 mS / cm) (20 mM histidine, pH 6.0) was supplied to a hollow fiber device using a pump P1 with a flow rate of 45 mL / min (1.5 LMH), and pump P4 was adjusted to 4.5 mL / min for the desired concentration factor along the hollow fiber membrane. For the shell side, dialysis buffer pumps P2 and P3 were adjusted to 63 mL / min and 22.5 mL / min, respectively. 【0095】 【Table 5】 【0096】 This demonstrates the development of an innovative one-step process of asymmetric dialysis, which aims for continuous UF and buffer exchange, eliminating the need for two-stage UF / DF. Careful manipulation of the inlet / outlet flow rates is performed to achieve product concentration, buffer exchange, and salt removal. Product concentrations of 105 g / L (3.8-fold), 200 g / L (10-fold), and 64 g / L (9.4-fold), starting from feed concentrations of 28 g / L, 20 g / L, and 7 g / L respectively, can be achieved as described above with a moderate (less than 6 psi) pressure profile across the cartridge. This method also achieves a 74% reduction in buffer utilization (0.026 L / g, mAb) compared to conventional batch UF-DF (0.1 L / g, mAb) with a maximum mAb productivity of 0.7 kg / m 2 / day. This results in a simplified process and a smaller footprint compared to current-generation technologies.

Claims

[Claim 1] A method for purifying a target protein using countercurrent concentration dialysis, (a) A first fluid solution containing the target protein and impurities is flowed through a first hollow fiber dialysis cassette at a first flow rate, wherein the dialysis cassette constitutes a dialysate inflow at a dialysate inflow rate and a dialysate outflow at a dialysate outflow rate, and the first fluid solution is countercurrent to the dialysate inflow and outflow. (b) The impurities are flowed through the semipermeable membrane of the dialysis cassette, wherein the amount of dialysate inflow is higher than the first flow rate, and a second fluid solution containing the target protein and a small amount of impurities flows out of the dialysis cassette at a second flow rate, and the amount of dialysate outflow is the sum of the amount of dialysate inflow and the difference between the first flow rate and the second flow rate, (c) The second fluid solution is flowed directly from the first dialysis cassette to the second dialysis cassette, (d) A method comprising repeating steps (a) and (b) using the second fluid solution and the second dialysis cassette, thereby forming a third fluid solution containing a lower amount of impurities compared to the first fluid solution and the second fluid solution. [Claim 2] The method according to claim 1, further comprising: flowing the third fluid solution directly from the second dialysis cassette to the third dialysis cassette; and repeating steps (a) and (b) to form a fourth fluid solution containing a lower amount of impurities compared to the first fluid solution, the second fluid solution, and the third fluid solution. [Claim 3] The method according to claim 2, further comprising: flowing the fourth fluid solution directly from the third dialysis cassette to the fourth dialysis cassette; and repeating steps (a) and (b) to form a fifth fluid solution containing a lower amount of impurities compared to the first fluid solution, the second fluid solution, the third fluid solution, and the fourth fluid solution. [Claim 4] The method according to claim 1, wherein the amount of dialysate flowing in is about 0.1 times, about 0.2 times, about 0.3 times, about 0.4 times, about 0.5 times, about 0.6 times, about 0.7 times, about 0.8 times, about 0.9 times, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.25 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 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 higher than the first flow rate. [Claim 5] The method according to claim 4, wherein the amount of dialysate flowing in is approximately 2.25 times higher than the first flow rate. [Claim 6] The method according to claim 1, wherein the second flow rate is approximately 0.1 times, approximately 0.15 times, approximately 0.2 times, approximately 0.25 times, approximately 0.3 times, approximately 0.35 times, approximately 0.4 times, approximately 0.45 times, approximately 0.5 times, approximately 0.55 times, approximately 0.6 times, approximately 0.65 times, approximately 0.7 times, or approximately 0.75 times the first flow rate. [Claim 7] The method according to claim 1, wherein the second flow rate is approximately 0.25 to approximately 0.5 times the first flow rate. [Claim 8] The method according to claim 1, wherein the first flow rate is approximately 0.01 mL / min to approximately 25 mL / min. [Claim 9] The method according to claim 8, wherein the first flow rate is approximately 0.5 mL / min, approximately 1 mL / min, approximately 2 mL / min, approximately 3 mL / min, approximately 4 mL / min, approximately 5 mL / min, approximately 6 mL / min, approximately 7 mL / min, approximately 8 mL / min, approximately 9 mL / min, approximately 10 mL / min, approximately 11 mL / min, approximately 12 mL / min, approximately 13 mL / min, approximately 14 mL / min, approximately 15 mL / min, approximately 16 mL / min, approximately 17 mL / min, approximately 18 mL / min, approximately 19 mL / min, approximately 20 mL / min, approximately 21 mL / min, approximately 22 mL / min, approximately 23 mL / min, approximately 24 mL / min, or approximately 25 mL / min. [Claim 10] The method according to claim 1, wherein the impurity includes a low molecular weight species. [Claim 11] The method according to claim 1, wherein the target protein is dialyzed and filtered. [Claim 12] The method according to claim 1, wherein the target protein is obtained from a bioreactor. [Claim 13] The method according to claim 1, for purifying a target protein in amounts of approximately 0.1 kg / day, approximately 0.5 kg / day, approximately 1 kg / day, approximately 2 kg / day, approximately 3 kg / day, approximately 4 kg / day, approximately 5 kg / day, approximately 6 kg / day, approximately 7 kg / day, approximately 8 kg / day, approximately 9 kg / day, or approximately 10 kg / day. [Claim 14] The method according to claim 1, wherein the target protein comprises an antibody, an antigen-binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof. [Claim 15] The method according to claim 14, wherein the protein comprises an antibody selected from IgM, IgA, IgE, IgD, and IgG. [Claim 16] The method according to claim 15, wherein the protein comprises an antibody, and the antibody is an IgG antibody selected from IgG1, IgG2, IgG3, and IgG4. [Claim 17] The method according to claim 16, wherein the antibody is a therapeutic antibody.