Use of multihydrophobic interaction chromatography for preparing polypeptides from mixtures

Abstract

The disclosure of the present invention describes a method for extracting a target polypeptide, preferably an antibody, from a mixture. The method comprises contacting the mixture with a hydrophobic interaction chromatography (HIC) apparatus comprising multiple chromatographic regions. The retention time of the mixture containing the target polypeptide in the first region may be approximately equal to the retention time of one or more mobile phases in the second region.

Description

Technology Field

[0001] Technology field

[0002] Related applications

[0003] This application claims priority to U.S. provisional patent application No. 62 / 693,024 filed on July 2, 2018, the entire contents of which are incorporated herein by reference.

[0004] The present disclosure generally relates to a method for preparing polypeptides. More particularly, the present disclosure relates to a method for preparing polypeptides from a mixture using a chromatographic method. Background Technology

[0005] background

[0006] Chromatography, for example, hydrophobic interaction chromatography (HIC), affinity chromatography, etc., can be performed as part of a method for manufacturing drug products. In some cases, chromatography may be particularly useful for the manufacture of drug products containing polypeptides. However, the equipment, materials, manufacturing time, and operating time for a standard batch HIC step or other batch chromatography steps can result in added costs or reduced efficiency in the method for manufacturing drug products. In particular, the time required to perform each stage in the HIC or other chromatographic separation process, the amount of buffer and / or separation medium used, and any non-automated aspects of the process can reduce the efficiency of drug product manufacturing.

[0007] The methods and systems disclosed herein can improve the efficiency and / or productivity of polypeptide manufacturing methods. The methods and systems disclosed herein can also improve the efficiency and / or productivity of drug product manufacturing methods and can solve one or more of the identified problems.

[0008] summation

[0009] An embodiment of the disclosure of the present invention directs a method for preparing a target polypeptide from a mixture containing a target polypeptide. The method may comprise contacting a mixture containing a target polypeptide with a first region of an HIC device, contacting a mobile phase with a second region of an HIC device, and passing the target polypeptide through the outlets of the first and second regions of the HIC device, wherein each of the first region and the second region may have one or more chromatographic columns and outlets. In some embodiments, the residence time of the mixture containing the target polypeptide in the first region may be configured to be approximately the same as the residence time of the mobile phase in the second region.

[0010] In some embodiments, the target polypeptide may be a monoclonal antibody. The target polypeptide may be produced at a productivity of 50 g / L·hr or more. Alternatively, or additionally, the mobile phase may comprise an equilibration buffer and a wash buffer. In some embodiments, the method of the disclosure of the present invention may further comprise passing an effluent containing the target polypeptide from a first region of the HIC device to a second region of the HIC device. In some embodiments, contacting the mobile phase with the second region of the HIC device may comprise contacting the wash buffer with the second region of the HIC device, and then regenerating the second region. In some embodiments, regenerating the second region may comprise contacting water with the second region of the HIC device, contacting an alkaline solution with the second region of the HIC device, contacting an alcohol solution with the second region of the HIC device, and contacting the equilibration buffer with the second region of the HIC device. The target polypeptide may be passed through the outlet of the second region of the HIC device after contacting the wash buffer with the second region of the HIC device. In some embodiments, one or more of UV absorption, electrical conductivity, or pH of the retention solution may be measured at the outlet of either the first region or the second region. The first region or the second region may include more than one chromatography column. In some embodiments, the HIC device may further include a third region having a chromatography column and an outlet. In some embodiments, the method may further include performing a regeneration cycle in the third region, wherein performing the regeneration cycle includes contacting the mobile phase with the third region, and wherein the duration of the regeneration cycle is configured to be approximately equal to the retention time of the mixture containing the target polypeptide in the first region.

[0011] In some embodiments of the present disclosure, a method for preparing a target polypeptide from a mixture containing a target polypeptide may comprise passing a mixture containing a target polypeptide through a first column of a plurality of chromatographic columns in an HIC apparatus, passing an effluent containing the target polypeptide through a second column from the first column of the plurality of columns, passing one or more mobile phases through a third column of the plurality of columns, and passing the target polypeptide through an outlet of each of the plurality of columns, wherein each of the plurality of columns includes an outlet connectable to another column of the plurality of columns, and the sum of the retention times of the mixture containing the target polypeptide in the first and second columns is substantially equal to the sum of the retention times of one or more mobile phases in the third column.

[0012] In some embodiments, the method may further comprise passing one or more mobile phases through each of the plurality of columns. In some embodiments, passing one or more mobile phases through the columns may comprise passing a wash buffer through the columns and, after passing the wash buffer through the columns, regenerating the columns, wherein regenerating the columns comprises passing water, an alkaline solution, an alcohol solution, or an equilibration buffer through the columns. In some embodiments, the step of passing the target polypeptide through the outlet of the columns may occur after the wash buffer has been passed through the columns. In some embodiments, one or more of UV absorption, electrical conductivity, or pH of the retention solution are measured at the outlet of either the first column or the second column. In some embodiments, the method may comprise producing the target polypeptide with a productivity of 50 g / L·hr or more. In an additional embodiment, the HIC device may include four columns, and the sum of the retention times of a mixture containing a target polypeptide in the first and second columns may be substantially equal to the sum of the regeneration times of the third and fourth columns.

[0013] Further embodiments of the disclosure of the present invention may include a method for producing an antibody using a plurality of chromatographic columns, wherein each of the plurality of chromatographic columns comprises a hydrophobic interaction medium. The method comprises, in a first step, loading a certain amount of a mixture containing an antibody into a first column of the plurality of columns, loading the certain amount of the mixture into a second column of the plurality of columns via the first column, and performing a non-loading step comprising at least one of washing, stripping, and equilibration processes in a third column of the plurality of columns; and, in a second step, loading a certain amount of a mixture containing an antibody into a second column, loading the certain amount of the mixture into a third column via the second column, and performing a non-loading step comprising at least one of washing, stripping, and equilibration processes in a first column. In the third stage, a certain amount of mixture containing antibodies is loaded into the third column, said certain amount of mixture is loaded into the third column via the second column, and a non-loading step including at least one of washing, stripping, and equilibration processes is performed in the second column.

[0014] In some embodiments, the method may further include periodically repeating the first, second, and third stages in succession, wherein each stage includes performing a loading and a non-loading step simultaneously. In some embodiments, the duration of one of the loading steps is configured to be approximately the same as the duration of the non-loading step. Brief explanation of the drawing

[0015] The attached drawings, introduced and configured as part of this specification, serve to illustrate various exemplary embodiments and, together with the detailed description, explain the principles of the disclosed embodiments. Any feature of the embodiments or examples described herein (e.g., compositions, formulations, methods, etc.) may be combined with any other embodiments or examples, and all such combinations are included in the disclosure of the invention. Furthermore, the disclosed systems and methods do not limit any single aspect or embodiment, nor do they limit any combination or permutation of such aspects and embodiments. For the sake of brevity, specific permutations and combinations are not discussed and / or illustrated individually herein. FIG. 1 is a schematic diagram illustrating a portion of a chromatography apparatus according to some embodiments of the disclosure of the invention. FIG. 2 is a schematic diagram of a chromatography apparatus according to some embodiments of the disclosure of the invention. FIG. 3a is a graphical diagram of an exemplary method for preparing a target polypeptide according to some embodiments of the disclosure of the invention; FIG. 3b-3d are brief examples illustrating a method for preparing the target polypeptide shown in FIG. 3a. FIG. 4 is a schematic diagram of a chromatography apparatus according to some embodiment of the disclosure of the present invention. FIG. 5a is a schematic graph of an exemplary method for preparing a target polypeptide according to some embodiment of the disclosure of the present invention; FIG. 5b-5e are brief examples illustrating the method for preparing the target polypeptide shown in FIG. 5a. FIG. 6 is a flowchart of a method for preparing a target polypeptide according to some embodiment of the disclosure of the present invention. FIG. 7a is a plot of high molecular weight percentage as a function of loading according to aspects of the disclosure of the present invention. FIG. 7b is a plot of host cell protein amount as a function of loading according to aspects of the disclosure of the present invention. FIG. 7c is a plot of high molecular weight percentage as a function of loading concentration according to aspects of the disclosure of the present invention.FIG. 8a is a plot of productivity as a function of the number of chromatography columns according to aspects of the disclosure of the present invention. FIG. 8b is a plot of productivity as a function of the number of chromatography columns according to aspects of the disclosure of the present invention. Specific details for implementing the invention

[0016] As used herein, the terms “comprises,” “comprising,” or any other variation thereof are intended to include non-exclusive inclusions, and accordingly, a process, method, product, or apparatus comprising elements enumerated may not include only these elements but may include other elements not explicitly enumerated or inherent in such process, method, product, or apparatus. The term “exemplary” is used in the sense of “example” rather than “ideal.” The terms “for example” and “such as,” and their grammatical equivalents, and the phrase “and without limitation” are understood as follows unless otherwise explicitly stated.

[0017] As used herein, the term “about” is intended to describe variation due to experimental error. When applied to numerical values, the term “about” may indicate a variation of + / - 10% from the disclosed numerical value (unless other variations are specified). One singular form (“a”, “an”) and the above (“the”) as used herein include plural objects unless otherwise specified in the context.

[0018] It should be noted that all numerical values ​​disclosed herein (including all disclosed values, limits, and ranges) may have a variation of + / - 10% from the disclosed numerical values ​​(unless other variations are specified). Additionally, in the claims, values, limits, and / or ranges mean values, limits, and / or ranges + / - 10%. Similarly, the phrase "approximately identical" as used herein may mean identical within a variation of + / - 10%. Furthermore, all ranges are understood to include endpoints, for example, 1 centimeter (cm) to 5 cm will include lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0019] details

[0020] The present disclosure is not limited to the specific composition, formulation, material manufacturer, drug product, device, system, experimental condition, or specific method disclosed herein, as numerous variations are possible within the understanding of those skilled in the art. The terms used herein are intended to describe only specific embodiments and are not intended to be limited.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. While all methods and materials similar or equivalent to those described herein may be used for the practice or testing of the disclosure of the invention, specific methods are described below. All publications mentioned are incorporated herein by reference.

[0022] As used herein, the term “contacting” refers to the meeting, joiner, interface, or other physical interaction of two or more surfaces, solutions, or compounds. Although a particular fluid is described herein as passing into, from, or through a region, it is understood that the fluid will necessarily contact any region, and said fluid passes into, from, or through said region. Similarly, introducing a fluid or component into a region constitutes a fluid or component that contacts the region.

[0023] As used herein, the term “polypeptide” refers to any amino acid polymer having more than about 20 amino acids covalently bonded through amide bonds. Proteins comprise one or more amino acid polymer chains (e.g., polypeptides). Thus, polypeptides can be proteins, and proteins can comprise multiple polypeptides to form single-function biomolecules.

[0024] Post-translational modifications can further alter or modify the structure of polypeptides. For example, disulfide bridges (e.g., SS bonds between cysteine ​​residues) may be present in some proteins. Some disulfide bridges are essential for the appropriate structure, function, and interactions of polypeptides, immunoglobulins, proteins, co-factors, substrates, etc. In addition to the formation of disulfide bonds, proteins may undergo other post-translational modifications. These modifications include lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and the formation of a glycosylphosphatidylinositol (GPI) anchor), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of a phosphate group to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobic, electrostatic surface properties, or other properties that determine the surface-to-surface interactions involved by the polypeptide.

[0025] As used herein, the term “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Protein-of-interest (POI) may include any polypeptide or protein that is preferably isolated and purified, but otherwise produced. POI may include a target polypeptide or other polypeptide produced by a cell containing an antibody.

[0026] As used herein, the term "antibody" comprises an immunoglobulin composed of four polypeptide chains: two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Typically, the antibody has a molecular weight greater than 100 kDa, e.g., 130 kDa to 200 kDa, e.g., about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions may be further subdivided into a hypervariability region, referred to as the complementarity determining region (CDR), interspersed with a more conserved region, referred to as the framework region (FR). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs are abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3).

[0027] For example, the class of immunoglobulins called immunoglobulin G (IgG) is common in human serum and comprises four polypeptide chains—two light chains and two heavy chains. Each light chain is bound to one heavy chain via a cystine disulfide bond, and the two heavy chains are bound to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, four subclasses exist: IgG 1, IgG 2, IgG 3, and IgG 4. Each subclass differs in their invariant regions and, consequently, may have different effector functions. In some embodiments described herein, the POI may comprise a target polypeptide comprising IgG. In at least one embodiment, the target polypeptide comprises IgG 4.

[0028] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or, in particular, a glycoprotein that binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, which include, for example, the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA may be known and / or readily available, for example, from commercial source DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. DNA may be sequenced or manipulated chemically or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains into a suitable batch, introduce codons, generate cysteine ​​residues, or modify, add, or delete amino acids, etc.

[0029] Target polypeptides are produced in recombinant cell-based manufacturing systems, e.g., insect baculovirus systems, yeast systems (e.g., Pichia ( Peach It can be prepared using mammalian systems (e.g., CHO derivatives such as CHO cells and CHO-K1 cells). The term "cell" includes any cell suitable for expressing the recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single-cell or multi-cell), and bacterial cells (e.g., *E. coli* ( E. coli ), Bacillus( Bacillus ) species, Streptomyces( Streptomyces ) strains of species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae ( S. cerevisiae ), Es Pombe ( S. pombe ), P. Pastoris( Pastor's P. ), p. methaneolica( P. methanolica )), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, trichoplucyanis( TrichoplusianIt includes non-human animal cells, human cells, or cell fusions, for example, hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a progressive cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, e.g., retinal cells (e.g., PER.C6™ cells) expressing viral genes. The target polypeptide or protein or polypeptide other than the POI produced by the cell may be referred to as a host-cell protein (HCP). Where the POI is manufactured in a host cell and / or purified from a host cell, the HCP may be characterized as a product- and process-related contaminant or impurity.

[0030] Some HCPs (e.g., enzymes) can be co-purified with POIs (e.g., target polypeptides) and may affect the components of mixtures, formulations, or drug products, including the POIs. For example, the presence of some HCPs may affect product stability, reduce the shelf life of drug products, or even cause the product to fail to meet compendial or regulatory particulate matter specifications (e.g., FDA specifications). As an additional example, some HCPs may cause clinical effects, such as immunogenic reactions upon administration. While HICs or other chromatography methods can be used, alone or in combination, to purify and / or separate POIs and remove HCPs from mixtures, formulations, or drug products, thereby reducing the potential effects of HCPs on drug products, the addition of HIC or affinity chromatography steps requires additional equipment, materials (e.g., hydrophobic interaction media), and preparations. This corresponds to added time, resources, experimentation, and costs. Therefore, it is desirable to perform an efficient method of carrying out a chromatography process to separate a POI (e.g., a target polypeptide) from one or more co-purified HCPs or other impurities.

[0031] As used herein, the term “chromatography” refers to any process of separating the components of a mixture by passing the mixture through a medium such that the components of the mixture pass through the medium at different rates, and includes, but is not limited to, column chromatography, planar chromatography, thin-layer chromatography, substitution chromatography, gas chromatography, affinity chromatography, ion-exchange chromatography, size-exclusion chromatography, reverse-phase chromatography, hydrophobic interaction chromatography (HIC), rapid protein liquid chromatography, high-performance liquid chromatography, countercurrent chromatography, periodic countercurrent chromatography, or chiral chromatography. While embodiments of the present invention may be disclosed for exemplary types of chromatographic processes (e.g., HIC) or apparatus, the embodiments disclosed herein are applicable to all types of chromatography.

[0032] As used herein, the term “water” may refer to any suitable type of laboratory-grade water, e.g., deionized water or injection water. In some embodiments, e.g., a chromatography device may come into contact with either deionized water or injection water. Any reference to the use of “water” herein may refer to deionized water, injection water, or any other type of laboratory-grade water.

[0033] The term “mobile phase” as used in the disclosure of the present invention may refer to any suitable fluid for contacting a chromatographic region or column as part of a separation or purification process. The mobile phase may include, for example, water, a buffer solution, an acidic solution, an alkaline solution, and / or a solution containing an alcohol. The terms “washing buffer,” “stripping buffer,” and “equilibration buffer” may additionally be used to describe a mobile phase having the specific characteristics described herein.

[0034] In some embodiments, a method for preparing a target polypeptide from a mixture containing a target polypeptide may include contacting the mixture with a chromatography apparatus. The chromatography apparatus may include a plurality of regions, wherein each region includes one or more chromatography columns, wherein one or more chromatography columns include a hydrophobic interaction medium. Such a chromatography apparatus may include a pre-fabricated apparatus (e.g., Cadence™ BioSMB (Pall Biosciences), BioSC® (novasep), Varicol® (novasep), or Octave (Semba® Biosciences)), hand-assembled apparatuses, or just two or more standard batch chromatography apparatuses used in a tandem.

[0035] Aspects of the disclosure of the present invention may provide various advantages for methods of preparing target polypeptides or other molecules. For example, the simultaneous use of multiple zones in a chromatography apparatus enables more efficient and fuller loading of individual columns, and / or performance of separation processes using less chromatographic medium than in standard chromatography processes. Additional advantages and benefits of aspects of the disclosure of the present invention will be apparent to those skilled in the art.

[0036] From now on, we will refer to the drawings of the disclosure of the present invention.

[0037] FIG. 1 illustrates a section (100) of a chromatographic column of an HIC apparatus according to some embodiment of the disclosure of the present invention. The chromatographic column comprises a hydrophobic interaction medium. The hydrophobic interaction medium comprises a support structure (110) and a hydrophobic moiety (120), wherein the hydrophobic moiety (120) is attached to the support structure (110). The medium may exist as a chromatographic medium, for example, in the form of beads or other particles, maintained in a packed bed column configuration, in the form of a membrane, or in any configuration capable of containing a mixture or other liquid comprising a target polypeptide (or other POI) and contaminants (e.g., HCP). Thus, exemplary hydrophobic interaction media may include agarose beads (e.g., Sepharose), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, etc.

[0038] The chromatography column of the HIC device disclosed in the present invention may be configured such that the hydrophobic interaction medium has a depth (e.g., bed height) of about 0.5 centimeters (cm) to about 40 cm. In some embodiments, for example, the chromatography column of the HIC device may have a bed height of about 0.5 cm to about 30 cm, about 0.5 cm to about 20 cm, about 0.5 cm to about 10 cm, about 0.5 cm to about 5 cm, about 1 cm to 20 cm, about 1 cm to about 10 cm, or about 1 cm to about 5 cm. In some embodiments, the chromatography column may be configured such that the internal diameter of the chromatography column is about 0.5 cm to about 150 cm. In some embodiments, for example, the inner diameter of the chromatography column is about 0.5 cm to about 140 cm, about 0.5 cm to about 120 cm, about 0.5 cm to about 100 cm, about 0.5 cm to about 80 cm, about 0.5 cm to about 60 cm, about 0.5 cm to about 40 cm, about 0.5 cm to about 20 cm, about 0.5 cm to about 10 cm, about 0.75 cm to about 8 cm, about 1 cm to about 6 cm, about 1 cm to about 5 cm, about 1 cm to about 3 cm, about 1.5 cm to about 5 cm, about 1.5 cm to about 3 cm, or about 1 cm to about 2 cm. For example, in some embodiments, the inner diameter of the chromatography column is about 0.5 cm, about 1 cm, about 5 cm, about 8 cm, about 10 cm, about 15 cm, about 20 cm, about 30 cm, about 40 cm, about 50 cm, about 60 cm, about 80 cm, about 100 cm, about 125 cm, or about 150 cm. In some embodiments, the chromatography column of the HIC apparatus according to the present disclosure is about 0.It has a total volume of 4 milliliters (mL) to about 175 L (e.g., total capacity for a mixture, mobile phase, or other material). In some embodiments, for example, the chromatographic column of the HIC apparatus according to the present disclosure is about 0.5 mL to about 150 L, about 0.5 mL to about 130 L, about 0.5 mL to about 115 L, about 0.5 mL to about 100 L, about 0.5 mL to about 80 L, about 0.5 mL to about 60 L, about 0.5 mL to about 40 L, about 0.5 mL to about 20 L, about 0.5 mL to about 15 L, about 0.5 mL to about 10 L, about 0.5 mL to about 5 L, about 0.5 mL to about 1 L, about 1 mL to about 750 mL, about 1 mL to about 600 mL, about 1 mL to about 500 mL, about 1 mL to about 300 mL, about 1 mL to about 250 mL, about 1 mL to about It has a total volume of 200 mL, or about 1 mL to about 150 mL. For example, in some embodiments, the chromatography column according to the present disclosure may have a total volume of about 0.5 mL, about 1 mL, about 5 mL, about 10 mL, about 50 mL, about 100 mL, about 150 mL, about 300 mL, about 400 mL, about 500 mL, about 1 L, about 5 L, about 10 L, about 50 L, about 80 L, about 100 L, about 120 L, or about 150 L.

[0039] In some embodiments, the hydrophobic moiety (120) binds to the hydrophobic region and hydrophobic surface of the polypeptide. The hydrophobic surface may be a part of the amino acid structure containing the peptide, the aforementioned or other post-translational modification, or a combination thereof. The degree of hydrophobicity of the hydrophobic interaction medium may be controlled by selecting a suitable hydrophobic moiety (120). The hydrophobic moiety (120) selected to bind to a specific target polypeptide or POI may be any currently known or future-developed hydrophobic moiety. In some embodiments, the hydrophobic moiety (120) may comprise methyl, propyl, isopropyl, butyl, hexyl, octyl, and / or phenyl groups. Those skilled in the art will recognize that the hydrophobicity of the selected hydrophobic moiety (120) may be variable based on the target polypeptide and / or HCP / other impurities of the provided application, as well as the type and degree of separation or purification desired in the chromatographic process.

[0040] A target polypeptide or other POI can be separated from the product and process-related contaminants and impurities (e.g., HCP) using a hydrophobic interaction medium. Also, referring to FIG. 1, in some embodiments, a mixture containing the target polypeptide (140) and other components, e.g., contaminants (130) (e.g., impurities, HCP, etc.) is loaded into an HIC device. The mixture may contain a solution (e.g., a buffer) designed to facilitate the binding of hydrophobic groups of the target polypeptide (140) to the hydrophobic moiety (120) of the hydrophobic interaction medium. Some target polypeptides (140) bind to the hydrophobic moiety (120) via intramolecular forces and attach to the medium, while other target polypeptides (140) may pass through a chromatography column. Additionally or alternatively, while the mixture passes through the column, some contaminants (130) from the mixture may bind to the hydrophobic moiety (120) via intramolecular forces and be attached to the hydrophobic interaction medium, while other contaminants (130) fail to bind to the hydrophobic moiety (120). In some embodiments, the target polypeptide (140) comprises a specific hydrophobic region comprising a constituent amino acid, a post-translational modification, or a combination thereof that can be attached to the hydrophobic moiety (120) having a higher affinity than the specific contaminant or impurity (e.g., HCP). As described in detail later, an additional mobile phase may then be introduced into the column to reduce the affinity between the target polypeptide (140) and the hydrophobic moiety (120) and allow the target polypeptide (140) to pass through the chromatographic column of the HIC device.

[0041] In an additional embodiment, the contaminant (130) may be attached to a hydrophobic moiety (120) having a higher affinity than the target polypeptide (140). Subsequently, an additional mobile phase may be introduced into the column to lower the affinity between the contaminant (130) and the hydrophobic moiety (120), which may allow the contaminant (130) to pass through the chromatography column of the HIC device.

[0042] The composition of the mixture containing the target polypeptide (140) may be changed by adding an additive containing a salt such as sodium, potassium, phosphate, tris(hydroxymethyl)aminomethane (tris), citrate, or acetate. By adding other additives, the hydrophobicity or other intramolecular interactions of the target polypeptide (140), contaminants (130), hydrophobic moiety (120), or a combination thereof may be altered.

[0043] An exemplary HIC device (200) is schematically illustrated in FIG. 2 according to some embodiments described herein. The HIC device (200) may include a first region (210), a second region (220), and a third region (230). Each of the first region (210), the second region (220), and the third region (230) may include one or more chromatographic columns, for example, the chromatographic columns described for FIG. 1. The first region (210) may have a first inlet (212) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the first region (210). The first area (210) may also have a first outlet (214) through which effluent (e.g., fluid passing through the first area (210)) may be passed from the HIC device (200) to be collected or disposed of. Effluent may also be passed from the first area (210) to the second area (220) via the first interconnect (216). The first area (210) may also receive effluent from the third area (230) via the third interconnect (236).

[0044] The second region (220) can receive effluent from the first region (210) via the first interconnect (216). The second region (220) may also have a second inlet (222) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the second region (220). The second region (220) may also have a second outlet (224) through which effluent (e.g., fluid passed through the second region (220)) can pass from the HIC device (200) to be collected or disposed of. Effluent may also pass from the second region (220) to the third region (230) via the second interconnect (226).

[0045] The third region (230) may receive effluent from the second region (220) via the second interconnect (226). The third region (230) may have a third inlet (232) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the third region (230). The third region (230) may also have an outlet (234) through which effluent (e.g., fluid passed through the third region (230)) may pass from the HIC device (200) to be collected or disposed of. Effluent may also pass from the third region (230) to the first region (220) via the third interconnect (236).

[0046] As will be understood by those skilled in the art, various components known to be used in chromatographic devices (e.g., filters, sensors, gauges, thermometers) may be introduced into the HIC device (200), but are not shown in the simplified schematic of FIG. 2. In some embodiments, one or more of UV absorption, electrical conductivity, or pH of the retained solution may be measured at one or more points within the HIC device (200). Suitable points for measuring UV absorption, electrical conductivity, or pH include at the inlet (212, 222, 232), within the region (210, 220, 230), at the interconnect (216, 226, 236), or at the outlet (214, 224, 234). The inlets (212, 222, 232), interconnects (216, 226, 236), and outlets (214, 224, 234) can be operated to move from an open arrangement to a closed arrangement; the open arrangement allows fluid to pass through the inlets (212, 222, 232), interconnects (216, 226, 236), or outlets (214, 224, 234), and the closed arrangement prevents fluid from passing through the inlets (212, 222, 232), interconnects (216, 226, 236), or outlets (214, 224, 234). The HIC device (200) may include one or more pumps that provide pressure to transfer fluid between regions (210, 220, 230), outlets (212, 222, 232), connects (216, 226, 236), and outlets (214, 224, 234). In some embodiments, one or more interconnects (216, 226, 236) may be moved to connect different regions (210, 220, 230). For example, during a process using the HIC device (200), it may be desirable to rearrange them, wherein the interconnect (226) passes effluent from region (220).In this situation, the interconnect (226) can be reconfigured to allow the effluent to pass from region (220) to region (210) without interfering with the chromatography process. This is just one example; generally, any interconnect (216, 226, 236) can be reconfigured to connect different regions without interfering with the ongoing chromatography process.

[0047] FIG. 3a is a graph diagram of a method according to some embodiment of the disclosure of the present invention. On the left axis of the graph, three individual columns are defined as labels C1, C2, and C3, representing the first, second, and third columns of an HIC device. The top axis represents time and extends infinitely to the left and right. The continuous occupation of each column is an example of the embodiment described herein; this arrangement reduces or eliminates idle time (e.g., "dead time") for the columns compared to conventional HIC methods. The time segments shown throughout FIG. 3a represent one exemplary cycle of a repeating pattern, which may be repeated before and / or after the time segments shown in FIG. 3a. The four times are labeled T1, T2, T3, and T4 and are examples of any line T0 that can be drawn vertically through the graph. In some embodiments, the interval between T1 and T2 is substantially the same as the interval between T2 and T3, and in some embodiments, is substantially the same as the interval between T3 and T4. In some embodiments, the interval between adjacent labeled times (e.g., T1 to T2 or T3 to T4) may be 30 seconds (s) or more, 90 minutes (min) or less, 30 s to 60 min, 30 s to 30 min, 30 s to 15 min, 30 s to 10 min, 30 s to 8 min, 30 s to 7 min, 30 s to 6 min, 30 s to 5 min, 30 s to 4 min, 30 s to 3 min, 1 min to 5 min, or 2 min to 5 min.Boxes (410, 412, 414, 415, 417, 419, 424, 420, 422, 424, 425, 427, 429, 430, 432, 434, 435, 437, and 439) represent events occurring within each column, C1, C2, and C3, within the time interval in which each box appears. For example, each box may indicate the presence of a mixture, mobile phase, or other resident liquid within the column in which the box appears.

[0048] Moving from left to right across FIG. 3a, as moving "forward" in time, from T1 to T2, a secondary load of the mixture may be present in the first column C1 (Box (410)). From T2 to T3, a primary load of the mixture may be present in the first column C1 (Box (412)), and from T3 to T4, one or more mobile phases may be present in the first column C1 (Box (414)). In some embodiments, the column may accommodate either the primary load of the mixture or the secondary load of the mixture. The "primary load" of the mixture refers to a load of the mixture passing into the column of the HIC device without first passing through another column of the HIC device. The “secondary load” of the mixture refers to a load of the mixture passed through another column of the HIC device before being introduced into the provided column (for example, effluent from the primary load of the mixture is introduced into another column as the secondary load of the mixture). Passing the effluent from one column containing the target peptide to another column can allow the columns to be fully loaded without concern for wasted overflow, can increase the utilization efficiency of each column, and can reduce the volume of hydrophobic interaction medium consumed. By passing the overflow through the hydrophobic interaction medium that was or can accommodate the primary load of the mixture containing the target polypeptide, the volume of hydrophobic interaction medium consumed relative to the amount of the load mixture processed can be reduced.

[0049] In some embodiments, contacting one or more mobile phases with the column may include contacting a washing buffer with the column, contacting a stripping buffer with the column, and / or contacting an equilibration buffer with the column. In some embodiments, the washing buffer may include one or more salts, for example, sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, Tris, or other salts.

[0050] In some embodiments, the stripping buffer may comprise water, an alkaline solution, or a solution containing an alcohol. The deionized water may have, for example, less than 5 volume percent (vol.%) dissolved ions, less than 1 vol.% dissolved ions, less than 0.1 vol.% dissolved ions, or even less than 0.01 vol.% dissolved ions. According to some embodiments, the alkaline solution may comprise one or more alkaline ionic compounds, for example, LiOH, NaOH, KOH, Ca(OH)2, NH4OH, or other alkaline compounds. The concentration of the alkaline compound in the stripping buffer may be, for example, in the range of about 0.1 N to about 1.5 N, about 0.1 N to about 1 N, about 0.1 N to about 1.5 N, about 0.5 N to about 1.5 N, about 0.1 N to about 0.8 N, about 0.1 N to about 0.6 N, about 0.1 N to about 0.5 N, about 0.1 N to about 0.4 N, or about 0.1 N to about 0.3 N. For example, the concentration of an alkaline compound in the stripping buffer may be about 0.1 N, about 0.2 N, about 0.3 N, about 0.4 N, about 0.5 N, about 0.6 N, about 0.7 N, about 0.8 N, about 0.9 N, about 1 N, about 1.1 N, about 1.2 N, about 1.3 N, about 1.4 N, or about 1.5 N. The stripping buffer containing alcohol may include methanol, ethanol, propanol, benzyl alcohol, or other alcohols. The concentration of alcohol in the stripping buffer is, based on the total weight of the stripping buffer, about 0.1 vol.% to about 30 vol.%, for example, about 0.5 vol.% to about 30 vol.%, about 0.5 vol.% to about 25 vol.%, about 0.5 vol.% to about 25 vol.%, about 0.5 vol.% to about 25 vol.%, about 1 vol.% to about 20 vol.%, about 1 vol.% to about 15 vol.%, about 1 vol.The range may be from about 10 vol.%, from about 10 vol.% to about 50 vol.%, from about 10 vol.% to about 40 vol.%, from about 10 vol.% to about 30 vol.%, from about 10 vol.% to about 25 vol.%, from about 15 vol.% to about 25 vol.%, or from about 20 vol.% to about 25 vol.%. For example, the concentration of alcohol in the stripping buffer may be about 0.1 vol.%, about 0.5 vol.%, about 1 vol.%, about 2 vol.%, about 3 vol.%, about 5 vol.%, about 10 vol.%, about 15 vol.%, about 20 vol.%, or about 25 vol.%.

[0051] In some embodiments, the equilibration buffer may have a composition similar to or identical to that of the wash buffer. In other embodiments, the composition of the equilibration buffer may be variable compared to that of the wash buffer. In some embodiments, the equilibration buffer may contain one or more salts, for example, sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, tris, or other salts.

[0052] Referring to FIG. 3a, one or more mobile phases contacted in the first column (414) may be divided into individual phases including a wash buffer (box (415)) in the first column, a stripping buffer (box (417)) in the first column, and an equilibration buffer (box (419)) in the first column. In the next column (indicated by C2), from T1 to T2, one or more mobile phases may be present in the second column (box (424)). This may also be divided into individual phases including a wash buffer (box (425)) in the second column, a stripping buffer (box (427)) in the second column, and an equilibration buffer (box (429)) in the second column. When moving to the right, the secondary load of the mixture from T2 to T3 may be in the second column (Box (420)), and the primary load of the mixture from T3 to T4 may be in the second column (Box (422)).

[0053] In the next column, the primary load of the mixture from T1 to T2 may be in the third column (Box (432)). Subsequently, from T2 to T3, one or more mobile phases may be in the third column (Box (434)), and from T3 to T4, a secondary load of the mixture may be in the third column (Box (430)). One or more mobile phases in the third column (Box (434)) may be divided into individual phases including a wash buffer (Box (435)) in the third column, a stripping buffer (Box (437)) in the third column, and an equilibration buffer (Box (439)) in the third column.

[0054] At the provided time T0, a vertical line can be illustrated through a graph such that each numbered box contacted by the vertical line from T0 represents the solution in the column at that time. Thus, for example, at time T1, a secondary load mixture is introduced into the first column C1 (box (410)), one or more mobile phases are passed into the second column C2 (box (424)), for example, a wash buffer C2 (box (425)), and the primary load mixture is passed into the third column C3 (box (432)). Subdivisions of a broader phase, for example, subdivisions (425, 427, and 429), are shown to occupy the same portion of one or more mobile phases in the second column (424), but in some embodiments, the subdivisions may occupy different portions of the broader phase. It should also be understood that the method illustrated in FIG. 3a is merely one exemplary proceeding according to an embodiment of the disclosure of the present invention. Other sequences, arrangements, and steps are thought to be within the scope of the disclosure of the present invention.

[0055] FIGS. 3b-3d illustrates an exemplary cycle for a method of preparing a target polypeptide from a mixture containing the previously described target polypeptide. FIG. 3b illustrates a series of events that may occur during the time intervals T1 to T2 of FIG. 3a. Accordingly, FIG. 3b illustrates an HIC apparatus in the first stage (301), where the first region (310) receives a secondary load (306) of the mixture containing the target polypeptide and elutes the effluent (307) of the secondary load, which may be collected or discarded. The second region (320) receives one or more mobile phases (315) and elutes the effluent (316) of one or more mobile phases, which may be collected or discarded. The third region (330) receives the primary load (305) of the mixture and passes the secondary load (306) of the mixture to another column.

[0056] FIG. 3c illustrates an HIC device in a second stage (302) (over intervals T2 to T3, as shown in FIG. 3a), where a first region (310) receives a primary load (305) of the mixture and passes a secondary load (306) of the mixture to another column. A second region (320) receives a secondary load (306) of the mixture and elutes an effluent (307) of the secondary load that can be collected or discarded. A third region (330) receives one or more mobile phases (315) and elutes an effluent (316) of one or more mobile phases that can be collected or discarded.

[0057] FIG. 3d illustrates an HIC device at the third stage (303) (over intervals T3 to T4, as shown in FIG. 3a), where a first region (310) receives one or more mobile phases (315) and elutes one or more effluents (316) of the mobile phases that may be collected or discarded. A second region (320) receives a primary load (305) of the mixture and passes a second load (306) of the mixture to another column. A third region (330) receives one or more mobile phases (315) and elutes one or more effluents (316) of the mobile phases that may be collected or discarded.

[0058] Another exemplary HIC device (500) is schematically illustrated in FIG. 4 according to some embodiments described herein. The HIC device (500) may include a first region (510), a second region (520), a third region (530), and a fourth region (540). The first region (510) may have a first inlet (512) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the first region (510). The first region (510) may also have a first outlet (514) through which an effluent (e.g., fluid passing through the first region (510)) may pass from the HIC device (500) to be collected or disposed of. The effluent can also pass from the first area (510) to the second area (520) via the first interconnect (516). The first area (510) can also receive the effluent from the fourth area (540) via the fourth interconnect (546).

[0059] The second region (520) can receive effluent from the first region (510) via the first interconnect (516). The second region (520) may also have a second inlet (522) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the second region (520). The second region (520) may also have a second outlet (524) through which effluent (e.g., fluid passing through the second region (520)) may pass from the HIC device (500) to be collected or disposed of. Effluent may also pass from the second region (520) to the third region (530) via the second interconnect (526).

[0060] The third region (530) can receive effluent from the second region (520) via the second interconnect (526). The third region (530) may have a third inlet (532) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the third region (530). The third region (530) may also have an outlet (534) through which effluent (e.g., fluid passing through the third region (530)) may pass from the HIC device (500) to be collected or disposed of. Effluent may also pass from the third region (530) to the fourth region (520) via the third interconnect (536).

[0061] The fourth region (540) can receive effluent from the third region (530) via the third interconnect (536). The fourth region (540) may have a fourth inlet (542) configured to allow a mixture containing a target polypeptide, one or more mobile phases, or other liquids to pass into the fourth region (540). The fourth region (540) may also have an outlet (544) through which effluent (e.g., fluid passing through the fourth region (540)) can pass from the HIC device (500) to be collected or disposed of. Effluent may also pass from the fourth region (540) to the first region (510) via the fourth interconnect (546).

[0062] Various known components used in chromatography devices (e.g., filters, sensors, gauges, thermometers) are not shown in the simplified schematic of FIG. 4 but may be introduced into the HIC device (500). In some embodiments, one or more of UV absorption, electrical conductivity, or pH of the retained solution may be measured at one or more points of the HIC device (500). Suitable points for measuring UV absorption, electrical conductivity, or pH include within the region (510, 520, 530, 540) at the inlet (512, 522, 532, 542), at the interconnect (516, 526, 536, 546), or at the outlet (514, 524, 534, 544). The inlets (512, 522, 532, 542), interconnects (516, 526, 536, 546) and outlets (514, 524, 534, 544) can be operated to move from an open arrangement to a closed arrangement; the open arrangement allows fluid to pass through the inlets (512, 522, 532, 542), interconnects (516, 526, 536, 546), or outlets (514, 524, 534, 544), and the closed arrangement prevents fluid from passing through the inlets (512, 522, 532, 542), interconnects (516, 526, 536, 546), or outlets (514, 524, 534, 544). The HIC device (500) may include one or more pumps that provide pressure to transfer fluid between regions (510, 520, 530, 540), inlets (512, 522, 532, 542), interconnects (516, 526, 536, 546) and outlets (514, 524, 534, 544). In some embodiments, one or more interconnects (516, 526, 536, 546) may be moved to connect different regions (510, 520, 530, 540).For example, during one or more chromatography processes using the HIC device (500), it may be desirable to rearrange the interconnect (536) so that it passes effluent from region (530). In this situation, the interconnect (536) may be reconfigured to pass effluent from region (530) to region (520) without interfering with the chromatography process. This is just one example; generally, any interconnect (516, 526, 536, 546) may be reconfigured to connect different regions (510, 520, 530, 540) without interfering with the ongoing chromatography process.

[0063] FIG. 5a is a graph diagram of one or more methods according to the present disclosure. On the left axis of the graph, four individual columns are defined by labels C1, C2, C3, and C4 representing the first, second, third, and fourth columns of the HIC device. The top axis represents time and extends infinitely to the left and right. The continuous occupation of each column is an example of the embodiments described herein, and this arrangement reduces or eliminates idle time (e.g., "float time") for the columns compared to conventional HIC methods. The segment of time shown represents one cycle of a repeating pattern, and it is understood that a pattern of numbered boxes as follows may be repeated on two aspects of the segment shown in FIG. 5a. Five times are labeled T1, T2, T3, T4, and T5, and are examples of any line T0 that can be drawn vertically through the graph. In some embodiments, the interval between T1 and T2 may be substantially the same as the interval between T2 and T3, in some embodiments, may be substantially the same as the interval between T3 and T4, and in some embodiments, may be substantially the same as the interval between T4 and T5. In some embodiments, the intervals between each of these times may be different. In some embodiments, the interval between adjacent labeled times (e.g., T1 to T2 or T4 to T5) may be 30 s or more, 90 min or less, 30 s to 60 min, 30 s to 30 min, 30 s to 15 min, 30 s to 10 min, 30 s to 8 min, 30 s to 7 min, 30 s to 6 min, 30 s to 5 min, 30 s to 4 min, 30 s to 3 min, 1 min to 5 min, or 2 min to 5 min.Boxes (710, 712, 714, 724, 720, 722, 734, 730, 732, 742, 744, and 740) represent mixtures, buffers, or other retained liquids within each column, C1, C2, C3, and C4.

[0064] Moving from left to right across FIG. 5a, from T1 to T2, a secondary load of the mixture may be present in the first column (representing the first column C1) (Box (710)). From T2 to T3, a primary load of the mixture may be present in the first column (Box (712)), and from T3 to T5, one or more mobile phases may be present in the first column (Box (714)).

[0065] Also, referring to FIG. 5a, one or more mobile phases (714) in the first column may be divided into individual phases including a wash buffer (box (715)) in the first column, a stripping buffer (box (717)) in the first column, and an equilibration buffer (box (719)) in the first column. In the next column (representing the second column C2), from T1 to T2, one or more mobile phases may be present in the second column (box (724)), continuing from T4 to T5 of the previous cycle. This may also be divided into individual phases including a wash buffer (box (725)) in the second column, a stripping buffer (box (727)) in the second column, and an equilibration buffer (box (729)) in the second column. If you move to the right, the secondary load of the mixture from T2 to T3 may be in the second column (Box (720)), and the primary load of the mixture from T3 to T4 may be in the second column (Box (722)).

[0066] In the next column (representing column C3), one or more mobile phases from T1 to T3 may be present in the third column (box (734)). One or more mobile phases (734) in the third column may be separated into individual phases including a wash buffer (box (735)) in the third column, a stripping buffer (box (737)) in the third column, and an equilibration buffer (box (739)) in the third column. Subsequently, from T3 to T4, a secondary load may be present in the third column (box (730)), and from T4 to T5, a primary load of the mixture may be present in the third column (box (732)).

[0067] In the next column (representing column C4), the primary load of the mixture from T1 to T2 may be in the fourth column (box (742)). Subsequently, from T2 to T4, one or more mobile phases may be in the fourth column (box (744)), and from T4 to T5, the secondary load of the mixture may be in the third column (box (740)). One or more mobile phases (744) in the fourth column may be divided into individual phases including a wash buffer (box (745)) in the third column, a stripping buffer (box (747)) in the third column, and an equilibration buffer (box (749)) in the third column.

[0068] At the provided time T0, a vertical line can be graphed so that each numbered box contacted by the vertical line from T0 represents the solution in the column at that time. Thus, for example, at time T1, a secondary load mixture is introduced into the first column (710), one or more mobile phases are present in the second column (724), for example, stripping buffer (727), and the primary load mixture is passed into the third column (732). It should be noted that while subdivisions of a broader phase, for example, subdivisions (725, 727, and 729), are shown to occupy the same portion of one or more mobile phases in the second column (724), in some embodiments, the subdivisions may occupy different portions of the broader phase. It should also be understood that the method illustrated in FIG. 5a is only one example of the method of the embodiment. Other sequences, arrangements, and steps are thought to be within the scope of the disclosure of the present invention.

[0069] FIGS. 5b-5e illustrate exemplary cycles for a method of preparing a target polypeptide from a mixture containing a previously described target polypeptide. FIG. 5b illustrates a series of events occurring during the interval between T1 and T2 of FIG. 5a. FIG. 5b illustrates an HIC apparatus in the first stage (601), where the first region (610) receives a secondary load (606) of a mixture containing a target polypeptide and elutes an effluent (607) of the secondary load that can be collected or discarded. The second region (620) receives one or more mobile phases (615) and elutes an effluent (616) of one or more mobile phases that can be collected or discarded. The third region (630) receives one or more mobile phases (615) and elutes an effluent (616) of one or more mobile phases that can be collected or discarded. The fourth section (640) receives the primary load (605) of the mixture and passes the secondary load (606) of the mixture to another column.

[0070] FIG. 5c illustrates an HIC apparatus in the second stage (602) (over intervals T2 to T3, as shown in FIG. 5a), where the first region (610) receives a primary load (605) of the mixture and passes a secondary load (606) of the mixture to another column containing a target polypeptide that can be collected or discarded. The second region (620) receives a secondary load (606) of the mixture containing the target polypeptide and elutes an effluent (607) of the secondary load that can be collected or discarded. The third region (630) receives one or more mobile phases (615) and elutes an effluent (616) of one or more mobile phases that can be collected or discarded. The fourth region (640) receives one or more mobile phases (615) and elutes an effluent (616) of one or more mobile phases that can be collected or discarded.

[0071] FIG. 5d illustrates an HIC device (over intervals T2 to T3, as shown in FIG. 5a) in the third stage (603), wherein the first region (610) receives one or more mobile phases (615) and elutes the effluent (616) of one or more mobile phases which may be collected or discarded. The second region (620) receives the primary load (605) of the mixture and passes the secondary load (606) of the mixture to another column containing a target polypeptide which may be collected or discarded. The third region (630) receives the secondary load (606) of the mixture containing the target polypeptide and elutes the effluent (607) of the secondary load which may be collected or discarded. The fourth area (640) accommodates one or more mobile phases (615) and elutes one or more mobile phase effluents (616) that can be collected or disposed of.

[0072] FIG. 5e illustrates an HIC apparatus in the fourth stage (604) (over intervals T2 to T3, as shown in FIG. 5a), wherein the first region (610) receives one or more mobile phases (615) and elutes one or more effluents (616) of the mobile phases that may be collected or discarded. The second region (620) receives one or more mobile phases (615) and elutes one or more effluents (616) of the mobile phases that may be collected or discarded. The third region (630) receives a primary load (605) of the mixture and passes a secondary load (606) of the mixture to another column containing a target polypeptide that may be collected or discarded. The fourth region (640) receives a secondary load (606) of the mixture containing the target polypeptide and elutes an effluent (607) of the secondary load that may be collected or discarded.

[0073] FIG. 6 illustrates a flowchart of an exemplary method (800) for preparing a target polypeptide from a mixture containing a target polypeptide. The method may include passing the mixture containing the target polypeptide through the first column of a plurality of columns (e.g., box (410) in FIG. 3a) (step 810). The method may further include passing the effluent containing the target polypeptide from the first column to the second column of the plurality of columns (e.g., second load (306) of the mixture, as shown in FIG. 3b) (step 820). The method may further include passing one or more mobile phases through the first column (e.g., box (414)) (step 830). In some embodiments, the method may further include passing the target polypeptide through the outlet of each of the plurality of columns (e.g., effluent (316) of one or more mobile phases, as shown in FIG. 3b-3d) (step 840). It is obvious to a person skilled in the art that, while comparing with Figs. 3a-3d, it can also be compared with Figs. 5a-5e.

[0074] In an embodiment of the disclosure of the present invention, a mixture containing a target polypeptide may also contain one or more HCPs. After preparing the target polypeptide from a mixture using the method of one or more embodiments, several effluent samples may be obtained. Samples may be collected from the effluent of one or more loads of the mixture and / or from the effluent of one or more mobile phases. For example, samples may be collected from the effluent of a primary load of the mixture or a secondary load of the mixture (or any other load of the mixture). In some embodiments, samples may be collected from the effluent of only one or more wash buffers. In other embodiments, samples may be collected from the effluent of other mobile phases and / or from the primary or secondary loads of the mixture. An aggregate collection of all collected samples containing the target polypeptide is referred to as a pool.

[0075] In some embodiments, one or more measurements may be collected to determine the efficiency of the method used to produce the target polypeptide. As used in the disclosure herein, efficiency refers to a combination of the following three different factors: high molecular weight molecular clearance factor (HMWCF), yield, and productivity. In some embodiments, a more efficient method has a higher HMWCF, a higher yield, and a higher productivity than a less efficient method. In other embodiments, a more efficient method has a higher productivity than a less efficient method, while simultaneously maintaining an HMWCF of 1.3 or higher and a yield of 80% or higher. In further embodiments, a more efficient method has a higher productivity than a less efficient method, while simultaneously maintaining an HMWCF of 1.5 or higher and a yield of 90% or higher.

[0076] The High molecular weight molecule clearance factor (HMW CF) is an approximation of the relative protein content in the collected pool compared to the loaded mixture. In some embodiments, analytical size exclusion chromatography may be performed to measure the percentage of samples (e.g., proteins) that produce high molecular weight molecules (HMW%). In other embodiments, centrifugation techniques may be used—when the sample is centrifuged, it is separated into strata based on the mass of the sample's components, and the heaviest stratum, or infranatant, contains the heaviest molecules, typically including proteins. HMW% can be calculated by weighing the mass of the precipitate of the centrifuged sample and dividing it by the total mass of the sample. Using either method, the HMW CF can be calculated according to Equation 1 shown below.

[0077] Mathematical formula (1)

[0078]

[0079] As shown in mathematical formula (1), HMW CF can be calculated by dividing the HMW% of the loaded mixture by the HMW% of the pool. In some embodiments, the method for producing a target polypeptide from a mixture has an HMW CF of 1.3 or more. In other embodiments, the method for producing a target polypeptide from a mixture has an HMW CF of 1.4 or more, 1.5 or more, 1.6 or more, 1.8 or more, or 2.0 or more.

[0080] Yield is a measure of the amount of target polypeptide collected from a pool, compared to how much target polypeptide is present in the loading mixture. The amount of target polypeptide in a sample can be quantified by UV absorption, electrical conductivity, or enzyme immunoassay (e.g., ELISA). Yield can be calculated according to Equation 2 shown below.

[0081] Mathematical formula (2)

[0082]

[0083] As shown in Equation 2, the yield can be calculated by dividing the mass of the target polypeptide loaded into the HIC device by the mass of the target polypeptide collected in the pool. Since the mass of the target polypeptide in the sample cannot be measured directly, the mass can be measured by multiplying the concentration (calculated by UV absorption, electrical conductivity, or enzyme immunoassay) by the volume. In some embodiments, the method for preparing the target polypeptide from the mixture has a yield of 55% or more. In other embodiments, the method for preparing the target polypeptide from the mixture has a yield of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0084] Productivity is the quantification of the time and cost required to produce an amount of target polypeptide. Productivity can be calculated according to Equation 3 as shown below.

[0085] Mathematical formula (3)

[0086]

[0087] As shown in Equation 3, productivity can be calculated by dividing the mass of the target polypeptide collected from the pool by the product of the volume of the hydrophobic interaction medium used and the time elapsed to produce the mass of the polypeptide collected from the pool (e.g., cycle time). In some embodiments, the method for producing the target polypeptide from the mixture has a productivity of about 35 g / L·hr or more. In other embodiments, the method for producing the target polypeptide from the mixture has a productivity of about 40 g / L·hr or more, about 50 g / L·hr or more, about 75 g / L·hr or more, about 100 g / L·hr or more, about 125 g / L·hr or more, about 150 g / L·hr or more, about 175 g / L·hr or more, about 200 g / L·hr or more, or about 220 g / L·hr or more.

[0088] Examples

[0089] The following examples are intended to illustrate the disclosure of the invention without actually limiting it. It should be understood that the disclosure of the invention includes additional aspects or embodiments consistent with the above detailed description and the following examples.

[0090] In the following examples, a target polypeptide was prepared from a mixture containing the target polypeptide and HCP. The target polypeptide was prepared using three different methods according to embodiments of the disclosure of the present invention. As a comparative example, the target polypeptide was also prepared using a conventional batch processing method.

[0091] Example 1

[0092] In the first example, a target antibody was prepared. A mixture containing 300 mL of 12.2 g / L target antibody was loaded into a three-column HIC apparatus at a loading flow rate of 1.67 mL / min, where each column had a bed height of 2.5 cm, an internal diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mmol (mM) solution of sodium citrate and was adjusted to a pH of 6.0 with a 2 M acetic acid solution. The loading buffer / mixture was loaded into the first and second columns of the three columns. The second column was loaded via the first column (i.e., the outlet from the first column allowed the loading buffer to pass into the second column). After loading the mixture into the first and second columns of the HIC device, the mixture was loaded into the second and third columns of the three columns, and the third column was loaded via the second column (i.e., the outlet from the second column allowed the loading buffer to pass into the third column).

[0093] While loading the loading buffer / mixture into the second and third columns, a series of mobile phases were passed through the first column of the HIC device to separate the target antibody from other components of the mixture in the first column and collect the target antibody, and subsequently, a series of stripping buffers were passed through the first column to regenerate the column. Washing and stripping of the first column occurred simultaneously during the loading of the second and third columns. After this step, the loading buffer / mixture was loaded into the third and first columns, and the first column was loaded via the third column (i.e., the outlet from the third column allowed the loading buffer to pass into the first column), while the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded in the second column, and subsequently, a series of stripping buffers were passed through the second column to regenerate the second column. Washing and stripping of the second column occurred simultaneously during the loading of the third and first columns. Finally, the loading buffer / mixture was reloaded into the first and second columns, and the second column was loaded via the first column as previously described, while the buffer was passed through the third column to separate and collect the target antibody from the mixture loaded in the third column, and subsequently, a series of stripping buffers were passed through the third column to regenerate the third column. Washing and stripping of the third column occurred simultaneously with the loading of the first and second columns. This process was repeated periodically twice.

[0094] The series of mobile phases included a wash buffer, a series of stripping buffers, and an equilibration buffer. The wash buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0. To wash each column, four column volumes of wash buffer were added to the column.

[0095] After applying wash buffer and collecting the effluent containing the target antibody from the column as part of the pool, a series of stripping buffers were passed through the column as part of the column regeneration process. The first stripping buffer contained deionized water, and two column volumes of this buffer were added to each column. The column volumes used herein refer to the volume of liquid that the provided column can hold. The next stripping buffer contained 1 N NaOH, and two column volumes of this buffer were added to each column after the first stripping buffer. The next stripping buffer contained deionized water, and two column volumes of this buffer were added to each column after the previous alkaline stripping buffer. The next stripping buffer contained 20 vol.% ethanol, and two column volumes of this buffer were added to each column after the previous deionized water stripping buffer. A final stripping buffer containing deionized water was added to the column (in an amount equal to two column volumes). After applying the stripping buffer, four column volumes of equilibration buffer were added to the column. The equilibration buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0.

[0096] After collecting the pool from the method carried out in Example 1, the HMW CF, yield, and productivity of the method were measured and calculated as described herein. The results are summarized in Table 1 below.

[0097] Example 2

[0098] In the second example, a target antibody was prepared. A mixture containing 729 mL of 12.4 g / L target polypeptide was loaded into a three-column HIC apparatus at a loading flow rate of 1.67 mL / min, where each column had a bed height of 2.5 cm, an internal diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mmol (mM) solution of sodium citrate and was adjusted to a pH of 6.0 with a 2 M acetic acid solution. The loading buffer / mixture was loaded into the first and second columns of the three columns. The second column was loaded via the first column (i.e., the outlet from the first column allowed the loading buffer to pass into the second column). After loading the mixture into the first and second columns of the HIC device, the mixture was loaded into the second and third columns of the three columns, and the third column was loaded via the second column (i.e., the outlet from the second column allowed the loading buffer to pass into the third column).

[0099] While loading the loading buffer / mixture into the second and third columns, a series of mobile phases were passed through the first column of the HIC device to separate the target antibody from other components of the mixture in the first column and collect the target antibody, and subsequently, a series of stripping buffers were passed through the first column to regenerate the column. Washing and stripping of the first column occurred simultaneously during the loading of the second and third columns. After this step, the loading buffer / mixture was loaded into the third and first columns, and the first column was loaded via the third column (i.e., the outlet from the third column allowed the loading buffer to pass into the first column), while the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded in the second column, and subsequently, a series of stripping buffers were passed through the second column to regenerate the second column. Washing and stripping of the second column occurred simultaneously during the loading of the third and first columns. Finally, the loading buffer / mixture was reloaded into the first and second columns, and the second column was loaded via the first column as previously described, while the buffer was passed through the third column to separate and collect the target antibody from the mixture loaded in the third column, and subsequently, a series of stripping buffers were passed through the third column to regenerate the third column. Washing and stripping of the third column occurred simultaneously with the loading of the first and second columns. This process was repeated periodically four times.

[0100] The series of mobile phases included a wash buffer, a series of stripping buffers, and an equilibration buffer. The wash buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0. To wash each column, four column volumes of wash buffer were added to the column.

[0101] After applying wash buffer and collecting the effluent containing the target antibody from the column as part of the pool, a series of stripping buffers were passed through the column as part of the column regeneration process. The first stripping buffer contained deionized water, and two column volumes of this buffer were added to each column. The column volumes used herein refer to the volume of liquid that the provided column can hold. The next stripping buffer contained 1 N NaOH, and two column volumes of this buffer were added to each column after the first stripping buffer. The next stripping buffer contained deionized water, and two column volumes of this buffer were added to each column after the previous alkaline stripping buffer. The next stripping buffer contained 20 vol.% ethanol, and two column volumes of this buffer were added to each column after the previous deionized water stripping buffer. A final stripping buffer containing deionized water was added to the column (in an amount equal to two column volumes). After applying the stripping buffer, four column volumes of equilibration buffer were added to the column. The equilibration buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0.

[0102] After collecting the pool from the method carried out in Example 2, the HMW CF, yield, and productivity were measured and calculated as described herein. The results are summarized in Table 1 below.

[0103] Example 3

[0104] In the third example, a target polypeptide was prepared. A mixture containing 726 mL of 12.4 g / L target polypeptide was loaded into a three-column HIC apparatus at a loading flow rate of 6.70 mL / min, where each column had a bed height of 2.5 cm, an internal diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mmol (mM) solution of sodium citrate and was adjusted to a pH of 6.0 with a 2 M acetic acid solution. The loading buffer / mixture was loaded into the first and second columns of the three columns. The second column was loaded via the first column (i.e., the outlet from the first column allowed the loading buffer to pass into the second column). After loading the mixture into the first and second columns of the HIC device, the mixture was loaded into the second and third columns of the three columns, and the third column was loaded via the second column (i.e., the outlet from the second column allowed the loading buffer to pass into the third column).

[0105] While loading the loading buffer / mixture into the second and third columns, a series of mobile phases were passed through the first column of the HIC device to separate the target antibody from other components of the mixture in the first column and collect the target antibody, and subsequently, a series of stripping buffers were passed through the first column to regenerate the column. Washing and stripping of the first column occurred simultaneously during the loading of the second and third columns. After this step, the loading buffer / mixture was loaded into the third and first columns, and the first column was loaded via the third column (i.e., the outlet from the third column allowed the loading buffer to pass into the first column), while the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded in the second column, and subsequently, a series of stripping buffers were passed through the second column to regenerate the second column. Washing and stripping of the second column occurred simultaneously during the loading of the third and first columns. Finally, the loading buffer / mixture was reloaded into the first and second columns, and the second column was loaded via the first column as previously described, while the buffer was passed through the third column to separate and collect the target antibody from the mixture loaded in the third column, and subsequently, a series of stripping buffers were passed through the third column to regenerate the third column. Washing and stripping of the third column occurred simultaneously with the loading of the first and second columns. This process was repeated periodically four times.

[0106] The series of mobile phases included a wash buffer, a series of stripping buffers, and an equilibration buffer. The wash buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0. To wash each column, four column volumes of wash buffer were added to the column.

[0107] After applying wash buffer and collecting the effluent containing the target antibody from the column as part of the pool, a series of stripping buffers were passed through the column as part of the column regeneration process. The first stripping buffer contained deionized water, and two column volumes of this buffer were added to each column. The column volumes used herein refer to the volume of liquid that the provided column can hold. The next stripping buffer contained 1 N NaOH, and two column volumes of this buffer were added to each column after the first stripping buffer. The next stripping buffer contained deionized water, and two column volumes of this buffer were added to each column after the previous alkaline stripping buffer. The next stripping buffer contained 20 vol.% ethanol, and two column volumes of this buffer were added to each column after the previous deionized water stripping buffer. A final stripping buffer containing deionized water was added to the column (in an amount equal to two column volumes). After applying the stripping buffer, four column volumes of equilibration buffer were added to the column. The equilibration buffer contained 40 mM Tris and 30 mM sodium citrate and was adjusted to a pH of 6.0.

[0108] After collecting the pool from the method carried out in Example 3, the HMW CF, yield, and productivity of the method were measured and calculated as described herein. The results are summarized in Table 1 below.

[0109] Comparative Examples

[0110] To compare the target polypeptide with the methods of Examples 1-3, it was prepared from a mixture using the conventional batch process described herein. The loading additive, washing buffer, stripping buffer, and equilibration buffer were the same as those used in the example methods, but the conventional batch methodology was used. 590 g of the 13.1 g / L loading mixture was added to a chromatography column. After passing the mixture through the column, 4 column volumes of washing buffer were added to the column, and the effluent was collected. After collecting the pool from the comparative example method, the HMW CF, yield, and productivity were characterized. The results are summarized in Table 1.

[0111]

[0112] As can be seen from the data in Table 1, Examples 2 and 3 have higher productivity than the batch method of the comparative examples. Additionally, Example 3 can achieve higher productivity than the other examples, yet still maintain an HMW CF of 1.5 or higher and a yield of 90% or higher.

[0113] Example 4

[0114] Target antibodies can be prepared using HIC at three different loading rates to compare impurity breakthroughs at variable rates. The columns were prepared as described in Table 2:

[0115]

[0116] The loading rates in the above sequence runs were 300 cm / h (3.93 mL / min, or a retention time in a 4.0 min column), 200 cm / hr (2.62 mL / min, or a retention time in a 6.0 min column), and 400 cm / hr (5.24 mL / min, or a retention time in a 3.0 min column). All runs were performed on the same column. Immersion was performed overnight in 0.5 N NaOH prior to the 400 cm / hr run.

[0117] High molecular weight percentages (HMW%) were plotted as a function of loading, as shown in Fig. 7a. The HMW% of the loaded material was 1.78%. The cumulative pool HMW% at 200 g / L resin and 400 g / L resin are shown in Table 3 below:

[0118]

[0119] Host cell proteins were quantified in parts per million for each loading rate using the F665 CHO HCP ELISA kit (Cygnus Technologies). The obtained amounts were plotted as a function of loading as shown in Fig. 7b. For comparison, host cell proteins were quantified from an anion exchange chromatography pool of the same loading material and were found to be present at 549.61 ppm.

[0120] Example 5

[0121] Target antibodies were prepared using HIC in two columns with different bed heights (20 cm and 2.5 cm as used in Example 4). Operations of both were performed such that the residence time in each column was 3 minutes (i.e., a linear rate of 400 cm / hr in the 20 cm bed height column). The HMW% for the loading material was 2.2%. The HMW% for the pool in each column was plotted as a function of loading concentration as shown in Fig. 7c.

[0122] Those skilled in the art will recognize that concepts based on the present disclosure can be readily used as a basis for designing other methods and systems to achieve the solutions and objectives of the disclosed invention. Accordingly, the claims should not be considered limited by the foregoing detailed description.

Claims

Claim 1 A method for preparing a target polypeptide from a mixture containing a target polypeptide, the method comprising: a step of contacting the mixture containing the target polypeptide with a first region of a hydrophobic interaction chromatography (HIC) apparatus, wherein the first region has one to four chromatographic columns, each chromatographic column having an outlet; a step of passing an effluent containing the target polypeptide from the first region of the HIC apparatus to a second region of the HIC apparatus; a step of contacting one or more mobile phases with a second region of the HIC apparatus, wherein the second region has one to four chromatographic columns, each chromatographic column having an outlet, and the one or more mobile phases comprising an equilibration buffer and a wash buffer; A method comprising: a step of passing a target polypeptide through an outlet of a first region of an HIC device, wherein the outlet of the first region of the HIC device is fluidly connected to either or both of a second region of the HIC device and outside the HIC device; and a step of passing a target polypeptide through an outlet of a second region of the HIC device, wherein the outlet of the second region of the HIC device is fluidly connected to either or both of another chromatography column of the HIC device and outside the HIC device; wherein each step of the method is performed simultaneously or sequentially, and the sum of the residence time of a mixture containing the target polypeptide in the first region and the residence time of a mixture containing the target polypeptide in the second region is configured to be within a variation of + / - 10% of the residence time of one or more mobile phases in the second region. Claim 2 A method according to claim 1, wherein the target polypeptide is a monoclonal antibody. Claim 3 delete Claim 4 A method according to claim 1, wherein the step of contacting one or more mobile phases with a second region of the HIC device comprises: contacting a washing buffer with a second region of the HIC device; and regenerating the second region after contacting the washing buffer with a second region of the HIC device, wherein regenerating the second region comprises: contacting water with a second region of the HIC device, contacting an alkaline solution with a second region of the HIC device, contacting an alcohol solution with a second region of the HIC device, and contacting an equilibration buffer with a second region of the HIC device. Claim 5 A method according to claim 4, wherein, after contacting the washing buffer with the second region of the HIC device, the target polypeptide is subsequently passed through the outlet of the second region of the HIC device. Claim 6 A method according to claim 1, wherein one or more of ultraviolet absorption, electrical conductivity, or pH of the retained solution are measured at the outlet of either the first region or the second region. Claim 7 A method according to claim 1, wherein the target polypeptide is produced with a productivity of 50 g / L·hr or more. Claim 8 A method according to claim 1, wherein the first region or the second region comprises 2 to 4 chromatography columns. Claim 9 The method of claim 1, wherein the HIC device further comprises a third region having 1 to 4 chromatographic columns, each of which comprises an outlet, and the method further comprises: performing a regeneration cycle in the third region, wherein performing the regeneration cycle comprises contacting one or more mobile phases in the third region and passing an effluent containing a target polypeptide from the second region of the HIC device to the third region of the HIC device, and the duration of the regeneration cycle is configured to be within a variation of + / - 10% of the residence time for a mixture containing a target polypeptide in the first region. Claim 10 A method for preparing a target polypeptide from a mixture containing a target polypeptide, the method comprising: a step of passing the mixture containing the target polypeptide through a first column of a plurality of chromatographic columns in a hydrophobic interaction chromatography (HIC) apparatus, wherein each of the plurality of chromatographic columns comprises: a first inlet fluidically connected to the outside of the HIC apparatus; a first outlet fluidly connected to a second inlet of another column of the plurality of chromatographic columns; and a second outlet fluidly connected to the outside of the HIC apparatus; a step of passing the effluent containing the target polypeptide through the first column of the plurality of chromatographic columns to the second column; a step of passing one or more mobile phases through a third column of the plurality of chromatographic columns, wherein the one or more mobile phases comprise an equilibration buffer and a washing buffer; and a step of passing the target polypeptide through the first outlet or the second outlet of each of the plurality of columns; A method wherein each step of the above method is performed simultaneously or sequentially, wherein the sum of the retention times of the mixture containing the target polypeptide in the first column and the second column is within a variation of + / - 10% of the sum of the retention times of one or more mobile phases in the third column. Claim 11 A method according to claim 10, further comprising the step of passing one or more mobile phases through each of the plurality of columns. Claim 12 A method according to claim 10, wherein passing one or more mobile phases through a column comprises: passing a washing buffer through the column; and regenerating the column after passing the washing buffer through the column, wherein regenerating the column comprises passing water, an alkaline solution, an alcohol solution, or an equilibration buffer through the column. Claim 13 A method according to claim 12, wherein the step of passing the target polypeptide through the first or second outlet of each of the first column, second column, and third column occurs after the washing buffer has passed through the column. Claim 14 A method according to claim 10, wherein one or more of ultraviolet absorption, electrical conductivity, or pH of the retained solution are measured at the outlet of either the first column or the second column. Claim 15 A method according to claim 10, wherein the target polypeptide is produced with a productivity of 50 g / L·hr or more. Claim 16 A method according to claim 12, wherein the HIC device comprises four columns, and the sum of the retention times of the mixture containing the target polypeptide in the first and second columns is within a variation of + / - 10% of the sum of the regeneration times of the third and fourth columns. Claim 17 A method for preparing an antibody using a plurality of chromatography columns, wherein each of the plurality of chromatography columns comprises a hydrophobic interaction medium, and the method comprises: in a first step: loading a quantity of a mixture containing the antibody into a first column of the plurality of columns; loading the quantity of the mixture into a second column of the plurality of columns via the first column; and performing a non-loading step in a third column of the plurality of columns, comprising at least one of washing, stripping, and equilibration processes; in a second step: loading a quantity of a mixture containing the antibody into the second column; loading the quantity of the mixture into a third column via the second column; and performing a non-loading step in a first column, comprising at least one of washing, stripping, and equilibration processes; and in a third step: loading a quantity of a mixture containing the antibody into a third column; and loading the quantity of the mixture into a first column via the third column. A method comprising performing a non-loading step in the second column including at least one of a washing, stripping, and equilibration process, wherein each of the stages comprises performing the loading and non-loading steps simultaneously, and wherein the duration of the loading step in each of the first, second, and third columns is configured to be within a variation of + / - 10% of the duration of the non-loading step in each of the first, second, and third columns. Claim 18 A method according to claim 17, further comprising periodically repeating the first, second, and third stages in succession. Claim 19 delete Claim 20 delete

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