Use of multiple hydrophobic interaction chromatography for preparing polypeptides from a mixture

Abstract

The methods and systems disclosed herein may improve the efficiency and / or productivity of polypeptide preparation methods. [Solution] Embodiments of the present disclosure relate to a method for preparing a target polypeptide from a mixture containing the target polypeptide. The method involves contacting the mixture with a hydrophobic interaction chromatography (HIC) apparatus having a plurality of chromatography zones. The method may further include passing the target polypeptide through the outlets of at least a first zone and a second zone of the HIC apparatus. The residence time of the mixture containing the target polypeptide in the first zone may be substantially the same as the residence time of one or more mobile phases in the second zone.

Description

Technical Field

[0001] The present disclosure generally relates to methods for preparing polypeptides. More specifically, the present disclosure relates to methods for preparing polypeptides from mixtures using chromatography methods.

Background Art

[0002] Chromatographies such as hydrophobic interaction chromatography (HIC) and affinity chromatography can be implemented as part of a pharmaceutical preparation process. In some cases, chromatography can be particularly useful for the preparation of pharmaceuticals containing polypeptides. However, the equipment, materials, preparation time, and implementation time for standard batch HIC steps or other batch chromatography steps can add to the cost of the pharmaceutical preparation process or reduce its efficiency. Specifically, the time required to perform each step of the HIC or other chromatography separation process, the amount of buffer and / or separation medium used, and any aspect of the process that is not automated can reduce the efficiency of pharmaceutical preparation.

[0003] The methods and systems disclosed herein can improve the efficiency and / or productivity of polypeptide preparation methods. The methods and systems disclosed herein can also improve the efficiency and / or productivity of pharmaceutical preparation methods and can address one or more of the problems identified above.

Summary of the Invention

[0004] Embodiments of the present disclosure may relate to a method for preparing a target polypeptide from a mixture containing the target polypeptide. This method may include contacting the mixture containing the target polypeptide with a first zone of a HIC apparatus, contacting a mobile phase with a second zone of the HIC apparatus, and passing the target polypeptide through the outlets of the first and second zones of the HIC apparatus, where each of the first and second zones may have one or more chromatography columns and outlets. In some embodiments, the residence time of the mixture containing the target polypeptide in the first zone may be configured to be substantially the same as the residence time of the mobile phase in the second zone.

[0005] In some embodiments, the target polypeptide may be a monoclonal antibody. The target polypeptide can be prepared with a productivity of 50 g / L·hour or higher. Alternatively, the mobile phase may further include an equilibration buffer and a wash buffer. In some embodiments, the method of the present disclosure may further include passing the effluent containing the target polypeptide from a first zone of the HIC apparatus to a second zone of the HIC apparatus. In some embodiments, contacting the mobile phase with the second zone of the HIC apparatus may include contacting the wash buffer with the second zone of the HIC apparatus, and regenerating the second zone after contacting the wash buffer with the second zone of the HIC apparatus. In some embodiments, regenerating the second zone may include contacting water with the second zone of the HIC apparatus, contacting an alkaline solution with the second zone of the HIC apparatus, contacting an alcohol solution with the second zone of the HIC apparatus, and contacting the equilibration buffer with the second zone of the HIC apparatus. After contacting the wash buffer with the second zone of the HIC apparatus, the target polypeptide can be passed through the outlet of the second zone of the HIC apparatus. In some embodiments, one or more of the ultraviolet absorptivity, electrical conductivity, or pH of the retained solution can be measured at the outlet of either the first or second zone. The first or second zone may include a plurality of chromatography columns. In some embodiments, the HIC apparatus may further include a third zone having chromatography columns and an outlet. In some embodiments, the method may further include performing a regeneration cycle in the third zone, which includes contacting the mobile phase with the third zone, and the duration of the regeneration cycle is configured to be substantially the same as the residence time of the mixture containing the target polypeptide in the first zone.

[0006] In some embodiments of the present disclosure, a method for preparing a target polypeptide from a mixture containing the target polypeptide may include passing the mixture containing the target polypeptide through a first column of a plurality of chromatography columns of an HIC apparatus, passing the effluent containing the target polypeptide from the first column through a second 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 the outlets 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 residence times of the mixture containing the target polypeptide in the first and second columns is substantially the same as the sum of the residence times of the mobile phases in the third column.

[0007] In some embodiments, this method may further include passing one or more mobile phases through each of a plurality of columns. In some embodiments, passing one or more mobile phases through the columns may include passing a wash buffer through the columns and, after passing the wash buffer through the columns, regenerating the columns, where regeneration of the columns includes 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 column may occur after passing the wash buffer through the columns. In some embodiments, one or more of the ultraviolet absorptivity, electrical conductivity, or pH of the retained solution is measured at the outlet of either the first or second column. In some embodiments, this method may include preparing the target polypeptide with a productivity of 50 g / L·hour or more. In further embodiments, the HIC apparatus may include four columns, and the sum of the residence times of the mixture containing the target polypeptide in the first and second columns may be substantially the same as the sum of the regeneration times of the third and fourth columns.

[0008] Further embodiments of the present disclosure may include a method for preparing an antibody using a plurality of chromatography columns, each of which comprises a hydrophobic interacting medium. The method may include, in a first step, loading a certain amount of a mixture containing the antibody onto a first column of the plurality of columns, loading a certain amount of the mixture onto a second column of the plurality of columns via the first column, and performing a non-loading step on a third column of the plurality of columns, comprising at least one of the washing, stripping, and equilibration processes; in a second step, loading a certain amount of a mixture containing the antibody onto a second column, loading a certain amount of the mixture onto a third column via the second column, and performing a non-loading step on the first column, comprising at least one of the washing, stripping, and equilibration processes; and in a third step, loading a certain amount of a mixture containing the antibody onto a third column, loading a certain amount of the mixture onto a third column via the second column, and performing a non-loading step on the second column, comprising at least one of the washing, stripping, and equilibration processes.

[0009] In some embodiments, the method may further include cyclically repeating the first, second, and third stages, each stage comprising simultaneously performing a load step and a non-load step. In some embodiments, one period of the load step is configured to be substantially the same as the period of the non-load step. [Brief explanation of the drawing]

[0010] The accompanying drawings incorporated herein and constituting part thereof illustrate various exemplary embodiments and, together with the specification, serve to illustrate the principles of the disclosed embodiments. Any feature of the embodiments or examples described herein (e.g., compositions, formulations, methods, etc.) can be combined with any other embodiments or examples, and all such combinations are incorporated herein. Furthermore, the systems and methods described herein are not limited to a single aspect or embodiment thereof, or any combination or permutation of such aspects and embodiments. For brevity, certain permutations and combinations are not discussed and / or illustrated herein separately. [Figure 1] This is a schematic diagram showing a portion of the zones of a chromatography apparatus according to some embodiments of the present disclosure. [Figure 2] This is a schematic diagram of a chromatography apparatus according to some embodiments of the present disclosure. [Figure 3A] This graph illustrates exemplary methods for preparing a target polypeptide according to several embodiments of the present disclosure. [Figure 3B] Figure 3A is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 3C] Figure 3A is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 3D] Figure 3A is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 4] This is a schematic diagram of a chromatography apparatus according to some embodiments of the present disclosure. [Figure 5A] This graph illustrates exemplary methods for preparing a target polypeptide according to some embodiments of the present disclosure. [Figure 5B] As shown in Figure 5A, this is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 5C] As shown in Figure 5A, this is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 5D]As shown in Figure 5A, this is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 5E] As shown in Figure 5A, this is a simplified diagram illustrating a method for preparing the target polypeptide. [Figure 6] This is a flowchart of a method for preparing a target polypeptide according to some embodiments of the present disclosure. [Figure 7A] This is a plot of the percentage of high molecular weight as a function of load, according to one aspect of the present disclosure. [Figure 7B] This is a plot of host cell protein quantity as a function of load, according to one aspect of the present disclosure. [Figure 7C] This is a plot of the percentage of high molecular weight as a function of the load concentration, according to one aspect of the present disclosure. [Figure 8A] This is a plot of productivity as a function of the number of chromatography columns, according to one aspect of the present disclosure. [Figure 8B] This is a plot of productivity as a function of the number of chromatography columns, according to one aspect of the present disclosure. [Modes for carrying out the invention]

[0011] Where used herein, “includes” or any other inflection is intended to include non-exclusive inclusion; that is, a process, method, article, or apparatus containing a list of elements may include not only those elements but also other elements not explicitly listed, or other elements specific to such process, method, article, or apparatus. The term “exemplary” is used in the sense of “example” and not “ideal.” The terms “for example” and “such as,” and their grammatical equivalents, are understood to be followed by the phrase “but not limited to,” unless otherwise specified.

[0012] As used herein, the term "about" means to account for variations due to experimental error. When applied to a numerical value, the term "about" may indicate a variation of + / - 10% from the disclosed numerical value (when no different variation is specified). As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0013] Note that all numerical values disclosed herein (including all disclosed values, upper and lower limits, and ranges) may have a variation of + / - 10% from the disclosed numerical value (unless a different variation is specified). Further, in the claims, a value, upper and lower limits, and / or a range mean that value, upper and lower limits, and / or that range + / - 10%. Similarly, the phrase "substantially the same" as used herein may mean equivalent within a variation of + / - 10%. Further, all ranges are understood to include their endpoints, for example, from 1 centimeter (cm) to 5 cm may include lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0014] [Detailed Description] The present disclosure is not limited to the specific compositions, formulations, material manufacturers, pharmaceuticals, devices, systems, experimental conditions, or particular methods disclosed herein, as many modifications are possible within the skill of those in the art. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but the specific methods are described herein. All publications mentioned are incorporated herein by reference.

[0016] As used herein, the term "contacting" refers to the touching, joining, interfacing, or other physical interaction between two or more surfaces, solutions, or compounds. A particular fluid may be described herein as passing through, passing from, passing into, or causing to pass through a region, but it is understood that the fluid necessarily contacts any region through which it passes. Similarly, introducing a fluid or component into a region will constitute the fluid or component contacting that region.

[0017] As used herein, the term "polypeptide" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. Proteins include one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide can be a protein, and a protein can include multiple polypeptides that form a single functional biomolecule.

[0018] Post-translational modifications can further modify or alter the structure of a polypeptide. For example, disulfide bridges (e.g., S-S bonds between cysteine residues) can be present in some proteins. Some disulfide bridges are essential for the proper structure, function, and interactions of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to the formation of disulfide bonds, proteins can undergo other post-translational modifications. These modifications include lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of glycosyl groups to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface-to-surface interactions in which the polypeptide is involved.

[0019] 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, and the like. The target protein (POI) may include any polypeptide or protein that is desired to be isolated, purified, or otherwise prepared. The POI may include the target polypeptide or other polypeptides, including antibodies produced by cells.

[0020] As used herein, the term “antibody” includes immunoglobulins, which consist of four polypeptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Typically, antibodies have a molecular weight greater than 100 kDa, for example, 130 kDa to 200 kDa, for example, about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs are sometimes abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs as LCDR1, LCDR2, and LCDR3).

[0021] For example, a class of immunoglobulins called immunoglobulin G (IgG) is common in human serum and consists of four polypeptide chains (two light chains and two heavy chains). Each light chain is linked to one heavy chain via a cystine disulfide bond, and the two heavy chains are linked to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass has a different constant region and, as a result, may have different effector functions. In some embodiments described herein, the POI may include a target polypeptide containing IgG. In at least one embodiment, the target polypeptide contains IgG4.

[0022] As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, which include, for example, manipulating and expressing DNA encoding an antibody variable domain and, optionally, a constant domain. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biological techniques for purposes such as arranging one or more variable and / or constant domains into a suitable configuration, introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids.

[0023] Target polypeptides can be produced using recombinant cell-based production systems such as insect baculovirus lines, yeast lines (e.g., Pichia species), and mammalian lines (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). The term "cell" includes any cell suitable for expressing recombinant nucleic acid sequences. Cells include prokaryotes and eukaryotes (single or multiple cells), bacterial cells (e.g., strains of Escherichia coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusiani, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotes and are 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, 293EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells include one or more viral genes, for example, retinal cells expressing the viral genes (e.g., PER.C6® cells). Proteins or polypeptides other than the target polypeptide or POI produced by the cells are sometimes called host cell proteins (HCPs).If POIs are produced in and / or purified from host cells, HCPs may be characterized as contaminants or impurities associated with the product and process.

[0024] Some HCPs (e.g., enzymes) can co-purify with POIs (e.g., target polypeptides) and affect the components of mixtures, formulations, or pharmaceuticals containing POIs. For example, the presence of certain HCPs may affect product stability, shorten the shelf life of a pharmaceutical, or even cause the product to fail to meet official or regulatory particulate matter specifications (e.g., U.S. Food and Drug Administration specifications). As a further example, some HCPs may cause clinical effects such as immunogenic reactions at administration. POIs can be purified and / or separated using HIC or other chromatography, either alone or in combination, to remove HCPs from mixtures, formulations, or pharmaceuticals and reduce their potential impact on pharmaceuticals. On the other hand, adding HIC or affinity chromatography steps requires additional equipment, materials (e.g., hydrophobic interaction media), and preparation. This translates to additional time, resources, experiments, and costs. Therefore, it is desirable to have an efficient method for performing chromatographic processes to separate POIs (e.g., target polypeptides) from one or more co-purified HCPs or other impurities.

[0025] As used herein, the term "chromatography" refers to any process for separating 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, reversed-phase chromatography, hydrophobic interaction chromatography (HIC), high-performance protein liquid chromatography, high-performance liquid chromatography, countercurrent chromatography, periodic countercurrent chromatography, or chiral chromatography. Embodiments disclosed herein may relate to exemplary types of chromatography processes (e.g., HIC) or apparatus, but embodiments disclosed herein may be applicable to any type of chromatography.

[0026] As used herein, the term “water” may refer to any suitable type of laboratory-grade water, such as deionized water or water for injection. In some embodiments, for example, a chromatography apparatus may be brought into contact with deionized water or water for injection. References to the use of “water” herein may refer to deionized water, water for injection, or another type of laboratory-grade water.

[0027] Where used in this disclosure, the term “mobile phase” may refer to any fluid suitable for contact with a chromatography zone or column as part of a separation or purification process. Mobile phases may include, for example, water, buffers, acidic solutions, alkaline solutions, and / or solutions containing alcohols. The terms “wash buffer,” “stripping buffer,” and “equilibrium buffer” may be used to describe mobile phases having specific properties, as further described herein.

[0028] In some embodiments, a method for preparing a target polypeptide from a mixture containing the target polypeptide may involve contacting the mixture with a chromatographic apparatus. The chromatographic apparatus may include multiple zones, each zone containing one or more chromatographic columns, one or more of which contain hydrophobic interacting media. Such chromatographic apparatuses may include pre-fabricated apparatuses (e.g., Cadence® BioSMB (Pall Biosciences), BioSC® (novasep), Varicol® (novasep), or Octave® (Semba Biosciences)), manually assembled apparatuses, or simply two or more more standard batch chromatographic apparatuses used in series.

[0029] Aspects of this disclosure may offer various benefits to processes for preparing target polypeptides or other molecules. For example, simultaneous use of multiple zones in a chromatography apparatus may enable more efficient and complete loading of individual columns and / or separation processes using less chromatographic medium than standard chromatography processes. Additional benefits and advantages of aspects of this disclosure will be apparent to those skilled in the art.

[0030] The drawings of this disclosure are referenced here. Figure 1 shows section 100 of a chromatography column in an HIC apparatus according to several embodiments of the present disclosure. The chromatography column includes a hydrophobic interacting medium. The hydrophobic interacting medium includes a support structure 110 and a hydrophobic portion 120, the hydrophobic portion 120 being fixed to the support structure 110. The medium can be in the form of a chromatography medium, e.g., beads or other particles held in a packed-bed column format, in the form of a membrane, or in any format capable of containing a mixture or other liquid containing a target polypeptide (or other POI) and contaminants (e.g., HCP). Thus, exemplary hydrophobic interacting mediums may include agarose beads (e.g., Sepharose), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, and the like.

[0031] The chromatography column of the HIC apparatus of this disclosure may be configured such that the hydrophobic interaction medium has a depth (e.g., bed height) of about 0.5 cm to about 40 cm. In some embodiments, for example, the chromatography column of the HIC apparatus may have bed heights 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 inner 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 approximately 0.5 cm to 140 cm, approximately 0.5 cm to 120 cm, approximately 0.5 cm to 100 cm, approximately 0.5 cm to 80 cm, approximately 0.5 cm to 60 cm, approximately 0.5 cm to 40 cm, approximately 0.5 cm to 20 cm, approximately 0.5 cm to 10 cm, approximately 0.75 cm to 8 cm, approximately 1 cm to 6 cm, approximately 1 cm to 5 cm, approximately 1 cm to 3 cm, approximately 1.5 cm to 5 cm, approximately 1.5 cm to 3 cm, or approximately 1 cm to 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 this disclosure has a total volume of about 0.4 milliliters (mL) to about 175 L (e.g., total volume for holding a mixture, mobile phase, or other substance).In some embodiments, for example, the chromatographic columns of the HIC apparatus according to this disclosure have a total volume of 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 200 mL, or about 1 mL to about 150 mL. For example, in some embodiments, the chromatography columns 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.

[0032] In some embodiments, the hydrophobic moiety 120 binds to the hydrophobic region and hydrophobic surface of the polypeptide. The hydrophobic surface may be part of the structure of the amino acids constituting the peptide, the aforementioned or other post-translational modifications, or a combination thereof. The degree of hydrophobicity of the hydrophobic interaction medium can be controlled by selecting an appropriate hydrophobic moiety 120. The hydrophobic moiety 120 can be selected to bind to a specific target polypeptide or POI and may be any hydrophobic moiety currently known or to be developed in the future. In some embodiments, the hydrophobic moiety 120 may include methyl, propyl, isopropyl, butyl, hexyl, octyl, and / or phenyl groups. Those skilled in the art will understand that the hydrophobicity of the selected hydrophobic moiety 120 may vary based on the target polypeptide and / or other impurities of the HCP / given application, as well as the type and degree of desired separation or purification from the chromatographic process.

[0033] Using a hydrophobic interaction medium, a target polypeptide or other POI can be separated from product and process-related contaminants and impurities (e.g., HCP). Continuing to refer to Figure 1, in some embodiments, a mixture containing the target polypeptide 140 and other components such as contaminants 130 (e.g., impurities, HCP, etc.) is loaded into a HIC apparatus. The mixture may contain a solution (e.g., a buffer) designed to facilitate the binding of the hydrophobic groups of the target polypeptide 140 to the hydrophobic moieties 120 of the hydrophobic interaction medium. Some of the target polypeptide 140 adheres to the medium by binding to the hydrophobic moieties 120 via intramolecular forces, while other target polypeptides 140 can pass through the chromatography column. In addition, or alternatively, while the mixture passes through the column, some contaminants 130 from the mixture may adhere to the hydrophobic interaction medium by binding to the hydrophobic moieties 120 via intramolecular forces, while other contaminants 130 cannot bind to the hydrophobic moieties 120. In some embodiments, the target polypeptide 140 includes specific hydrophobic regions from constituent amino acids, from post-translational modifications, or from a combination thereof that allows it to adhere to the hydrophobic moiety 120 with a higher affinity than certain contaminants or impurities (e.g., HCP). As will be described in more detail later, an additional mobile phase is then introduced into the column to reduce the affinity between the target polypeptide 140 and the hydrophobic moiety 120, allowing the target polypeptide 140 to pass through the chromatographic column of the HIC apparatus.

[0034] In a further embodiment, the contaminant 130 may adhere to the hydrophobic moiety 120 with a higher affinity than the target polypeptide 140. Subsequently, an additional mobile phase can be introduced into the column to reduce the affinity between the contaminant 130 and the hydrophobic moiety 120, allowing the contaminant 130 to pass through the chromatographic column of the HIC apparatus.

[0035] The composition of the mixture containing the target polypeptide 140 can be altered by adding additives, for example, salts such as sodium, potassium, phosphate, tris(hydroxymethyl)aminomethane (Tris), citrate, or acetate. Other additives can be added to alter the hydrophobicity or other intramolecular interactions of the target polypeptide 140, the contaminant 130, the hydrophobic moiety 120, or combinations thereof.

[0036] An exemplary HIC apparatus 200 according to several embodiments described herein is schematically shown in Figure 2. The HIC apparatus 200 may include a first zone 210, a second zone 220, and a third zone 230. Each of the first zone 210, the second zone 220, and the third zone 230 may include one or more chromatography columns, such as the chromatography columns described with respect to Figure 1. The first zone 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 through the first zone 210. The first zone 210 may also have a first outlet 214 through which effluent from the HIC apparatus 200 (e.g., fluid that has passed through the first zone 210) can be passed for collection or disposal. The effluent can also pass from the first zone 210 to the second zone 220 via a first interconnect 216. The first zone 210 can also receive spills from the third zone 230 via the third interconnection 236.

[0037] The second zone 220 can receive effluent from the first zone 210 via the first interconnect 216. The second zone 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 through the second zone 220. The second zone 220 may also have a second outlet 224 through which effluent from the HIC device 200 (e.g., fluid that has passed through the second zone 220) can pass for collection or disposal. The effluent can also pass from the second zone 220 to the third zone 230 via the second interconnect 226.

[0038] The third zone 230 can receive effluent from the second zone 220 via the second interconnect 226. The third zone 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 through to the third zone 230. The third zone 230 may also have an outlet 234 through which effluent from the HIC device 200 (e.g., fluid that has passed through the third zone 230) can pass for collection or disposal. The effluent can also pass from the third zone 230 to the first zone 220 via the third interconnect 236.

[0039] As those skilled in the art will understand, various components known to be used in chromatography apparatuses (e.g., filters, sensors, gauges, thermometers) can be incorporated into the HIC apparatus 200, but are not shown in the schematic diagram of Figure 2. In some embodiments, one or more of UV absorptivity, electrical conductivity, or pH or stagnant solution can be measured at one or more locations within the HIC apparatus 200. Suitable locations for measuring UV absorptivity, electrical conductivity, or pH include inlets 212, 222, 232, within zones 210, 220, 230, interconnections 216, 226, 236, or outlets 214, 224, 234. The inlets 212, 222, 232, interconnects 216, 226, 236, and outlets 214, 224, 234 may be operable to move from an open configuration to a closed configuration, the open configuration allowing fluid to pass through the inlets 212, 222, 232, interconnects 216, 226, 236, or outlets 214, 224, 234, and the closed configuration preventing 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 for transferring fluid between zones 210, 220, 230, inlets 212, 222, 232, interconnects 216, 226, 236, and outlets 214, 224, 234. In some embodiments, one or more interconnects 216, 226, 236 can be moved to connect different zones 210, 220, 230. For example, during a process using the HIC apparatus 200, it may be desirable to reposition the location through which interconnect 226 passes effluent from zone 220. In these situations, interconnect 226 can be reconfigured to pass effluent from zone 220 to zone 210 without interrupting the chromatography process. This is just one example. In most cases, interconnects 216, 226, 236 can be reconfigured to connect different zones without interrupting the ongoing chromatography process.

[0040] Figure 3A graphically illustrates a method according to several embodiments of the present disclosure. On the left axis of the graph, three separate rows are defined by labels C1, C2, and C3, representing the first, second, and third columns of the HIC apparatus. The top axis represents time and extends infinitely to the left and right. The continuous occupancy periods of each column are illustrative examples of the embodiments described herein. This arrangement reduces or eliminates idle time (e.g., “dead time”) of the columns compared to conventional HIC methods. The time segments shown throughout Figure 3A represent one exemplary cycle of a repeating pattern, which may repeat before and / or after the time segments shown in Figure 3A. Four time segments are labeled T1, T2, T3, and T4, which 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, it is substantially the same as the interval between T3 and T4. In some embodiments, the interval between adjacent labeled times (e.g., between T1 and T2, or between T3 and T4) may be 30 seconds (s) or more, 90 minutes (min) or less, 30 seconds to 60 minutes, 30 seconds to 30 minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30 seconds to 8 minutes, 30 seconds to 7 minutes, 30 seconds to 6 minutes, 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 1 minute to 5 minutes, or 2 minutes to 5 minutes. 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 the C1, C2, and C3 columns at the time interval in which each box appears. For example, each box could represent the presence of a mixture, a mobile phase, or other stagnant liquids in the row of the column in which it appears.

[0041] Moving forward in time from T1 to T2 across Figure 3A from left to right, the second loading of the mixture may be in the first column C1 (box 410). From T2 to T3, the first loading of the mixture may be in the first column C1 (box 412), and from T3 to T4, one or more mobile phases may be in the first column C1 (box 414). In some embodiments, a column can receive either the first loading or the second loading of the mixture. The “first loading” of the mixture refers to the loading of the mixture that passes through the column of the HIC apparatus without first passing through another column of the HIC apparatus. The “second loading” of the mixture refers to the loading of the mixture that has passed through another column of the HIC apparatus before being introduced into a given column (for example, the effluent from the first loading of the mixture is introduced into another column as the second loading of the mixture). Passing effluent containing the target peptide from one column to another allows the columns to be fully loaded without worrying about wasting overflow, improving the utilization efficiency of each column and reducing the amount of hydrophobic interaction medium consumed. Passing overflow over the hydrophobic interaction medium that has received or can receive the initial load of the mixture containing the target polypeptide can reduce the amount of hydrophobic interaction medium consumed relative to the amount of loading mixture being processed.

[0042] 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, such as sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, Tris, or other salts.

[0043] In some embodiments, the stripping buffer may include water, an alkaline solution, or a solution containing alcohol. For example, deionized water may contain dissolved ions of less than 5 volume percent (vol.%), less than 1 vol.%, less than 0.1 vol.%, or less than 0.01 vol.%. According to some embodiments, the alkaline solution may contain one or more alkaline ionic compounds such as LiOH, NaOH, KOH, Ca(OH)2, NH4OH, or other alkaline compounds. The concentration of the alkaline compound in the stripping buffer may be in the range of, for example, about 0.1N to about 1.5N, about 0.1N to about 1N, about 0.1N to about 1.5N, about 0.5N to about 1.5N, about 0.1N to about 0.8N, about 0.1N to about 0.6N, about 0.1N to about 0.5N, about 0.1N to about 0.4N, or about 0.1N to about 0.3N. For example, the concentration of the alkaline compound in the stripping buffer may be about 0.1N, about 0.2N, about 0.3N, about 0.4N, about 0.5N, about 0.6N, about 0.7N, about 0.8N, about 0.9N, about 1N, about 1.1N, about 1.2N, about 1.3N, about 1.4N, or about 1.5N. The alcohol-containing stripping buffer may contain methanol, ethanol, propanol, benzyl alcohol, or other alcohols. The alcohol concentration in the stripping buffer may range from approximately 0.1 vol.% to approximately 30 vol.%, for example, approximately 0.5 vol.% to approximately 30 vol.%, approximately 0.5 vol.% to approximately 25 vol.%, approximately 0.5 vol.% to approximately 25 vol.%, approximately 0.5 vol.% to approximately 25 vol.%, approximately 1 vol.% to approximately 20 vol.%, approximately 1 vol.% to approximately 15 vol.%, approximately 1 vol.% to approximately 10 vol.%, approximately 10 vol.% to approximately 50 vol.%, approximately 10 vol.% to approximately 40 vol.%, approximately 10 vol.% to approximately 30 vol.%, approximately 10 vol.% to approximately 25 vol.%, approximately 15 vol.% to approximately 25 vol.%, or approximately 20 vol.% to approximately 25 vol.%, based on the total weight of the stripping buffer.For example, the alcohol concentration in the stripping buffer may be approximately 0.1 vol.%, 0.5 vol.%, 1 vol.%, 2 vol.%, 3 vol.%, 5 vol.%, 10 vol.%, 15 vol.%, 20 vol.%, or 25 vol.%.

[0044] In some embodiments, the equilibration buffer may be similar to or identical in composition to the washing buffer. In other embodiments, the equilibration buffer may have a different composition compared to the washing buffer. In some embodiments, the equilibration buffer may contain one or more salts, such as sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, Tris, or other salts. Referring to Figure 3A, contact with one or more mobile phases in the first column 414 may be divided into separate phases, each including the washing buffer for the first column (box 415), the stripping buffer for the first column (box 417), and the equilibration buffer for the first column (box 419). In the next row (representing C2), from T1 to T2, one or more mobile phases may be in the second column (box 424). This, too, may be divided into separate phases, each including the washing buffer for the second column (box 425), the stripping buffer for the second column (box 427), and the equilibration buffer for the second column (box 429). Moving to the right, from T2 to T3, the second loading of the mixture may be in the second column (box 420), and from T3 to T4, the first loading of the mixture may be in the second column (box 422).

[0045] In the following row, from T1 to T2, the initial loading of the mixture may be in the third column (box 432). Then, from T2 to T3, one or more mobile phases may be in the third column (box 434), and from T3 to T4, the second loading 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 separate phases, each including the washing buffer for the third column (box 435), the stripping buffer for the third column (box 437), and the equilibration buffer for the third column (box 439).

[0046] A vertical line can be drawn through the graph such that, at a given time T0, each numbered box touched by the vertical line from T0 represents the solution in the column at that point in time. Thus, for example, when the second loading mixture is introduced into the first column C1 at T1 (box 410), one or more mobile phases are passed through the second column C2 (box 424). For example, the washing buffer is passed through C2 (box 425) and the initial loading mixture is passed through the third column C3 (box 432). For example, the broader phase subdivisions such as subdivisions 425, 427, and 429 appear to occupy equal parts of one or more mobile phases in the second column 424, but in some embodiments, the subdivisions may occupy inequal parts of the broader phase. It should also be understood that the method shown in Figure 3A is also just one exemplary progression according to embodiments of the present disclosure. Other sequences, configurations, and steps are considered to be within the scope of the present disclosure.

[0047] Figures 3B-3D show an exemplary cycle of a method for preparing a target polypeptide from a mixture containing the target polypeptide, as described above. Figure 3B shows a series of events that may occur between the time interval T1 and T2 in Figure 3A. Thus, Figure 3B shows the HIC apparatus in the first stage 301, where the first zone 310 receives a second load of the mixture 306 containing the target polypeptide and elutes the effluent of the second load 307, which can be collected or discarded. The second zone 320 receives one or more mobile phases 315 and elutes the effluent of one or more mobile phases 316, which can be collected or discarded. The third zone 330 receives an initial load of the mixture 305 and passes a second load of the mixture 306 through another column.

[0048] Figure 3C shows the HIC apparatus in the second stage 302 (spanning interval T2 to T3 as shown in Figure 3A), where the first zone 310 receives the initial load of mixture 305 and passes the second load of mixture 306 through another column. The second zone 320 receives the second load of mixture 306 and elutes the effluent of the second load 307, which can be collected or discarded. The third zone 330 receives one or more mobile phases 315 and elutes the effluent of one or more mobile phases 316, which can be collected or discarded.

[0049] Figure 3D shows the HIC apparatus in the third stage 303 (spanning interval T3 to T4 as shown in Figure 3A), where the first zone 310 receives one or more mobile phases 315 and elutes one or more mobile phases 316 effluents that can be collected or discarded. The second zone 320 receives the initial load of mixture 305 and passes a second load of mixture 306 through another column. The third zone 330 receives one or more mobile phases 315 and elutes one or more mobile phases 316 effluents that can be collected or discarded.

[0050] Another exemplary HIC apparatus 500 according to some embodiments described herein is schematically shown in Figure 4. The HIC apparatus 500 may include a first zone 510, a second zone 520, a third zone 530, and a fourth zone 540. The first zone 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 through the first zone 510. The first zone 510 may also have a first outlet 514 through which effluent from the HIC apparatus 500 (e.g., fluid that has passed through the first zone 510) can be collected or disposed of. The effluent can also pass from the first zone 510 to the second zone 520 via a first interconnect 516. The first zone 510 can also receive effluent from the fourth zone 540 via a fourth interconnect 546.

[0051] The second zone 520 can receive effluent from the first zone 510 via the first interconnect 516. The second zone 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 through the second zone 520. The second zone 520 may also have a second outlet 524 through which effluent from the HIC device 500 (e.g., fluid that has passed through the second zone 520) can pass for collection or disposal. The effluent can also pass from the second zone 520 to the third zone 530 via the second interconnect 526.

[0052] The third zone 530 can receive effluent from the second zone 520 via the second interconnect 526. The third zone 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 through the third zone 530. The third zone 530 may also have an outlet 534 through which effluent from the HIC device 500 (e.g., fluid that has passed through the third zone 530) can pass for collection or disposal. The effluent can also pass from the third zone 530 to the fourth zone 520 via the third interconnect 536.

[0053] The fourth zone 540 can receive effluent from the third zone 530 via the third interconnect 536. The fourth zone 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 through the fourth zone 540. The fourth zone 540 may also have an outlet 544 through which effluent from the HIC device 500 (e.g., fluid that has passed through the fourth zone 540) can pass for collection or disposal. The effluent can also pass from the fourth zone 540 to the first zone 510 via the fourth interconnect 546.

[0054] Various components known to be used in chromatography apparatuses (e.g., filters, sensors, gauges, thermometers) can be incorporated into the HIC apparatus 500, but are not shown in the simplified schematic diagram of Figure 4. In some embodiments, one or more of UV absorptivity, electrical conductivity, or pH, or a retained solution, can be measured at one or more locations in the HIC apparatus 500. Suitable locations for measuring UV absorptivity, electrical conductivity, or pH include inlets 512, 522, 532, 542, interconnects 516, 526, 536, 546, or outlets 514, 524, 534, 544 within zones 510, 520, 530, 540. Inlets 512, 522, 532, 542, interconnects 516, 526, 536, 546, and outlets 514, 524, 534, 544 may be operable to move from an open configuration to a closed configuration. An open configuration allows fluid to pass through inlets 512, 522, 532, 542, interconnects 516, 526, 536, 546, or outlets 514, 524, 534, 544, while a closed configuration prevents fluid from passing through 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 zones 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 zones 510, 520, 530, 540. For example, during one or more chromatography processes using the HIC apparatus 500, it may be desirable to reposition interconnect 536 to pass effluent from zone 530. In these situations, interconnect 536 may be reconfigured to pass effluent from zone 530 to zone 520 without interrupting the chromatography process. This is just one example, and in most cases, any of interconnects 516, 526, 536, 546 can be reconfigured to connect different zones 510, 520, 530, 540 without interrupting the ongoing chromatography process.

[0055] Figure 5A is a graph illustrating one or more methods according to the present disclosure. On the left axis of the graph, four separate rows are defined by labels C1, C2, C3, and C4, representing the first, second, third, and fourth columns of the HIC apparatus. The upper axis represents time and extends infinitely to the left and right. The continuous occupancy periods of each column are illustrative of the embodiments described herein, and this arrangement reduces or eliminates the idle time (e.g., “dead time”) of the columns compared to conventional HIC methods. The time segments shown represent one cycle of a repeating pattern, and understanding the pattern of numbered boxes described below can be repeated on both sides of the segments shown in Figure 5A. Five times are labeled T1, T2, T3, T4, and T5, which 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; in some embodiments, it is substantially the same as the interval between T3 and T4; and in some embodiments, it is 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., between T1 and T2 or between T4 and T5) may be 30 seconds or more, 90 minutes or less, 30 seconds to 60 minutes, 30 seconds to 30 minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30 seconds to 8 minutes, 30 seconds to 7 minutes, 30 seconds to 6 minutes, 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 1 minute to 5 minutes, or 2 minutes to 5 minutes. Boxes 710, 712, 714, 724, 720, 722, 734, 730, 732, 742, 744, and 740 represent the mixtures, buffers, or other retained liquids in columns C1, C2, C3, and C4, respectively.

[0056] As we move across Figure 5A from left to right, moving the first row (representing the first column C1) from T1 to T2, the second loading of the mixture may be in the first column (box 710). From T2 to T3, the initial loading of the mixture may be in the first column (box 712), and from T3 to T5, one or more mobile phases may be in the first column (box 714).

[0057] Continuing to refer to Figure 5A, one or more mobile phases in the first column 714 may be divided into separate phases, each containing the first column wash buffer (box 715), the first column stripping buffer (box 717), and the first column equilibration buffer (box 719). In the next row (representing the second column C2), from T1 to T2, one or more mobile phases may be in the second column (box 724), continuing from T4 to T5 of the previous cycle. This too may be divided into separate phases, each containing the second column wash buffer (box 725), the second column stripping buffer (box 727), and the second column equilibration buffer (box 729). Moving to the right, from T2 to T3, the second loading of the mixture may be in the second column (box 720), and from T3 to T4, the first loading of the mixture may be in the second column (box 722).

[0058] In the next row (representing column C3), from T1 to T3, one or more mobile phases may be in the third column (box 734). One or more mobile phases in the third column 734 may be divided into separate phases, each containing the third column's washing buffer (box 735), the third column's stripping buffer (box 737), and the third column's equilibration buffer (box 739). Next, from T3 to T4, the second load may be in the third column (box 730), and from T4 to T5, the first load of the mixture may be in the third column (box 732).

[0059] In the next row (representing column C4), the initial loading of the mixture may be in the fourth column (box 732) from T1 to T2. Then, from T2 to T4, one or more mobile phases may be in the fourth column (box 744), and from T4 to T5, the second loading of the mixture may be in the third column (box 740). One or more mobile phases in the fourth column 744 may be divided into separate phases, each containing the washing buffer (box 745) of the third column, the stripping buffer (box 747) of the third column, and the equilibration buffer (box 749) of the third column.

[0060] A vertical line can be drawn through the graph such that, at a given time T0, each numbered box touched by the vertical line from T0 represents the solution in the column at that point in time. Thus, for example, when the second loading mixture is introduced into the first column 710 at time T1, one or more mobile phases, e.g., stripping buffer 727, are in the second column 724, and the initial loading mixture is passed through the third column 732. For example, broader phase subdivisions such as subdivisions 725, 727, and 729 appear to occupy equal parts of one or more mobile phases in the second column 724, although in some embodiments, subdivisions may occupy unequal parts of broader phases. It should be understood that the method shown in Figure 5A is also only one example of a method in the embodiments of this disclosure. Other sequences, configurations, and steps are considered to be within the scope of this disclosure.

[0061] Figures 5B–5E illustrate an exemplary cycle of a method for preparing a target polypeptide from a mixture containing the target polypeptide, as described above. Figure 5B shows a series of events that may occur between the time interval T1 and T2 in Figure 5A. Figure 5B shows the HIC apparatus in the first stage 601, where the first zone 610 receives a second load of the mixture 606 containing the target polypeptide and elutes the effluent of the second load 607, which can be collected or discarded. The second zone 620 receives one or more mobile phases 615 and elutes the effluent of one or more mobile phases 616, which can be collected or discarded. The third zone 630 receives one or more mobile phases 615 and elutes the effluent of one or more mobile phases 616, which can be collected or discarded. The fourth zone 640 receives the initial load of the mixture 605 and passes the second load of the mixture 606 through another column.

[0062] Figure 5C shows the HIC apparatus in the second stage 602 (spanning interval T2 to T3 as shown in Figure 5A), where the first zone 610 receives the initial load of mixture 605 and passes a second load of mixture 606 containing the target polypeptide, which can be collected or discarded, through another column. The second zone 620 receives the second load of mixture 606 containing the target polypeptide and elutes the effluent of the second load 607, which can be collected or discarded. The third zone 630 receives one or more mobile phases 615 and elutes the effluent of one or more mobile phases 616, which can be collected or discarded. The fourth zone 640 receives one or more mobile phases 615 and elutes the effluent of one or more mobile phases 616, which can be collected or discarded.

[0063] Figure 5D shows the HIC apparatus in the third stage 603 (spanning interval T2 to T3 as shown in Figure 5A), where the first zone 610 receives one or more mobile phases 615 and elutes one or more mobile phases 616 effluent which can be collected or discarded. The second zone 620 receives the initial load of mixture 605 and passes a second load of mixture 606 through another column containing the target polypeptide which can be collected or discarded. The third zone 630 receives the second load of mixture 606 containing the target polypeptide and elutes the effluent of the second load 607 which can be collected or discarded. The fourth zone 640 receives one or more mobile phases 615 and elutes one or more mobile phases 616 effluent which can be collected or discarded.

[0064] Figure 5E shows the HIC apparatus in the fourth stage 604 (spanning interval T2 to T3 as shown in Figure 5A), where the first zone 610 receives one or more mobile phases 615 and elutes one or more mobile phases 616 effluent which can be collected or discarded. The second zone 620 receives one or more mobile phases 615 and elutes one or more mobile phases 616 effluent which can be collected or discarded. The third zone 630 receives the initial load of mixture 605 and passes a second load of mixture 606 through another column containing the target polypeptide which can be collected or discarded. The fourth zone 640 receives the second load of mixture 606 containing the target polypeptide and elutes the effluent of the second load 607 which can be collected or discarded.

[0065] Figure 6 shows a flowchart of an exemplary method 800 for preparing a target polypeptide from a mixture containing the target polypeptide. This method may include passing the mixture containing the target polypeptide through a first column of a plurality of columns (e.g., box 410 in Figure 3A) (step 810). This method may further include passing the effluent containing the target polypeptide from the first column to a second column of a plurality of columns (e.g., a second load of mixture 306 as shown in Figure 3B) (step 820). This method may further include passing one or more mobile phases through the first column (e.g., box 414) (step 830). In some embodiments, this method may further include passing the target polypeptide through the respective outlets of the plurality of columns (e.g., effluents of one or more mobile phases 316 as shown in Figures 3B-3D) (step 840). While the comparison is made with respect to Figures 3A-3D, it will be apparent to those skilled in the art that comparisons with respect to Figures 5A-5E can also be made.

[0066] In embodiments of this disclosure, the mixture containing the target polypeptide may also contain one or more HCPs. After preparing the target polypeptide from the mixture using the method of one or more embodiments, several effluent samples can be obtained. Samples can 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 the first load of the mixture or the second load of the mixture (or any other load of the mixture). In some embodiments, samples may be collected only from the effluent of one or more wash buffers. In other embodiments, samples may be collected from the effluent of other mobile phases and / or from the first or second load of the mixture. The collections of all collected samples containing the target polypeptide are collected together and referred to as a pool.

[0067] In some embodiments, one or more measurements can be performed to confirm the efficiency of the method used to prepare the target polypeptide. As used in this disclosure, efficiency refers to a combination of three different factors: high molecular weight clearance (HMW CF), yield, and productivity. In some embodiments, more efficient methods have higher HMW CF, higher yield, and higher productivity than less efficient methods. In other embodiments, more efficient methods maintain an HMW CF of 1.3 or higher and a yield of 80% or higher while having higher productivity than less efficient methods. In further embodiments, more efficient methods maintain an HMW CF of 1.5 or higher and a yield of 90% or higher while having higher productivity than less efficient methods.

[0068] High molecular weight clearance (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 can be performed to determine the percentage of the sample attributable to high molecular weight molecules (e.g., proteins) (HMW%). In other embodiments, centrifugation techniques can be used. Centrifugation of a sample separates it into layers based on the mass of its components, with the heaviest layer, the supernatant, generally containing the heaviest molecules, including proteins. HMW% can be calculated by taking the supernatant of the centrifuged sample and dividing it by the total mass of the sample. Regardless of which method is used, HMW CF can be calculated according to Equation 1, as shown below.

[0069]

number

[0070] As shown in 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 preparing the target polypeptide from the mixture has an HMW CF of 1.3 or more. In other embodiments, the method for preparing the target polypeptide from the 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.

[0071] The yield is a measure of the amount of target polypeptide collected in the pool compared to the amount of target polypeptide present in the loading mixture. The amount of target polypeptide in the sample can be quantified by UV absorptivity, electrical conductivity, or enzyme immunoassay (e.g., ELISA). The yield can be calculated according to Equation 2, as shown below.

[0072]

number

[0073] As shown in Equation 2, the yield can be calculated by dividing the mass of the target polypeptide loaded into the HIC instrument by the mass of the target polypeptide collected in the pool. Since the mass of the target polypeptide in the sample cannot be directly measured, the mass can be determined by multiplying the volume by the concentration (calculated by UV absorptivity, electrical conductivity, or enzyme immunoassay). 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.

[0074] Productivity is a quantification of the time and cost required to prepare a certain amount of target polypeptide. Productivity can be calculated according to Equation 3, as shown below.

[0075]

number

[0076] As shown in Equation 3, productivity can be calculated by dividing the mass of the target polypeptide collected in the pool by the product of the volume of the hydrophobic interaction medium used and the time spent to prepare the mass of polypeptide collected in the pool (e.g., cycle time). In some embodiments, the method for preparing the target polypeptide from the mixture has a productivity of about 35 g / L·hour or more. In other embodiments, the method for preparing the target polypeptide from the mixture has a productivity of about 40 g / L·hour or more, about 50 g / L·hour or more, about 75 g / L·hour or more, about 100 g / L·hour or more, about 125 g / L·hour or more, about 150 g / L·hour or more, about 175 g / L·hour or more, about 200 g / L·hour or more, or about 220 g / L·hour or more.

[0077] Examples The following embodiments are intended to illustrate the disclosure without being inherently limiting. It is understood that the disclosure also includes additional aspects and embodiments that are not inconsistent with the foregoing description and the following examples.

[0078] In the following examples, the target polypeptide was prepared from a mixture containing the target polypeptide and HCP. The target polypeptide was prepared using three different methods according to the embodiments of this disclosure. The target polypeptide was also prepared using a conventional batch processing method as a comparative example.

[0079] Example 1 In the first example, the target antibody was prepared. 300 mL of a 12.2 g / L mixture containing the target antibody was loaded into a 3-column HIC instrument at a loading flow rate of 1.67 mL / min. Each column had a bed height of 2.5 cm, an inner diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mM sodium citrate solution and was adjusted to pH 6.0 with 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 loading buffer was passed through the outlet of the first column to the second column). After loading the mixture into the first and second columns of the HIC instrument, the mixture was loaded into the second and third columns of the three columns, except that the third column was loaded via the second column (i.e., the loading buffer was passed through the outlet of the second column to the third column).

[0080] While the loading buffer / mixture was being loaded onto the second and third columns, a series of mobile phases were passed through the first column of the HIC instrument to separate the target antibody from the other components of the mixture in the first column and collect the target antibody. Subsequently, a series of stripping buffers were passed through the first column to regenerate the column. Loading of the second and third columns was performed simultaneously with washing and stripping of the first column. After this step, the loading buffer / mixture was loaded onto the third and first columns, except that the first column was loaded via the third column (i.e., the loading buffer was passed through the first column via the outlet from the third column). During this time, the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded onto the second column, and then the second column was regenerated by passing a series of stripping buffers through the second column. Loading of the third and first columns was performed simultaneously with washing and stripping of the second column. Finally, the loading buffer / mixture was loaded onto the first and second columns again, and the second column was loaded from the first column as described above, while the buffer was passed through the third column to separate and collect the target antibody from the mixture. Subsequently, the third column was regenerated by passing a series of stripping buffers through it. Loading of the first and second columns was performed simultaneously with washing and stripping of the third column. This process was repeated periodically twice.

[0081] The mobile phase consisted of 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 pH 6.0. Four column volumes of wash buffer were added to each column to wash it.

[0082] After applying a washing 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. As used herein, column volume refers to the amount of liquid that a given column can hold. The next stripping buffer contained 1N 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. The final stripping buffer containing deionized water was added to the column (an amount equal to two column volumes). After applying the stripping buffers, 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 pH 6.0.

[0083] After collecting the pool from the method performed in Example 1, the HMW CF, yield, and productivity of this method were measured and calculated as described above. The results are summarized in Table 1 below.

[0084] Example 2 In the second example, the target antibody was prepared. 729 mL of a 12.4 g / L mixture containing the target polypeptide was loaded into a 3-column HIC instrument at a loading flow rate of 1.67 mL / min. Each column had a bed height of 2.5 cm, an inner diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mM sodium citrate solution and was adjusted to pH 6.0 with 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 loading buffer was passed through the outlet of the first column to the second column). After loading the mixture into the first and second columns of the HIC instrument, the mixture was loaded into the second and third columns of the three columns, except that the third column was loaded via the second column (i.e., the loading buffer was passed through the outlet of the second column to the third column).

[0085] While the loading buffer / mixture was being loaded onto the second and third columns, a series of mobile phases were passed through the first column of the HIC instrument to separate the target antibody from the other components of the mixture on the first column and collect the target antibody. Subsequently, a series of stripping buffers were passed through the first column to regenerate it. Loading of the second and third columns was performed simultaneously with washing and stripping of the first column. After this step, the loading buffer / mixture was loaded onto the third and first columns, except that the first column was loaded via the third column (i.e., the loading buffer was passed through the first column via the outlet from the third column). During this time, the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded onto the second column, and then the second column was regenerated by passing a series of stripping buffers through it. Loading of the third and first columns was performed simultaneously with washing and stripping of the second column. Finally, the loading buffer / mixture was loaded onto the first and second columns again, except that the second column was loaded from the first column as described above. During this process, the buffer was passed through the third column to separate and collect the target antibody from the mixture loaded onto the third column. Subsequently, a series of stripping buffers were passed through the third column to regenerate it. Loading of the first and second columns was performed simultaneously with washing and stripping of the third column. This process was repeated cyclically four times.

[0086] The mobile phase consisted of 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 pH 6.0. To wash each column, four columns' worth of wash buffer was added to the column.

[0087] After applying a washing 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. As used herein, column volume refers to the amount of liquid that a given column can hold. The next stripping buffer contained 1N 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. The final stripping buffer containing deionized water was added to the column (an amount equal to two column volumes). After applying the stripping buffers, 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 pH 6.0.

[0088] After collecting the pool from the method performed in Example 2, HMW CF, yield, and productivity were measured and calculated as described above. The results are summarized in Table 1 below. Example 3 In the third example, the target polypeptide was prepared. 726 mL of a 12.4 g / L mixture containing the target polypeptide was loaded into a 3-column HIC instrument at a loading flow rate of 6.70 mL / min. Each column had a bed height of 2.5 cm, an inner diameter of 1.6 cm, and a column volume of 5 mL. The loading buffer / mixture contained a 30 mM sodium citrate solution and was adjusted to pH 6.0 with 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 loading buffer was passed through the outlet of the first column to the second column). After loading the mixture into the first and second columns of the HIC instrument, the mixture was loaded into the second and third columns of the three columns, except that the third column was loaded via the second column (i.e., the loading buffer was passed through the outlet of the second column to the third column).

[0089] While the loading buffer / mixture was being loaded onto the second and third columns, a series of mobile phases were passed through the first column of the HIC instrument to separate the target antibody from the other components of the mixture on the first column and collect the target antibody. Subsequently, a series of stripping buffers were passed through the first column to regenerate it. Loading of the second and third columns was performed simultaneously with washing and stripping of the first column. After this step, the loading buffer / mixture was loaded onto the third and first columns, except that the first column was loaded via the third column (i.e., the loading buffer was passed through the first column via the outlet from the third column). During this time, the buffer was passed through the second column to separate and collect the target antibody from the mixture loaded onto the second column, and then the second column was regenerated by passing a series of stripping buffers through it. Loading of the third and first columns was performed simultaneously with washing and stripping of the second column. Finally, the loading buffer / mixture was loaded onto the first and second columns again, except that the second column was loaded from the first column as described above. During this process, the buffer was passed through the third column to separate and collect the target antibody from the mixture loaded onto the third column. Subsequently, a series of stripping buffers were passed through the third column to regenerate it. Loading of the first and second columns was performed simultaneously with washing and stripping of the third column. This process was repeated cyclically four times.

[0090] The mobile phase consisted of 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 pH 6.0. To wash each column, four columns' worth of wash buffer was added to the column.

[0091] After applying a washing 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. As used herein, column volume refers to the amount of liquid that a given column can hold. The next stripping buffer contained 1N 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. The final stripping buffer containing deionized water was added to the column (an amount equal to two column volumes). After applying the stripping buffers, 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 pH 6.0.

[0092] After collecting the pool from the method performed in Example 3, HMW CF, yield, and productivity were measured and calculated as described above. The results are summarized in Table 1 below. Comparative Example For comparison with the methods of Examples 1-3, the target polypeptide was prepared from the 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 examples, but the conventional batch method was employed. 590 g of the 13.1 g / L loading mixture was added to the chromatography column. After the mixture passed through the column, four columns' worth of washing buffer was added to the column, and the effluent was collected. After collecting the pool from the comparative example method, HMW was used. CF, yield, and productivity were characterized. The results are summarized in Table 1.

[0093] [Table 1]

[0094] As can be seen from the data in Table 1, Examples 2 and 3 have higher productivity than the batch method of the comparative example. Furthermore, Example 3 was able to achieve higher productivity than the other examples while maintaining an HMW CF of 1.5 or higher and a yield of 90% or higher.

[0095] Example 4 To compare impurity breakthrough at various rates, target antibodies were prepared using HIC at three different loading rates. Columns were prepared as shown in Table 2.

[0096] [Table 2]

[0097] The loading rates, in the order they were performed, were 300 cm / hour (3.93 mL / min, i.e., residence time in the column of 4.0 minutes), 200 cm / hour (2.62 mL / min, i.e., residence time in the column of 6.0 minutes), and 400 cm / hour (5.24 mL / min, i.e., residence time in the column of 3.0 minutes). All tests were performed on the same column. Before performing the 400 cm / hour test, the sample was immersed overnight in 0.5N NaOH.

[0098] As shown in Figure 7A, the high molecular weight percentage (HMW%) was plotted as a function of the load. The HMW% of the load material was 1.78%. The cumulative pooled HMW% for 200 g / L resin and 400 g / L resin are shown in Table 3 below.

[0099] [Table 3]

[0100] For each loading rate, host cell proteins were quantified in parts per million using the F665 CHO HCP ELISA kit (Cygnus Technologies). The obtained amounts were plotted as a function of loading, as shown in Figure 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.

[0101] Example 5 The target antibody was prepared using HIC in two columns with different bed heights (20 cm and 2.5 cm, as used in Example 4). Both runs were performed so that the residence time in each column was 3 minutes (i.e., the linear flow rate in the 20 cm bed height column was 400 cm / hour). The HMW% of the loading material was 2.2%. As shown in Figure 7C, the HMW% of the pool in each column was plotted as a function of loading concentration.

[0102] Those skilled in the art will understand that the underlying concepts of this disclosure can be readily used as a basis for designing other methods and systems for carrying out the solutions and objectives of this disclosure. Therefore, the claims should not be considered limited by the foregoing description.

Claims

[Claim 1] The invention described in the specification.

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