Method for unified concentration and buffer exchange

Abstract

Provided herein are methods of purifying a protein of interest using asymmetric dialysis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 366,147, filed on June 10, 2022, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] As biologics enter the forefront of drug development, the need for improved manufacturing processes is growing. With the expected increase in demand for recombinant protein therapeutics, more cost-effective and flexible manufacturing processes are needed. In fact, various economic analyses estimate that process development and clinical manufacturing costs can account for 40% to 60% of drug development costs. Together with commercial manufacturing, which is primarily driven by consumable downstream processing costs, this can amount to up to 25% of the sales revenue of a biologic. Therefore, more efficient downstream processing is needed. Summary of the invention

[0004] The present disclosure relates to a method for purifying a protein of interest using countercurrent concentrating dialysis, the method comprising: (a) passing a first mobile solution comprising the protein of interest and impurities through a first hollow fiber dialysis cassette at a first flow rate, wherein the dialysis cassette comprises a dialysate inflow having a dialysate inflow flow rate and a dialysate effluent having a dialysate effluent flow rate; and wherein the first mobile solution is countercurrent to the dialysate inflow and the dialysate effluent; (b) passing the impurities through a semipermeable membrane of the dialysis cassette, wherein the dialysate inflow flow rate is higher than the first flow rate, wherein a second mobile solution comprising the protein of interest and reduced levels of impurities exits the dialysis cassette at a second flow rate, and wherein the dialysate effluent flow rate is the sum of the dialysate inflow flow rate and the difference between the first flow rate and the second flow rate; (c) optionally passing the second mobile solution from the first dialysis cassette directly into a second dialysis cassette; and (d) optionally repeating steps (a) and (b) with the second mobile solution and the second dialysis cassette to form a third mobile solution having reduced levels of impurities compared to the first mobile solution and the second mobile solution.

[0005] In one aspect, the method further comprises passing the third mobile solution from the second dialysis cassette directly into the third dialysis cassette, and repeating steps (a) and (b), thereby forming a fourth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, and the third mobile solution. On the other hand, the method further comprises passing the fourth mobile solution from the third dialysis cassette directly into the fourth dialysis cassette, and repeating steps (a) and (b), thereby forming a fifth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, the third mobile solution, and the fourth mobile solution.

[0006] In one aspect, the dialysate inflow flow 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 times, 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 the first flow rate. On the other hand, the dialysate inflow flow rate is about 2.25 times of the first flow rate. On the other hand, 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 of the first flow rate. On the other hand, the second flow rate is between about 0.25 times and about 0.5 times of the first flow rate. On the other hand, the first flow rate is between about 0.01 ml / min and about 25 ml / min. On the other hand, 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.

[0007] In one aspect, impurities comprise low molecular weight species. In another aspect, low molecular weight species are ionic impurities (such as salts of inorganic acids / bases) and other species (amino acids), culture additives, metal salts, carbohydrates (<1000 kDa) and chelating agents (such as EDTA).

[0008] In one aspect, the protein of interest is diafiltered.

[0009] In one aspect, the protein of interest is obtained from a bioreactor.

[0010] In one aspect, 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 is purified.

[0011] In one aspect, the protein of interest comprises 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 comprises an antibody selected from IgM, IgA, IgE, IgD, and IgG. In another aspect, the protein comprises 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

[0012] Figure 1 A schematic diagram of a single-pass asymmetric dialysis system is shown. A feed containing a monoclonal antibody (mAb) is pumped into the hollow fiber module using 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.

[0013] Figure 2 Shown is the relationship between α' and buffer consumption per gram of mAb during asymmetric dialysis of a 20 g / L mAb feed with a target concentration factor of 10x.

[0014] Figure 3 A schematic diagram of the complete continuous downstream process combined with asymmetric dialysis is shown. DETAILED DESCRIPTION

[0015] The present disclosure provides an efficient method for removing contaminants using a series asymmetric continuous countercurrent concentration dialysis during protein purification without the need for chromatography. Therefore, the present disclosure provides a method for purifying a protein of interest using about 1 / 10 the amount of water and solution used in a chromatographic process.

[0016] I. Definitions

[0017] In order to more easily understand the present disclosure, some terms are first defined. As used in this specification, unless otherwise explicitly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0018] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "a feed medium" should be understood to mean one or more feed media. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0019] As used herein, the term "and / or" should be understood as specifically disclosing each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" 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" is intended to cover 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).

[0020] It should be understood that wherever herein aspects are described with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for the skilled person.

[0022] Units, prefixes and symbols are expressed in their SI recognized form. Numerical ranges include numerical values ​​that limit the range. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below can be more fully defined by reference to the specification in its entirety.

[0023] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0024] The term "about" or "substantially comprising" means that a value or composition is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, these terms may mean values ​​of up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value or composition.

[0025] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0026] The terms "polypeptide" or "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention; the intervention is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification operation, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies and polypeptides containing an Fc domain (e.g., immunoadhesins).

[0027] As used herein, the term "protein of interest" is used in its broadest sense and includes any protein (natural or recombinant) present in a mixture that needs to be purified. 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 aspects, a protein of interest refers to any protein that can be produced by the methods described herein. In some aspects, a protein of interest is an antibody. In some aspects, a protein of interest is a recombinant protein.

[0028] The terms "purification", "separation" or "isolation" as used interchangeably herein refer to increasing the purity of a protein of interest from a composition or sample comprising the protein of interest and one or more impurities. Typically, the purity of a protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0029] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or base that must be added to cause a unit pH change. Buffer solutions resist changes in pH under the action of their acid-base conjugate components. Buffer solutions used with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0030] As used herein, the term "impurity" is used in its broadest sense to encompass any undesirable component, pollutant or compound in a mixture. In cell culture, cell lysate or clarified body (e.g., clarified cell culture supernatant), pollutants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in cell culture medium. Host cell pollutant proteins include, but are not limited to, those produced naturally or recombinantly by host cells, and proteins (e.g., proteolytic fragments) and other process-related pollutants associated with or derived from proteins of interest. In certain embodiments, another means such as centrifugation, sterile filtration, deep filtration and tangential flow filtration are used to separate pollutant precipitates from cell culture.

[0031] The term "HMW species" refers to any one or more unwanted proteins present in a mixture. High molecular weight species may include dimers, trimers, tetramers or other polymers. These species are generally considered to be product-related impurities and may be covalently or non-covalently attached and may also, for example, consist of misfolded monomers in which hydrophobic amino acid residues are exposed to polar solvents and may cause aggregation.

[0032] The term "LMW species" refers to any one or more unwanted species present in a mixture. Low molecular weight species are generally considered product-related impurities and may include truncated species, or half molecules of compounds that are expected to dimerize, such as monoclonal antibodies.

[0033] The term "host cell protein" or HCP refers to an undesirable protein produced by the host cell that is unrelated to the production of the expected protein of interest. Undesirable host cell proteins can be secreted into the upstream cell culture supernatant. Undesirable host cell proteins can also be released during cell lysis. Cells used for upstream cell culture require proteins for growth, transcription, and protein synthesis, and these unrelated proteins are undesirable in the final drug product.

[0034] As used herein, the term "fed-batch culture" or "fed-batch culture process" refers to a method of culturing cells in which additional components are provided to the culture at a certain time after the start of the culture process. A fed-batch culture can be started with a basal medium. A culture medium in which additional components are provided to the culture at a certain time after the start of the culture process is a feed medium. The fed-batch culture is usually stopped at a certain time point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0035] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution through or through a cell population at a steady rate. Because perfusion systems typically involve retaining cells within a culture unit, perfusion cultures characteristically have relatively high cell densities, but culture conditions are difficult to maintain and control. In addition, because cells are grown at high density and retained within the culture unit, the growth rate typically decreases over time, causing the cell growth to enter a late exponential phase or even a stationary phase. This continuous culture strategy typically involves culturing mammalian cells, such as non-fixation-dependent cells, expressing a polypeptide and / or virus of interest during a production phase in a continuous cell culture system.

[0036] The term "ultrafiltration" refers to a separation process, such as membrane-based, that separates molecules in a solution based on size, thereby enabling separation of different molecules or concentration of similar molecules.

[0037] The term "tangential flow filtration" refers to a specific filtering method in which a solution containing a solute passes tangentially through an ultrafiltration membrane, and a solute of a relatively low molecular weight is passed through the membrane by applying pressure. The solution containing a relatively high molecular weight solute passing tangentially through the ultrafiltration membrane is retained, so the solution is referred to as "retentate" in this article. The relatively low molecular weight solute passing through the ultrafiltration membrane is referred to as "permeate" in this article. Therefore, the retentate is concentrated by flowing along the surface of the ultrafiltration membrane under pressure (e.g., tangentially). The ultrafiltration membrane has a pore size with a certain cutoff value. In some aspects, the cutoff value is about 50kDa or less. In some aspects, the cutoff value is 30kD or less.

[0038] The term "diafiltration" or "DF" refers to the removal, replacement or reduction of the concentration of solvents, buffers and / or salts from a solution or mixture containing proteins, peptides, nucleic acids or other biomolecules, for example using an ultrafiltration membrane.

[0039] "Antibodies" (Ab) shall include, but are not limited to, glycoprotein immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an 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 (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. In some aspects, the antibody is a full-length antibody.

[0040] Immunoglobulins can be derived from any commonly known isotype, 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 the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. The term "antibody" may include multivalent antibodies (e.g., trivalent antibodies) that can bind more than two antigens. A trivalent antibody is an IgG-type bispecific antibody composed of two regular Fab arms fused to an asymmetric third Fab-sized binding module via a flexible linker peptide. This third module replaces the IgG Fc region and consists of a heavy chain variable region fused to a CH3 with a "knob" mutation and a light chain variable region fused to a CH3 with a matching "hole". The hinge region does not contain a disulfide bond to facilitate antigen access to the third binding site. In the absence of explicit indication, the term "antibody" includes monospecific, bispecific or multispecific antibodies, as well as single-chain antibodies, unless the context indicates otherwise.

[0041] As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC and CH1 domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs), and (vii) a combination of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, which enables them to become a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called 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). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for usefulness in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0042] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity with other antigens (such as PD-L1 molecules from different species). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0043] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs, thereby generating two antigen-binding sites with specificity for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0044] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and which exhibit a single binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma technology, recombinant technology, transgenic technology, or other techniques known to those skilled in the art.

[0045] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence, wherein the first amino acid sequence is not naturally linked to the second amino acid sequence in nature. Amino acid sequences that are normally found in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that are normally found in the same protein can be placed in a fusion polypeptide in a new arrangement, for example, the fusion of the Factor VIII domain of the present disclosure with the Ig Fc domain. For example, a fusion protein is formed by chemical synthesis, or by forming and translating a polynucleotide in which the peptide regions are encoded in a desired relationship. A chimeric protein can also include a second amino acid sequence linked to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0046] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0047] As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of definable culture conditions include temperature, gas mixture, time, and medium formulation.

[0048] As used herein, the term "seeding" refers to adding cells to a culture medium to initiate a culture.

[0049] As used herein, the term "induction" or "induction phase" or "growth phase" of cell culture refers to the initial inoculation of a bioreactor (e.g., a seed bioreactor) at the start of upstream cell culture, and includes an exponential cell growth period (e.g., logarithmic phase) during which cells primarily divide rapidly. During this period, the rate of increase in viable cell density is higher than at any other time point.

[0050] As used herein, the term "production phase" of cell culture refers to the period of time during which cell growth is static or maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time. Logarithmic cell growth has ended, and protein production is the main activity during the production phase. At this point, culture medium is usually supplemented to support continued protein production and obtain the desired glycoprotein product.

[0051] As used herein, the term "expression" or "expresses" is used to refer to transcription and translation occurring in a cell. The expression level of a product gene in a host cell can be determined based on 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.

[0052] As used herein, the terms "culture medium" and "cell culture medium" as well as "feed medium" and "fermentation medium" refer to nutrient solutions used to grow and / or maintain cells, particularly mammalian cells. Without limitation, these solutions typically provide at least one component from one or more of the following categories: (1) an energy source, typically in the form of a carbohydrate such as glucose; (2) all essential amino acids, typically a basic set of twenty amino acids plus cysteine; (3) vitamins and / or other organic compounds at lower required 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 required at typically very low concentrations (often in the micromolar range). The nutrient solution may be optionally supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, such as serum, insulin, transferrin, and epidermal growth factor; (2) salts, such as magnesium salts, calcium salts, and phosphates; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, such as peptone or peptone mixtures obtained from purified gelatin, plant materials, or animal by-products; (6) antibiotics, such as gentamicin; (7) cell protectants, such as pluronic polyols; and (8) galactose. Commercially available culture media such as Ham's F10 (Sigma), Minimum 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 culture medium for the host cells. Any other necessary supplements may also be included at appropriate concentrations.

[0053] Various aspects of the disclosure are described in more detail in the following subsections.

[0054] II. Countercurrent dialysis

[0055] The present disclosure provides an efficient method for removing contaminants using serial countercurrent dialysis during protein purification without the need for chromatography. Therefore, the present disclosure provides a method for purifying a protein of interest using about 1 / 10 the amount of water and solution used in a chromatographic process.

[0056] In some aspects, the present disclosure provides a method for purifying a protein of interest using countercurrent concentrating dialysis, the method comprising: (a) passing a first mobile solution comprising the protein of interest and impurities into a first hollow fiber dialysis cassette at a first flow rate, wherein the dialysis cassette comprises a dialysate inflow having a dialysate inflow flow rate and a dialysate effluent having a dialysate effluent flow rate; and wherein the first mobile solution is countercurrent to the dialysate inflow and the dialysate effluent; (b) passing the impurities through a semipermeable membrane of the dialysis cassette, wherein the dialysate inflow flow rate is higher than the first flow rate, wherein a second mobile solution comprising the protein of interest and reduced levels of impurities exits the dialysis cassette at a second flow rate, and wherein the dialysate effluent flow rate is the sum of the dialysate inflow flow rate and the difference between the first flow rate and the second flow rate; (c) optionally passing the second mobile solution from the first dialysis cassette directly into a second dialysis cassette; and optionally repeating steps (a) and (b) with the second mobile solution and the second dialysis cassette to form a third mobile solution having reduced levels of impurities compared to the first mobile solution and the second mobile solution.

[0057] In some aspects, the method further comprises passing the third mobile solution from the second dialysis cassette directly into the third dialysis cassette and repeating steps (a) and (b) to form a fourth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, and the third mobile solution.

[0058] In some aspects, the method further comprises passing a fourth mobile solution from the third dialysis cassette directly into a fourth dialysis cassette and repeating steps (a) and (b) to form a fifth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, the third mobile solution, and the fourth mobile solution. In some aspects, a 1.0 m2 area can be used. 2 , 2.0m 2 , 2.5m 2 、3.6m 2 、5.4m 2 、7m 2 、8m 2 or 10m 2 Hollow fiber membranes for asymmetric dialysis at various manufacturing scales.

[0059] In some aspects, the dialysate inflow flow 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 times, 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 the first flow rate. In some aspects, the dialysate inflow flow rate is about 2.25 times the first flow rate.

[0060] In some aspects, 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 aspects, the second flow rate is between about 0.25 times and about 0.5 times the first flow rate.

[0061] In some aspects, the first flow rate is between about 0.01 ml / min and about 25 ml / min. In some aspects, 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 aspects, 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.

[0062] In some aspects, asymmetric continuous countercurrent concentrating dialysis reduces the level of impurities present in a solution containing a protein of interest. In some aspects, molecules with a molecular weight of 100 kDa, 200 kDa, 300 kDa, 500 kDa, 700 kDa, or 1000 kDa are removed from the mAb feed.

[0063] In some aspects, impurities include host cell proteins (HCP). In some aspects, asymmetric continuous countercurrent concentration dialysis 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%.

[0064] In some aspects, impurities include low molecular weight substances. In some aspects, 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 low molecular weight substances from a solution containing a protein of interest.

[0065] In some aspects, the impurities include DNA. In some aspects, the asymmetric continuous countercurrent concentrating dialysis reduces the amount of DNA to about 20pg / mL or less, about 18pg / mL or less, about 16pg / mL or less, about 14pg / mL or less, about 12pg / mL or less, about 10pg / mL or less, about 8pg / mL or less, about 6pg / mL or less, about 4pg / mL or less, or about 2pg / mL or less.

[0066] In some aspects, the contaminants comprise residual Protein A. In some aspects, the asymmetric continuous countercurrent concentrating dialysis reduces 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%.

[0067] In some aspects, the protein of interest is diafiltered. In some aspects, 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 aspects, the protein of interest is diafiltered into a buffer having a pH of about 6. In some aspects, the protein of interest is diafiltered into a buffer having a conductivity of about 1.0 mS / cm. In some aspects, 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 aspects, 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 aspects, the method is used to exchange other carbohydrates (such as sucrose, trehalose, lactose, maltose, etc.) or sugar alcohols (such as mannitol, sorbitol, xylitol, lactitol, maltitol, etc.) into a product containing the protein of interest.

[0068] In some aspects, obtain the protein of interest from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 10g / L to about 100g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 15g / L to about 95g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 20g / L to about 40g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 25g / L to about 35g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 30g / L to about 35g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 25g / L to about 30g / L from a bioreactor. In some aspects, obtain the protein of interest at a concentration of about 25g / L to about 30g / L from a bioreactor.

[0069] In some aspects, the protein of interest is obtained after an ultrafiltration step. In some aspects, the ultrafiltration step is combined with a dialysis step and performed simultaneously (e.g., a series asymmetric continuous countercurrent concentration dialysis, Figure 3 ).

[0070] In some aspects, 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 a protein of interest.

[0071] III. Protein of Interest

[0072] In some aspects, the methods disclosed herein can be applied to any protein product (e.g., a protein of interest). In some aspects, the protein product is a therapeutic protein. In some aspects, the therapeutic protein is selected from antibodies or their antigen binding fragments, Fc fusion proteins, anticoagulants, coagulation factors, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, receptors, and thrombolytic agents. In some aspects, the protein product is an antibody or its antigen binding fragment. In some aspects, the protein is a recombinant protein.

[0073] In other embodiments, the protein of interest is produced in a host cell. In some embodiments, the protein of interest is produced in a culture comprising a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell, a HEK293 cell, a mouse myeloma (NS0), a baby hamster kidney cell (BHK), a monkey kidney fibroblast (COS-7), a Madin-Darby bovine kidney cell (MDBK), or any combination thereof. In some embodiments, the starting mixture can be a cell culture fluid, a cell culture supernatant, a regulated cell culture supernatant, a cell lysate, and a clarified body of harvest.

[0074] 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 ("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.

[0075] In some aspects, the mixture comprising protein product and pollutant comprises the product of the previous purification step. In some aspects, the mixture is the crude product of the previous purification step. In some aspects, the mixture is a solution comprising the crude product of the previous purification step and buffer (e.g., starting buffer). In some aspects, the mixture is included in the crude product of the previous purification step reconstructed in the starting buffer.

[0076] In some aspects, the source of the protein product is bulk protein. In some aspects, the source of the protein product is a composition comprising the protein product and non-protein components. The non-protein components may include DNA and other contaminants.

[0077] In some aspects, the source of the protein product is from an animal. In some aspects, the animal is a mammal, such as a non-primate (e.g., a dairy cow, a pig, a horse, a cat, a dog, a rat, etc.) or a primate (e.g., a monkey or a human). In some aspects, the source is a tissue or a cell from a human. In some aspects, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, dairy cows, cats or dogs). In some aspects, such terms refer to pets or farm animals. In some aspects, such terms refer to people.

[0078] In some aspects, the protein product purified by the methods described herein is a fusion protein. "Fusion" or "fusion protein" comprises a first amino acid sequence connected in frame with a second amino acid sequence, and the first amino acid sequence is not naturally connected to the second amino acid sequence in nature. The amino acid sequences that are usually present in a single protein can be gathered together in a fusion polypeptide, or the amino acid sequences that are usually present in the same protein can be placed in a fusion polypeptide in a new arrangement. For example, by chemical synthesis, or by forming and translating a polynucleotide (wherein the peptide region is encoded with a desired relationship) to form a fusion protein. The fusion protein may also include a second amino acid sequence that is connected to the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond. When transcribed / translated, a single protein is produced. In this way, a variety of proteins or fragments thereof can be incorporated into a single polypeptide. "Operably connected" is intended to represent the functional connection between two or more elements. For example, the operable connection between two polypeptides fuses two polypeptides in frame together to produce a single polypeptide fusion protein. In a specific aspect, the fusion protein also includes a third polypeptide, as discussed in further detail below, and the third polypeptide may include a linker sequence.

[0079] In some aspects, the protein purified by the method described herein is an antibody.Antibody may include, for example, monoclonal antibodies, recombinant 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-antibody heavy chain pairs, intracellular antibodies, heterologous conjugated antibodies, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F (ab') 2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies) and the antigen-binding fragments of any of the above.In some aspects, antibody as described herein refers to polyclonal antibody groups. The antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some aspects, the antibody described herein is an IgG antibody or its class (e.g., human IgG1 or IgG4) or subclass. In one aspect, the antibody is a humanized monoclonal antibody. In some aspects, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In some aspects, the antibody described herein is an IgG1 or IgG4 antibody.

[0080] The present disclosure relates to methods disclosed herein, wherein 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 aspects, the protein of interest is a full-length IgG antibody. In some aspects, the antibody is an IgG1, IgG2, IgG3, and / or IgG4, or a hybrid thereof. In some aspects, the antibody is a therapeutic antibody.

[0081] In some aspects, the method disclosed herein is accomplished using bacterial cells, yeast cells, insect cells or mammalian cells. In some aspects, the mammalian cell is a Chinese hamster ovary cell. In some aspects, the protein of interest is prepared by the method disclosed herein.

[0082] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting.

[0083] Example

[0084] Example 1 - Systems and methods for unified concentration and buffer exchange

[0085] Proteins and membranes

[0086] The feed containing monoclonal antibody (mAb) was adjusted to a salinity of pH 5.0, 50 mM sodium acetate buffer, and 200 mM NaCl (conductivity, 20 + 3 mS / cm) to simulate a cation exchange elution cell. The mAh concentration in the feed was adjusted between 7 g / L and 30 g / L. The hollow fiber modules used in this study were composed of polysulfone or polyethersulfone. The polysulfone membranes used were Optiflux180NR (Fresenius, USA) and Renaflow HF1200 (Minntech, USA), with membrane surface areas of 1.8 m 2 and 1.2m 2 . Polyethersulfone membrane Minikros 0.16m 2 (S04-E030-05-N) was purchased from Repligen, USA. New hollow fiber modules were used for each study except for the Minikros modules, which were cleaned with 0.1 N NaOH for 20 min before reuse.

[0087] Asymmetric dialysis method

[0088] The hollow fiber modules are mounted in a vertical orientation, with the pump P1 ( Figure 1 ) introduce feed from the bottom port on the lumen side. The shell side port (shell outlet) near the feed port is attached to pump P2. The distal shell side port (shell inlet) and the lumen side port (lumen outlet) are attached to pumps P3 and P4, respectively. Dialysis buffer is introduced into the shell side using pump P3, while pump P2 regulates the flow rate at the shell outlet. The concentrated and buffer exchanged product is collected at pump P4. Peristaltic pumps P1, P2, P3 and P4 equipped with appropriate pump heads and tubing are calibrated by timed collection using a digital balance before starting the process.

[0089] The pressure was monitored using Pendotech pressure sensors placed just before and after the inlet / outlet ports. Prior to the experiment, the hollow fiber module (1.8 mM) was flushed with 20 mM histidine pH 5.7 ± 1 dialysis buffer. 2, OptiFlux 180NR) shell and lumen compartments. All solutions used in the experiments were filtered through a 0.45μm PES filter. In a typical experimental setup with a 20ml / min (flux 0.7LMH) feed flow (P1), pump P4 was adjusted to 5mL / min to obtain a 4x concentration factor along the hollow fiber membrane. At the same time on the shell side, pumps P2 and P3 were adjusted to 60mL / min and 45mL / min, respectively. Therefore, in order to allow simultaneous product concentration and buffer exchange, the flow rates on all pumps were adjusted. The accompanying inlet and outlet flow rates of the feed and exchange buffers were paired proportionally, but not matched (Table 1). All experiments were performed at room temperature (22±2°C) without deliberately controlling the temperature. For some experiments, vitamin B12 was dissolved in the pretreated mAb feed as a model impurity. All experiments were performed in single-pass mode without recirculation of feed or dialysis buffer.

[0090] Small samples were collected periodically from the lumen outlet to monitor offline pH, conductivity, mAb, and histidine concentrations.

[0091] Table 1. Flow rate parameters for a typical asymmetric dialysis setup

[0092]

[0093] In some experiments, pump P4 was not used or was replaced by a back pressure regulator to obtain the desired concentration factor.

[0094] Strategies to reduce buffer consumption

[0095] The amount of buffer used in asymmetric dialysis depends on the operation α', where

[0096]

[0097] Several values ​​of α' were evaluated to reduce buffer consumption. At α' = 5, buffer consumption was reduced by 75% without affecting process performance. For an asymmetric dialysis process with an α' value of 22.5, 20 g / L feed mAb, and a concentration factor of 10x, the resulting buffer consumption would be >0.1 L / g mAb ( Figure 2 ).

[0098] result

[0099] Example 2

[0100] In this example, 30 g / L mAb feed (pH 5, 200 mM NaCl) was supplied to the hollow fiber using pump P1 at a flow rate of 20 ml / min (0.7 LMH), and pump P4 was adjusted to 5 mL / min to obtain the desired 4x 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 Fresenius and Repligen hollow fibers showed comparable desalting and buffer exchange performance.

[0101] Table 2: Asymmetric dialysis performance of 30 g / L mAb feed

[0102]

[0103]

[0104] Example 3

[0105] In this example, 20 g / L mAb feed (pH 5, 200 mM NaCl) was supplied to the hollow fiber using pump P1 at a flow rate of 45 ml / min (1.5 LMH), pump P4 was adjusted to 4.5 mL / min to obtain the desired 10x concentration factor along the hollow fiber membrane. For the shell side, 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.

[0106] Example 4

[0107] In this example, to reduce buffer consumption, a lower α', i.e. 5, was selected, wherein 20 g / L mAb feed (pH 5, 200 mM NaCl) was supplied to the hollow fiber device using pump P1 at a flow rate of 45 ml / min (1.5 LMH), and pump P4 was adjusted to 4.5 mL / min to obtain the desired 10x 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.

[0108] Example 5

[0109] In this example, asymmetric dialysis with α' of 5 was used to remove relatively large model impurities / tracers from the feed. For this evaluation, a 20 g / L mAb feed (pH 4.9, 200 mM NaCl) with 3.2 g / L vitamin B12 (approximately 1,356 kDa) was supplied to the hollow fiber device using pump P1 at a flow rate of 45 ml / min (1.5 LMH), and pump P4 was adjusted to 4.5 mL / min to obtain 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. Based on empirical experimental data related to this evaluation, the fresh dialysis buffer flow rate was adjusted to pH 5.6 to achieve a target product pH of 6.0.

[0110] Table 3: Asymmetric dialysis performance of 20 g / L mAb feed with model impurity vitamin B12

[0111]

[0112] Example 6

[0113] In this embodiment, use α 'to be 5 and adopt the asymmetric dialysis containing the dialysis buffer of glucose (4.6g / L), nonionic molecules (such as carbohydrate, glucose) are exchanged in product.For this assessment, use pump P1 with the flow velocity of 45ml / min (1.5LMH) that 20g / L mAb is fed (pH 4.9, 200mM NaCl) is supplied to hollow fiber device, pump P4 is adjusted to 4.5mL / min to obtain the concentration factor along the expectation of hollow fiber membrane.For shell side, dialysis buffer pump P2 and P3 are adjusted to 63mL / min and 22.5mL / min respectively.

[0114] Table 4: Asymmetric dialysis performance of 20 g / L mAb feed with dialysis buffer containing glucose

[0115]

[0116] Example 7

[0117] In this embodiment, α' is used as 5 and adopts the asymmetric dialysis of dialysis buffer 20mM histidine pH 5.9, 100mM NaCl, ionic compounds (such as NaCl) are exchanged into the product. For this evaluation, low conductivity (1.2mS / cm) 20g / L mAh feed (20mM histidine pH 6.0) is supplied to the hollow fiber device using pump P1 with a flow rate of 45ml / min (1.5LMH), and pump P4 is adjusted to 4.5mL / min to obtain the desired concentration factor along the hollow fiber membrane. For the shell side, dialysis buffer pumps P2 and P3 are adjusted to 63mL / min and 22.5mL / min respectively.

[0118]

[0119] Therefore, the above data demonstrate the development of an innovative one-step process, asymmetric dialysis, which can be used for continuous UF and buffer exchange, eliminating the need for two-step UF / DF. The inlet / outlet flow rates are carefully manipulated to achieve product concentration, buffer exchange and desalination. As described above, a moderate (<6psi) pressure distribution across the cartridge can be used to achieve product concentrations of 105g / L (3.8x), 200g / L (10x), and 64g / L (9.4x) starting from feed concentrations of 28g / L, 20g / L and 7g / L, respectively. Compared with conventional batch UF-DF (0.1L / g, mAb), this method also reduces buffer utilization by 74% (0.026L / g, mAb), and mAb productivity is as high as 0.7kg / m 2 / day. This provides a simplified and smaller footprint compared to current generation techniques.

Claims

1. A method for purifying a protein of interest using countercurrent concentrating dialysis, the method comprising: (a) passing a first flowing solution comprising the protein of interest and impurities into a first hollow fiber dialysis cassette at a first flow rate, wherein the dialysis cassette comprises a dialysate inflow having a dialysate inflow flow rate and a dialysate effluent having a dialysate effluent flow rate; and wherein the first flowing solution is countercurrent to the dialysate inflow and the dialysate effluent; (b) passing the impurities through a semipermeable membrane of the dialysis cassette, wherein the dialysate inflow flow rate is greater than the first flow rate, wherein a second flowing solution comprising the protein of interest and a reduced level of the impurities exits the dialysis cassette at a second flow rate, and wherein the dialysate outflow flow rate is the sum of the dialysate inflow flow rate and the difference between the first flow rate and the second flow rate; (c) optionally passing the second mobile solution from the first dialysis cassette directly into a second dialysis cassette; as well as (d) optionally repeating steps (a) and (b) with the second moving solution and the second dialysis cassette, thereby forming a third moving solution having a reduced level of impurities compared to the first moving solution and the second moving solution.

2. The method of claim 1 , further comprising passing the third mobile solution from the second dialysis box directly into a third dialysis box and repeating steps (a) and (b) to form a fourth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, and the third mobile solution.

3. The method of claim 2, further comprising passing the fourth mobile solution from the third dialysis box directly into a fourth dialysis box and repeating steps (a) and (b) to form a fifth mobile solution having a reduced level of impurities compared to the first mobile solution, the second mobile solution, the third mobile solution, and the fourth mobile solution.

4. The method of any one of claims 1 to 3, wherein 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 times, 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 the first flow rate.

5. The method of claim 4, wherein the dialysate inflow flow rate is about 2.25 times the first flow rate.

6. The method of any one of claims 1 to 5, wherein 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.

7. The method of any one of claims 1 to 6, wherein the second flow rate is between about 0.25 and about 0.5 times the first flow rate.

8. The method of any one of claims 1 to 7, wherein the first flow rate is between about 0.01 ml / min and about 25 ml / min.

9. The method of claim 8, wherein 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.

10. The method according to any one of claims 1 to 9, wherein the impurities comprise low molecular weight species.

11. The method according to any one of claims 1 to 10, wherein the protein of interest is subjected to diafiltration.

12. The method according to any one of claims 1 to 11, wherein the protein of interest is obtained from a bioreactor.

13. The method of any one of claims 1 to 12, wherein 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 is purified.

14. The method of any one of claims 1 to 13, wherein the protein of interest comprises an antibody, an antigen binding fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof.

15. The method of claim 14, wherein the protein comprises an antibody selected from the group consisting of IgM, IgA, IgE, IgD and IgG.

16. The method of claim 15, wherein the protein comprises an antibody, and the antibody is an IgG antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

17. The method of claim 16, wherein the antibody is a therapeutic antibody.

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