Polypeptide purification method including two IMAC steps and apparatus therefor

Abstract

The present application relates to systems and methods for purifying polypeptides, such as His-tagged polypeptides, using immobilized metal chelate affinity chromatography (IMAC). The method includes loading a composition containing the polypeptide onto an IMAC matrix, eluting the polypeptide from a first IMAC matrix, performing a buffer exchange to remove the elution buffer from the first eluate, loading the first eluate from the buffer exchange onto a second IMAC matrix, eluting the polypeptide from the second IMAC matrix, and optionally purifying the eluate from the second IMAC matrix using size exclusion chromatography. In some embodiments, the method is fully automated.

Description

[Technical Field]

[0001] Field The present application relates to systems and methods for purifying polypeptides, such as His-tagged polypeptides, using immobilized metal chelate affinity chromatography (IMAC), for example, using two different IMAC matrices in a high-throughput setting. In some embodiments, the IMAC matrix comprises a chelating moiety that is bound to nickel. [Background technology]

[0002] background Drug discovery and basic research involving polypeptides often require reliable, high-quality protein reagents that can be rapidly purified, especially in high-throughput settings. Purification of secreted His-tagged proteins typically involves concentration of the culture medium containing the His-tagged protein, ultrafiltration to exchange the buffer, a single immobilized metal affinity chromatography (IMAC) step to selectively bind proteins with His tags, often using a nickel-containing Ni-NTA matrix, and elution of the His-tagged protein from the IMAC column (Figure 1). However, this method requires a time-intensive step of ultrafiltration to exchange the buffer for one compatible with the Ni-NTA matrix, which significantly increases the time required to prepare pure protein. New systems and methods are needed to efficiently and reliably purify proteins to meet the reagent specifications of modern drug discovery and polypeptide research. Summary of the Invention

[0003] overview The present disclosure relates to novel purification methods involving two different IMAC matrices for purifying His-tagged polypeptides or other polypeptides that selectively bind to metal ion chelating matrices. Certain embodiments herein include, for example, methods for purifying a polypeptide, the methods comprising: (a) loading a composition containing the polypeptide onto a first immobilized metal affinity chromatography (IMAC) matrix; (b) eluting the polypeptide from the first IMAC matrix with an elution buffer to form a first eluate containing the polypeptide; (c) performing a buffer exchange to remove the elution buffer from the first eluate; (d) loading the first eluate from step (c) onto a second IMAC matrix; and (e) eluting the polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate containing the polypeptide. In some embodiments, the methods further comprise performing size exclusion chromatography (SEC) on the second eluate. In some cases, the method further comprises detecting the polypeptide in the first eluate or the second eluate or after SEC, optionally by electrophoresis or mass spectrometry. In some embodiments, the first IMAC matrix comprises agarose beads and a chelating ligand bound to nickel. In some embodiments, the first IMAC matrix retains nickel upon exposure to cell culture medium. In some embodiments, the second IMAC matrix comprises agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel. In some cases, the method further comprises regenerating the first IMAC matrix and / or the second IMAC matrix by washing with a regeneration buffer. In some such cases, the regeneration buffer comprises sodium hydroxide. In some embodiments, the polypeptide comprises a polyhistidine tag (His-tag). In some embodiments, the polypeptide is an antibody, a cellular receptor, an intracellular protein, a secreted protein, or a membrane protein. In some embodiments, the polypeptide is a secreted protein. In some embodiments, buffer exchange comprises passing the first eluate through a desalting column.In some embodiments, the first elution buffer and the second elution buffer are the same. In some cases, the composition comprises cell culture medium. In some cases, the cell culture medium is a conditioned cell culture medium. In some cases, the method further comprises washing the first IMAC matrix with a wash buffer after step (a) and before step (b). In some cases, the method further comprises washing the second IMAC matrix with a wash buffer after step (d) and before step (e). In some cases, a controller directs flow through a column comprising the first IMAC matrix and the second IMAC matrix, and the method is automated. In some cases, the method further comprises determining elution time windows for the first eluate and the second eluate. The disclosure herein also relates to polypeptides purified according to the methods herein.

[0004] The present disclosure also includes a system for purifying a polypeptide. In some embodiments, the system includes: (a) an injection valve for injecting a composition containing the polypeptide into the system, the injection valve being connected to a first immobilized metal affinity chromatography (IMAC) matrix and a pump to control the flow of the sample through the first IMAC matrix; (b) a column valve for controlling the flow paths of an equilibration buffer, an elution buffer, and an SEC buffer; (c) a desalting column configured to receive the flow-through or eluate from the first IMAC matrix; (d) a second IMAC matrix configured to receive the flow-through or eluate from the desalting column; (e) a size-exclusion chromatography (SEC) column configured to receive the flow-through or eluate from the second IMAC matrix; and (f) a detection device for detecting the polypeptide in the system. In some cases, the injection valve and the column valve can be automatically controlled. In some cases, the first IMAC matrix and / or the second IMAC matrix are IMAC columns. In some cases, the detection device is a UV spectrometer or a mass spectrometer.

[0005] The present disclosure further includes, among other things, a method for purifying a His-tagged polypeptide, the method comprising: (a) loading a composition comprising the His-tagged polypeptide onto a first immobilized metal affinity chromatography (IMAC) matrix; (b) eluting the His-tagged polypeptide from the first IMAC matrix with an elution buffer to form a first eluate comprising the polypeptide; (c) performing buffer exchange to remove the elution buffer from the first eluate using a desalting column; (d) loading the first eluate from step (c) onto a second IMAC matrix, the second IMAC matrix comprising agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel; and (e) eluting the His-tagged polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate comprising the His-tagged polypeptide. In some embodiments, the method further comprises performing size exclusion chromatography (SEC) on the second eluate to collect the purified polypeptide. In some cases, the method further includes detecting the purified polypeptide collected from the SEC, optionally by electrophoresis or mass spectrometry. In some cases, the first IMAC matrix includes agarose beads and a chelating ligand bound to nickel. In some cases, the first IMAC matrix retains nickel upon exposure to cell culture medium. In some cases, the second IMAC matrix includes agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel. The present disclosure also includes polypeptides purified according to such methods. In some embodiments, the polypeptide is a secreted polypeptide.

[0006] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the claims.

[0007] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate specific embodiments and, together with the description, serve to further explain certain principles described herein. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows a flow chart for a typical conventional purification of secreted His-tagged polypeptides.

[0009] [Figure 2A] 1 shows the elution profile of a His-tagged polypeptide from a Ni Excel column at different concentrations of imidazole in conditioned medium.

[0010] [Figure 2B] A gel electrophoresis panel of the main fractions from tests with 0 mM imidazole, 10 mM imidazole, and 20 mM imidazole is shown.

[0011] [Figure 3A] 1 shows the elution profile of a His-tagged polypeptide from a Ni Excel column.

[0012] [Figure 3B] FIG. 3B shows a gel electrophoresis panel of the main NiExcel peak elution fractions from the study of FIG. 3A.

[0013] [Figure 4] 1 shows the elution profile for a two-step purification method for a His-tagged polypeptide, and an expanded plot of the portion of the profile where the His-tagged polypeptide and impurities elute, as shown in the graph below.

[0014] [Figure 5A]A flowchart of a portion of an exemplary two-step purification method herein is shown, which involves the use of a Ni Excel column to purify a polypeptide from BEVS culture medium, followed by further purification on a Superdex® SEC column, and regeneration of the Ni Excel column.

[0015] [Figure 5B] Shown is an overlay of elution profiles for the same polypeptide and multiple runs on gel electrophoresis obtained from purification of a highly expressed His-tagged polypeptide in BEVS medium as shown in Figure 5A.

[0016] [Figure 6A] 1 shows the Ni Excel elution profile of a low-expressing His-tagged polypeptide in BEVS medium.

[0017] [Figure 6B] Figure 6A shows the gel electrophoresis results of the low-expressing His-tagged polypeptide and demonstrates the low purity of the Ni-Excel elution, which is compatible with BEVS medium, indicating that the Superdex® SEC column alone is not sufficient to remove all of the contaminants in the Ni-Excel eluted material.

[0018] [Figure 7A] 1 shows the elution profile of a highly expressed His-tagged polypeptide in CHO medium purified using a Ni Excel column without further SEC purification.

[0019] [Figure 7B] Gel electrophoresis of the eluate from a Ni Excel column loaded with a highly expressed His-tagged polypeptide in CHO medium as shown in Figure 7A shows that the Ni Excel column pulls down some impurities as well as the desired His-tagged polypeptide.

[0020] [Figure 8A]7A and 7B show elution profiles after SEC purification of the His-tagged polypeptides of FIGS. 7A and 7B.

[0021] [Figure 8B] 8B shows gel electrophoresis results of the eluate from the SEC purification in FIG. 8A, demonstrating that impurities are not efficiently removed by SEC.

[0022] [Figure 9] FIG. 1 shows one embodiment of a system for purifying His-tagged polypeptides by a two-step process using two IMACs, in this case Ni Excel, followed by NiNTA, followed by SEC.

[0023] [Figure 10] Figure 9 shows the elution profile and gel electrophoresis results of the two-step purification process and four-column system shown in Figure 9. Low-expressing His-tagged polypeptides in BEVS medium were purified.

[0024] [Figure 11A] FIG. 1 shows the purification of a protein expressed in insect cells in a baculovirus expression system (BEVS) buffer using a four-column system: NiExcel, desalting, NiNTA, and Superdex® 75, and provides a flowchart of the purification process. [Figure 11B]

[0023] Figure 1 shows the purification of a protein expressed in insect cells in a baculovirus expression system (BEVS) buffer using a four-column system: NiExcel, desalting, NiNTA, and Superdex® 75. The elution profile and electrophoresis gel of fractions eluted from the columns are shown.

[0025] [Figure 12A] 1 shows the purification of a protein expressed in CHO cells in a four-column, NiExcel, desalting, NiNTA, and Superdex® 200 system and provides a flow chart of the purification process. [Figure 12B]1 shows the purification of a protein expressed in CHO cells in a four-column system, NiExcel, desalting, NiNTA, and Superdex® 200, showing the elution profile and electrophoresis gel of fractions eluted from the columns. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of Specific Embodiments 1.Definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0027] In this application, the use of "or" means "and / or" unless expressly stated otherwise. In the context of multiple dependent claims, the use of "or" refers back to multiple preceding independent or dependent claims in the alternative only. Also, terms such as "element" or "component" encompass both elements and components comprising a single unit and elements and components comprising multiple subunits, unless otherwise specified.

[0028] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0029] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure that can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

[0030] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0031] As used herein, a "composition" containing a polypeptide to be purified broadly includes at least one polypeptide of interest and one or more contaminants or impurities, such as other polypeptides and / or other non-polypeptide molecules. In some embodiments, the composition is derived from a cell culture (i.e., a culture from cells used to express the polypeptide), such as a cell lysate or clarified cell lysate, or other type of sample that is expressed in cells and provides the polypeptide to be purified. In other cases, the composition may be derived from a polypeptide synthesis reaction. In still other cases, the composition may be derived from an organism, such as a plant or animal, used to express the polypeptide of interest, such as a sample collected from the organism. The composition may be "partially purified" (i.e., has been subjected to one or more prior purification steps, such as chromatography), or may be obtained directly from the host cell or organism that produces the polypeptide (e.g., the composition is a homogenate or harvested cell culture fluid or lysate).

[0032] "Contaminants" or "impurities" are materials that differ from the desired polypeptide product. Contaminants may be, but are not limited to, variants, fragments, aggregates, or derivatives of the desired polypeptide (e.g., variants without His-tags where the desired polypeptide is His-tagged), other polypeptides, nucleic acids, endotoxins, nucleic acids, lipid and membrane components, cellular debris, small molecule chemicals, and other materials that may be found in the preparation of the polypeptide in a cell or synthetically.

[0033] "Affinity chromatography" or "affinity-based capture" refers to a method of separation based on specific interactions between molecules of an affinity column and a particular polypeptide to be purified, such as binding of the polypeptide to a ligand, binding of a His tag or other peptide tag to a metal ion, or to a specific antibody disposed on a chromatography matrix, etc.

[0034] As used herein, "immobilized metal affinity chromatography (IMAC)" refers to a type of affinity-based capture in which polypeptides are separated from contaminants according to their affinity for metal ions coordinated to ligands on an insoluble matrix. In some embodiments, the IMAC matrix is provided in a column, i.e., an "IMAC column." In some embodiments, the metal ions are nickel ions.

[0035] The term "matrix" is used herein to refer to a chromatography material, such as an affinity chromatography material. In some embodiments, the matrix may comprise beads or particles comprising a material to which a polypeptide can selectively bind, such as comprising a chelating ligand bound to nickel. In some embodiments, the matrix of affinity chromatography material may be disposed in a column through which the material to be purified can flow. In other cases, the matrix of affinity chromatography material may be disposed in a spin column, or may be disposed on a plate or chip or other device. In some cases, the matrix may comprise particles, such as beads or magnetic particles, that can be separated from a solution, for example, by the introduction of a magnet.

[0036] "Size exclusion chromatography (SEC)" is a chromatographic method that separates molecules according to size.

[0037] As used herein, "eluate" refers to material eluted from a chromatography matrix or column by application of an elution buffer.

[0038] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural and / or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. These terms also include polymers of amino acids that have modifications, such as glycosylation, sialylation, or that are complexed with other molecules.

[0039] In some embodiments, the polypeptide or protein to be purified is an antibody. The term "antibody" is used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, diabodies, and antigen-binding fragments such as Fv, scFv, Fab, and (Fab')2, so long as they exhibit antibody-antigen binding activity.

[0040] The term "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0041] The terms "isolated" or "purified" polypeptide or protein refer to a polypeptide that has been at least partially separated from one or more contaminants. In some embodiments, the polypeptide is purified to greater than 80%, 90%, 95%, or 99% purity, as determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing protein and antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0042] The term "unpurified polypeptide" or "unpurified protein," as used herein with respect to the methods herein, is a polypeptide or protein that has not been subjected to any chromatography or other process intended to at least partially separate the polypeptide from contaminants. For example, an unpurified polypeptide may be found in a cell lysate, a solution following peptide synthesis, etc.

[0043] The term "buffer exchange" refers to the process of replacing the buffer in which a polypeptide to be purified is found, such as to reduce the concentration of salt in the buffer or to remove components that may interfere with one or more chromatographic processes. In some embodiments, buffer exchange may be performed on a filter or column, such as a "desalting column."

[0044] A "desalting column" is a size-exclusion chromatography column with a molecular weight cutoff lower than the molecular weight of the polypeptide to be purified, so that a higher salt buffer can be removed from the polypeptide to be purified. In some embodiments, such a column may have a molecular weight cutoff of, for example, 17,000 to 90,000 Da, depending on the polypeptide to be purified.

[0045] As used herein, an "equilibration buffer" is an aqueous buffer that is compatible with the polypeptide to be purified and that promotes interaction and binding between metal ions and the polypeptide in a chromatography column, such as an IMAC column.

[0046] An "elution buffer" is used to elute (i.e., remove) a polypeptide bound to a column or matrix, such as an IMAC or SEC matrix. The elution buffer may elute the polypeptide from the matrix based on, for example, conductivity, pH, charge, ionic strength, etc.

[0047] A "regeneration buffer" may be used to regenerate a chromatography matrix, such as an IMAC matrix, so that it may be reused.

[0048] As used herein, an "automated" or "automatically controlled" process is one that can be performed by a computer-controlled system with appropriate software, as opposed to a system that requires active manual intervention during or between at least one step, e.g., to move an analyte-containing sample from one part of the system to another. In some embodiments, the process is automated by software that controls the movement or position of one or more pumps, valves, and / or tees during the course of the process, which in turn controls the flow of buffers and elution through the system.

[0049] 2. Method This disclosure relates to methods and systems for purifying, for example, polypeptides. As described in more detail in the Examples below, His tags and His tag affinity resins are often used to generate highly pure protein reagents. However, His-tagged secreted proteins in cell culture media cannot be directly loaded onto many nickel-containing IMAC matrices because the media chelates nickel from the resin, thereby limiting options for how such proteins can be purified. A traditional low-throughput method to solve this problem is to perform a buffer exchange before loading the conditioned media onto the IMAC matrix. Each buffer exchange is a time-consuming, highly manual process that makes the purification of secreted His-tagged proteins low-throughput. Certain IMAC matrices, such as Ni-excel, are not stripped by the conditioned media, making them suitable for direct loading without the need for buffer exchange. However, we have found that such matrices nonspecifically bind contaminating proteins in cell culture media compositions, preventing the impurities from being fully removed by further purification steps. For example, even with an additional size exclusion (SEC) step, uptake of mammalian or insect cell culture media, such as CHO medium, and baculovirus (BEVS) containing His-tagged proteins at low expression levels directly onto Ni-Excel resulted in very low-purity material. For polypeptides expressed in CHO medium, both high and low expression showed significant nonspecific binding that could not be completely removed by SEC. We tested the addition of low concentrations of imidazole to the conditioned medium before uptake and found that this did not result in an increase in purity and also resulted in a significant decrease in protein yield.

[0050] The methods disclosed herein solve these myriad problems in purifying His-tagged polypeptides from cell culture media, such as mammalian cell culture media, such as BEVS media and CHO media, and insect cell culture media. In some embodiments, the methods include (a) loading a composition comprising the polypeptide onto a first immobilized metal affinity chromatography (IMAC) matrix, (b) eluting the polypeptide from the first IMAC matrix with an elution buffer to form a first eluate comprising the polypeptide, (c) performing a buffer exchange to remove the elution buffer from the first eluate, (d) loading the first eluate from step (c) onto a second IMAC matrix, and (e) eluting the polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate comprising the polypeptide.

[0051] In some embodiments, the composition is derived from a cell culture, i.e., a host cell expressing the polypeptide to be purified. Thus, the composition from which the polypeptide is to be purified may be a cell lysate, a clarified cell lysate, or the like. Thus, in some embodiments, the composition comprises a cell culture medium. In some embodiments, the cell culture medium may be an artificial medium, a serum-containing medium, a serum-free medium, a chemically defined medium, or a protein-free medium. Examples of cell culture media include, but are not limited to, media used with mammalian cells and insect cells. Examples of such media include baculovirus expression vector system (BEVS) medium, Chinese hamster ovary (CHO) medium, N293 cell medium, RPMI 1640 medium, and Dulbecco's modified Eagle's medium (DMEM). In some embodiments, the cell culture medium may be a conditioned cell culture medium. The conditioned cell culture medium contains proteins and / or cytokines secreted by the cells. In some embodiments, the composition may also contain non-protein contaminants, such as nucleic acids and lipids. In other embodiments, the composition may be from a protein synthesis chemical reaction and may contain contaminants such as incompletely reacted polypeptide components.

[0052] In some embodiments, the polypeptide comprises a polyhistidine tag (His-tag) or other suitable tag that allows specific binding to an IMAC matrix, such as a matrix containing a chelating ligand that binds to nickel. His-tagged polypeptides can be produced in any suitable cell or added during a peptide synthesis reaction. Examples of cells capable of expressing His-tagged polypeptides include, but are not limited to, bacteria, insect cells, E. coli, or mammalian cells. In some embodiments, His-tagged polypeptides are produced in insect cells using BEVS medium or in CHO cells using CHO medium. CHO medium generally comprises Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum or equivalent serum and may further comprise L-glutamine and antibiotics such as penicillin and streptomycin to prevent bacterial growth. In some cases, CHO medium comprises DMEM, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. BEVS medium generally contains RPMI 1640 medium supplemented with FBS or an equivalent serum, e.g., 10% FBS in some cases, and may contain surfactants such as Pluronic® F68, L-glutamine, penicillin, and streptomycin. In some cases, BEVS medium also contains 100 U / mL penicillin and 100 μg / mL streptomycin. Media such as BEVS medium and CHO medium also contain essential amino acids and buffers to closely mimic the composition of mammalian cells.

[0053] The polypeptide to be purified may be any His-tagged polypeptide capable of binding to the IMAC matrix used herein. Examples of polypeptides include, but are not limited to, antibodies, cellular receptors, intracellular proteins, secreted proteins, membrane proteins, etc. In some cases, the polypeptide may be an antigen or target for antibody binding. In some embodiments, the polypeptide is secreted from cells in cell culture, i.e., is a secreted polypeptide.

[0054] The loading of the polypeptide-containing composition onto the first IMAC matrix can be accomplished by any known means available to those skilled in the art. For example, the composition can be loaded in an automated manner, in which a pump draws the composition from a reservoir containing the composition and pumps the fluid onto a column containing the first IMAC matrix. In some embodiments, the composition can be loaded into a tube (i.e., a loop), and the pump directs buffer from the reservoir through the tube containing the composition to the column containing the first IMAC matrix. Alternatively, the composition can be loaded (i.e., injected) into the first IMAC matrix manually or by other methods that do not utilize a pump, or generally by gravity flow.

[0055] In some embodiments, the first IMAC matrix contains a chelating ligand that binds to nickel ions. Polypeptides containing a His tag or similar tag that bind to the nickel-containing matrix are contacted with the first IMAC matrix and allowed to bind to the matrix until they are eluted using an elution buffer. In some embodiments, the first IMAC matrix contains agarose beads and a chelating ligand that binds to nickel. In some embodiments, the chelating ligand on the first IMAC matrix is an oxidation-resistant proteinaceous ligand that binds polyhistidine. In some embodiments, the chelating ligand contains a sulfopropyl, sulfoethyl, carboxymethyl, trimethylammonium, or diethylaminoethyl group.

[0056] In some embodiments, the first IMAC matrix retains nickel when exposed to BEVS or CHO medium. In some cases, the cell culture medium can contain a stripping agent that, when exposed to a nickel-containing IMAC matrix, results in the nickel being washed or leached from the matrix. In some embodiments, the first IMAC matrix is any matrix described in EP 1276716, which is incorporated herein by reference in its entirety. In some embodiments, the first IMAC matrix is a matrix sold under the tradename Ni Sepharose™ Excel or HisTrap™ Excel (Sigma-Aldrich Co.) (i.e., Ni Excel herein) or an equivalent of such a matrix.

[0057] In some embodiments, the first IMAC matrix is contained within a column. In some embodiments, the first IMAC column has a bed volume of 2 to 20 ml. In some embodiments, the first IMAC column has a bed volume of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. In other embodiments, the first IMAC matrix is contained within a spin column and may have a bed volume of, for example, 0.5, 0.75, 1, or 2 ml.

[0058] In some embodiments, after the polypeptide is loaded onto the first IMAC matrix, the first IMAC matrix can be washed with a wash buffer. In some embodiments, the wash buffer comprises imidazole. The amount of imidazole in the wash buffer can range from 0 to 20 mM. In some embodiments, the amount of imidazole in the wash buffer can range from 5 to 15 mM. In some embodiments, the amount of imidazole in the wash buffer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM. In some embodiments, the wash buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the wash buffer can range from 20 to 70 mM. In some embodiments, the amount of Na3PO4 in the wash buffer can range from 40 to 60 mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be 45, 50, or 55 mM. In some embodiments, the wash buffer comprises sodium chloride (NaCl). The amount of NaCl in the wash buffer can range from 5 to 35 mM. In some embodiments, the amount of NaCl in the wash buffer can range from 15 to 25 mM. In some embodiments, the wash buffer can contain a surfactant. In some embodiments, the amount of NaCl in the wash buffer can be 15, 20, or 25 mM. In some embodiments, the wash buffer contains 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole. In some embodiments, the wash buffer contains 50 mM Na3PO4, 20 mM NaCl, 10 mM imidazole, and 0.1% Triton X-114. In some embodiments, the amount of wash buffer used to wash the first IMAC column can range from 1 to 5 times the column bed volume. In some embodiments, the IMAC matrix can be first washed with a buffer without a surfactant, followed by a buffer containing a surfactant.

[0059] The polypeptide can be eluted from the first IMAC matrix using an elution buffer. In some embodiments, the elution buffer comprises imidazole. The amount of imidazole in the elution buffer can range from 0 to 1000 mM. In some embodiments, the amount of imidazole in the elution buffer can range from 100 to 1000 mM, 100 to 800 mM, 200 to 600 mM, or 300 to 500 mM. In some embodiments, the amount of imidazole in the elution buffer can be 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 mM. In some embodiments, the elution buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the elution buffer can range from 20 to 70 mM. In some embodiments, the amount of Na3PO4 in the elution buffer can range from 40 to 60 mM. In some embodiments, the amount of Na3PO4 in the elution buffer can be 45, 50, or 55 mM. In some embodiments, the elution buffer comprises sodium chloride (NaCl). The amount of NaCl in the elution buffer can range from 5 to 35 mM. In some embodiments, the amount of NaCl in the elution buffer can range from 15 to 25 mM. In some embodiments, the amount of NaCl in the elution buffer can be 15, 20, or 25 mM. In some embodiments, the elution buffer comprises 50 mM Na3PO4, 20 mM NaCl, and 400 mM imidazole. In some embodiments, the volume of elution buffer used to elute the polypeptide from the first IMAC column can range from 1 to 5 times the bed volume of the column.

[0060] In some embodiments, the method includes buffer exchange to separate the elution buffer in the first eluate from the polypeptide of interest. In this step, for example, if imidazole is utilized in the elution buffer or a high salt concentration is used in the elution buffer, such imidazole or salt can be substantially removed in preparation for loading the first eluate onto a second IMAC matrix. In some embodiments, the buffer exchange includes passing the first eluate through a desalting column. The polypeptide passes through the desalting column faster than lower molecular weight molecules, thus allowing the polypeptide to be collected while retaining lower molecular weight species such as imidazole. In some embodiments, the desalting column has a bed volume of 2 to 20 ml. In some embodiments, the desalting column has a bed volume of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. Other means for achieving buffer exchange include using dialysis or ultrafiltration, using membranes or filters that trap polypeptides but allow buffer components and small molecules to pass through.

[0061] After the buffer exchange step, the first eluate is loaded onto a second IMAC matrix. The loading of the first eluate onto the second IMAC matrix can be performed similarly to the way the composition was loaded onto the first IMAC matrix. In some embodiments, the eluate from the desalting column is collected in a tube (i.e., a loop) that is in line with the second IMAC matrix. Once the eluate is collected in the tube, a pump can direct the fluid to the second IMAC matrix.

[0062] In some embodiments, the second IMAC matrix contains nickel. In some embodiments, the second IMAC matrix contains agarose beads and a nitrilotriacetic acid chelating moiety (Ni NTA) that binds to nickel. The nitrilotriacetic acid (NTA) chelating moiety is attached to the agarose beads. In other embodiments, the first IMAC and second IMAC are the same. Any His-tagged polypeptide that can bind to the nickel chelate in the first eluate can be bound to the nickel on the second IMAC matrix. In some embodiments, the second IMAC matrix is contained within a column. In some embodiments, the second IMAC column has a bed volume of 2 to 20 ml. In some embodiments, the second IMAC column has a bed volume of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. In other embodiments, the second IMAC matrix is contained within a spin column and may comprise, for example, a bed volume of 0.5, 0.75, 1, or 2 ml.

[0063] In some embodiments, after the polypeptide is incorporated into the second IMAC matrix, the second IMAC matrix can be washed with a wash buffer. In some embodiments, the wash buffer comprises imidazole. The amount of imidazole in the wash buffer can range from 0 to 20 mM. In some embodiments, the amount of imidazole in the wash buffer can range from 5 to 15 mM. In some embodiments, the amount of imidazole in the wash buffer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM. In some embodiments, the wash buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the wash buffer can range from 20 to 70 mM. In some embodiments, the amount of Na3PO4 in the wash buffer can range from 40 to 60 mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be 45, 50, or 55 mM. In some embodiments, the wash buffer comprises sodium chloride (NaCl). The amount of NaCl in the wash buffer can range from 5 to 35 mM. In some embodiments, the amount of NaCl in the wash buffer can range from 15 to 25 mM. In some embodiments, the amount of NaCl in the wash buffer can be 15, 20, or 25 mM. In some embodiments, the wash buffer comprises 50 mM Na3PO4, 20 mM NaCl, 10 mM imidazole, and 0.1% Triton X-114. In some embodiments, the wash buffer can contain a surfactant. In some embodiments, the wash buffer comprises 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole. In some embodiments, the amount of wash buffer used to wash the second IMAC column can range from 1 to 5 times the bed volume of the column. In some embodiments, the IMAC matrix can be washed first with a buffer without a surfactant, followed by a buffer containing a surfactant.

[0064] The polypeptide can be eluted from the second IMAC matrix using an elution buffer. In some embodiments, the elution buffer comprises imidazole. The amount of imidazole in the elution buffer can range from 0 to 1000 mM. In some embodiments, the amount of imidazole in the elution buffer can range from 100 to 1000 mM, 100 to 800 mM, 200 to 600 mM, or 300 to 500 mM. In some embodiments, the amount of imidazole in the elution buffer can be 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 mM. In some embodiments, the elution buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the elution buffer can range from 20 to 70 mM. In some embodiments, the amount of Na3PO4 in the elution buffer can range from 40 to 60 mM. In some embodiments, the amount of Na3PO4 in the elution buffer can be 45, 50, or 55 mM. In some embodiments, the elution buffer comprises sodium chloride (NaCl). The amount of NaCl in the elution buffer can range from 5 to 35 mM. In some embodiments, the amount of NaCl in the elution buffer can range from 15 to 25 mM. In some embodiments, the amount of NaCl in the elution buffer can be 15, 20, or 25 mM. In some embodiments, the elution buffer comprises 50 mM Na3PO4, 20 mM NaCl, and 400 mM imidazole. In some embodiments, the volume of elution buffer used to elute the polypeptide from the second IMAC column can range from 1 to 5 times the bed volume of the column. In some embodiments, the elution buffer used for the first IMAC matrix and the second IMAC matrix is the same.

[0065] In some embodiments, the time at which the polypeptide elutes from the first and second IMAC columns can be determined empirically using UV absorbance of the eluate fractions at a wavelength suitable for observing the polypeptide, such as 280 nm. The elution time window can be determined, and the correct fractions can be collected. For example, during a particular elution time window, the eluate can be collected in a tube (i.e., a loop) for processing through the next column. Outside the elution time window, the eluate can be directed to a waste collector. In some embodiments, the eluate can be further analyzed, for example, by gel electrophoresis or mass spectrometry or other detection techniques, to assess its purity and yield.

[0066] If necessary, the second eluate can be further purified using other chromatographic methods, such as size exclusion chromatography (SEC) or anion or cation exchange. In some embodiments, the method includes performing SEC on the second eluate. Performing SEC can include loading the second eluate onto an SEC column. The specific pore size of the solid phase in the SEC column can be selected based on the molecular weight of the polypeptide. For example, in some embodiments, Superdex® 75 or Superdex® 200, or equivalent, can be used as the SEC matrix. In some embodiments, the eluate from the SEC matrix can be collected into different fractions for subsequent analysis, such as on a 96-well plate.

[0067] In some embodiments, the method includes detecting the purified polypeptide collected from either the first IMAC eluate or the second IMAC eluate, or from the SEC eluate if SEC is performed. Various methods can be used to detect elution of the polypeptide from the SEC column. In some embodiments, UV spectroscopy can be used to detect elution of the polypeptide from the SEC column. Other methods include, but are not limited to, electrophoresis or mass spectrometry.

[0068] In some embodiments, the method includes regenerating the first IMAC matrix and / or the second IMAC matrix, and any additional chromatography matrix, such as SEC, if used, by washing with a regeneration buffer. In some embodiments, the regeneration buffer comprises sodium hydroxide. In some embodiments, the regeneration buffer comprises 0.1-1 M sodium hydroxide. In some embodiments, the regeneration buffer comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 M sodium hydroxide. In some embodiments, equilibration buffer is then run over the matrix to remove such sodium hydroxide and prepare the matrix for a new polypeptide purification process.

[0069] In some embodiments, a method for purifying a His-tagged polypeptide described herein includes (a) loading a composition containing the His-tagged polypeptide onto a first immobilized metal affinity chromatography (IMAC) matrix, (b) eluting the His-tagged polypeptide from the first IMAC matrix with an elution buffer to form a first eluate containing the polypeptide, (c) performing buffer exchange to remove the elution buffer from the first eluate using a desalting column, (d) loading the first eluate from step (c) onto a second IMAC matrix, the second IMAC matrix comprising agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel, and (e) eluting the His-tagged polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate containing the His-tagged polypeptide. In some such embodiments, the first IMAC matrix comprises a chelating ligand bound to nickel ions. Polypeptides containing a His tag or similar tag that bind to a nickel-containing matrix are contacted with a first IMAC matrix and allowed to bind to the matrix until they are eluted using an elution buffer. In some embodiments, the first IMAC matrix comprises agarose beads and a chelating ligand that binds to nickel. In some embodiments, the chelating ligand on the first IMAC matrix is an oxidation-resistant proteinaceous ligand that binds polyhistidine. In some embodiments, the chelating ligand comprises a sulfopropyl, sulfoethyl, carboxymethyl, trimethylammonium, or diethylaminoethyl group. In some embodiments, the first IMAC matrix retains nickel when exposed to BEVS or CHO medium. For example, in some cases, the cell culture medium can contain a stripping agent that, when exposed to a nickel-containing IMAC matrix, results in the nickel being washed or leached from the matrix.In some embodiments, the first IMAC matrix is any matrix described in EP 1276716, which is incorporated herein by reference for all that it contains. In some embodiments, the first IMAC matrix is a matrix sold under the trade name Ni Sepharose™ Excel or HisTrap™ Excel (Sigma-Aldrich Co.) (i.e., Ni Excel herein) or an equivalent of such a matrix.

[0070] In some embodiments, the first IMAC matrix and the second IMAC matrix are contained within a column. In some embodiments, the first IMAC matrix and the second IMAC matrix are columns having a bed volume of 2 to 20 ml. In some embodiments, the first IMAC column and the second IMAC column have a bed volume of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. Such a process may further include a post-chromatographic SEC step using the two IMAC matrices.

[0071] In some embodiments, where the IMAC matrix and / or the SEC matrix are provided on columns, a controller directs flow through columns containing the first IMAC matrix and the second IMAC matrix, and the method is automated. In some embodiments, a controller directs flow through columns containing the first IMAC matrix, a desalting column, a second IMAC matrix, and an SEC column, and the method is automated.

[0072] The flow rates of the composition, buffer, and eluent through the columns can be selected based on the size of the column. The flow rates through the columns (IMAC, desalting, SEC) can be the same or different. In some embodiments, the flow rates through the first and second IMAC columns can be 2-8 ml / min. In some embodiments, the flow rates through the first and second IMAC columns can be 5 ml / min. In some embodiments, the flow rate through the desalting column can be 5-15 ml / min. In some embodiments, the flow rate through the first and second IMAC columns can be 10 ml / min.

[0073] In some embodiments, the methods described herein are advantageous over conventional methods using a single IMAC column in that they eliminate the need to first buffer exchange a starting composition containing cell culture medium, such as BEVS or CHO medium, before the composition is loaded onto an IMAC matrix, such as a column. Such a buffer exchange process to remove cell culture medium can be relatively time-consuming. At the same time, the processes described herein enable the purification of more His-tagged polypeptides than conventional 1-IMAC methods, as seen in the examples and figures of the present disclosure. The methods described herein are compatible with both highly and poorly expressed polypeptides from a particular cell culture host cell. The methods described herein may also be partially or fully automated. Automated methods may also enable high-throughput protein preparations that would otherwise be impossible, particularly when starting compositions, such as cell lysates, must be loaded onto an IMAC matrix in batches or when cell lysates must first be buffer-exchanged prior to chromatography to be compatible with the IMAC composition. Furthermore, as described in more detail in the following sections, systems can be designed to implement the methods described herein so that several large-scale polypeptide expressions can be purified in parallel with minimal hands-on effort.

[0074] 3. System The present disclosure also relates to systems capable of performing the methods described herein, including those described in the preceding sections. In some cases, the system includes: (a) an injection valve for injecting a composition containing a polypeptide into the system, the injection valve being connected to a first immobilized metal affinity chromatography (IMAC) matrix and a pump to control the flow of the sample through the first IMAC matrix; (b) a column valve for controlling the flow paths of an equilibration buffer, an elution buffer, and an SEC buffer; (c) a desalting column or similar device used for buffer exchange configured to receive the flow-through or eluate from the first IMAC matrix; (d) a second IMAC matrix configured to receive the flow-through or eluate from the desalting column; and (f) a detection device, such as a UV spectrometer or mass spectrometer, for detecting the polypeptide in the system. In some cases, the system further includes an additional chromatography column for further purification of the polypeptide, such as (e) a size-exclusion chromatography (SEC) column configured to receive the flow-through or eluate from the second IMAC matrix. In some cases, the injection valve and column valve can be automatically controlled.

[0075] 9 shows an embodiment of an exemplary system 10 for purifying a polypeptide according to the methods herein. For example, system 10 includes four separate columns: two IMAC columns; (a) an injection valve 11 for injecting a polypeptide-containing composition 12 into system 10, said injection valve 11 being connected to a first immobilized metal affinity chromatography (IMAC) column 13 and a pump to control the flow of the sample through first IMAC column 13; (b) a column valve 14 for controlling the flow path of equilibration buffer 15, elution buffer 16, and SEC buffer; (c) a desalting column 17 configured to receive the flow-through or eluate from first IMAC column 13; (d) a second IMAC column 18 configured to receive the flow-through or eluate from desalting column 17; (e) a size-exclusion chromatography (SEC) column 19 configured to receive the flow-through or eluate from second IMAC column 18; and (f) a detection device 20 for detecting polypeptides within the system. Optionally, the system may also include a mixer (M in FIG. 9) for mixing a mobile phase buffer gradient if such a gradient is utilized in the chromatographic method.

[0076] In some embodiments, the system incorporates the controller, pump, and interface of a commercially available multi-column chromatography system, such as the AKTA chromatography system (Cytiva). Each column can be connected in series to the tubing of such a chromatography system. In some embodiments, the injection valves and column valves can be automatically controlled to direct buffers through the appropriate columns at the appropriate times.

[0077] As previously mentioned, the systems disclosed herein may have certain advantages over systems utilizing only one IMAC matrix. For example, the entire starting composition may be incorporated into the IMAC matrix without breaking it down into batches. In some embodiments, it is not necessary to first exchange the buffer of the starting composition, including cell culture medium such as BEVS or CHO medium, before the composition is incorporated into the IMAC matrix, while at the same time enabling the purification of more His-tagged polypeptides than traditional 1-IMAC methods, as seen in the examples and figures of the present disclosure. The systems disclosed herein may be used with both highly and poorly expressed polypeptides from a particular cell culture host cell. Automated systems may also enable high-throughput protein preparations that would otherwise be impossible, particularly when starting compositions such as cell lysates must be incorporated into an IMAC column or other matrix in batches, or when the cell lysate must first be buffer-exchanged prior to chromatography to make it compatible with the IMAC composition. Furthermore, a system such as that shown in Figure 9 may implement the methods herein so that several large-scale polypeptide expressions can be purified in parallel with minimal hands-on effort. For example, a 5 mL Ni Excel column used in the methods and systems herein has been used in approximately 30 1 L runs of compositions in CHO or BEVS media over a 3 month period without color change or loss of capacity. [Example]

[0078] Example 1. Imidazole effect on protein yield from Ni Excel The effect of adding up to 20 mM imidazole on protein purity and yield from a Ni Excel column (Cytiva catalog number 17371201) was tested by loading a composition of a polypeptide to be purified in CHO medium onto the column with 0, 10, or 20 mM imidazole. Imidazole can generally be added when performing metal ion affinity chromatography to purify a specific polypeptide, as it can aid in the removal of impurities resulting from nonspecific binding to the chromatography matrix. Filtered medium (over a 0.2 micron filter) was loaded directly onto the column. No additives were added. The column was pre-equilibrated with 50 mM sodium phosphate, 200 mM NaCl, and 10 mM imidazole, pH 8, 2 mM NaN3. This same buffer was then used to wash the column. A second wash was performed with 50 mM Tris Na3PO4, 200 mM NaCl, 10 mM imidazole, 2 mM NaN3, and 0.1% Triton-X (TX) 114. Bound polypeptides were eluted with a buffer containing 50 mM Tris Na3PO4, 200 mM NaCl, 400 mM imidazole, and 2 mM NaN3. The results in Figures 2A and 2B show that increasing the imidazole concentration resulted in a loss of protein yield, as indicated by the decrease in size of the elution trace shown in Figure 2A, but no significant improvement in purity, as shown in Figure 2B. Gel electrophoresis of collected fractions is shown in Figure 2A.

[0079] The effect of washing a Ni Excel column (Cytiva) with different concentrations of imidazole on protein purity and yield was further tested using a 1 L starting composition of highly expressed, secreted His-tagged protein produced in baculovirus (BEVS) medium and loaded onto a 5 mL Ni Excel column in imidazole-free buffer. Wash buffers containing 10, 20, 30, 40, or 50 mM imidazole were tested. Each wash buffer had a pH of 8 and contained 50 mM sodium phosphate and 200 mM NaCl. The His-tagged protein was eluted from the Ni Excel column using an elution buffer containing 50 mM sodium phosphate, 200 mM NaCl, and 300–400 mM imidazole at pH 8. The Ni Excel column was regenerated using 0.5 M NaOH. A buffer containing 50 mM sodium phosphate, pH 8, 200 mM NaCl, and 10 mM imidazole was selected as the wash buffer.

[0080] Figure 3A shows the UV trace of the solution exiting the Ni Excel column. The peak at approximately 240 min corresponds to the eluted protein from the Ni Excel column. No decrease in yield was observed up to a concentration of 50 mM imidazole in the wash buffer. Figure 3B shows the elution profile of the fraction of eluted His-tagged polypeptide, corresponding to approximately 220 to 260 min shown in the trace in Figure 3A. Elution was tested with both 300 and 400 mM imidazole in the elution buffer. A buffer containing 50 mM sodium phosphate, pH 8, 200 mM NaCl, and 400 mM imidazole gave a sharper peak in the gel and was selected as the elution buffer.

[0081] Example 2. Two-step automated purification using Ni Excel and SEC (BEVS medium) A two-step automated purification system containing a Ni Excel column (Cytiva, catalog no. 17371201) and a Superdex 200 (SEC) column was tested. Figure 5A shows a flow chart of the steps and some features of the two-step procedure. Highly expressing, secreted His-tagged proteins were produced in BEVS medium and loaded onto a 5 mL Ni Excel column in a 1 L volume. The wash buffer contained 50 mM sodium phosphate, 200 mM NaCl, and 10 mM imidazole at pH 8, and the elution buffer contained 50 mM sodium phosphate, 200 mM NaCl, and 400 mM imidazole at pH 8. Figure 4 shows the separation of the His-tagged protein of interest from previously eluted impurities. The total run time for the nickel and SEC column purification was 7 hours, requiring minimal hands-on time.

[0082] In BEVS medium, when the expression level of the His-tagged protein was high, the protein eluted from the column was sufficiently pure to reach >95% purity by size exclusion (SEC) (see Figure 5B). This purification process was also performed with a low-expressing secreted protein in BEVS medium, as shown in Figures 6A-6B. As shown in these figures, this polypeptide could not be purified to a sufficiently high purity using a Ni Excel column, where SEC was sufficient to remove the remaining impurities (see Figures 6A and 6B). This may be because Ni Excel exhibits less specific binding to non-His-tagged proteins than other IMAC matrices, such as Ni NTA.

[0083] Example 3. Two-Step Automated Purification (CHO Medium)

[0084] In this example, we also tested the direct uptake of His-tagged proteins in CHO culture media. Specifically, the filtered medium (filtered through a 0.2 micron filter) containing the His-tagged heterodimeric polypeptide to be purified was directly loaded onto a Ni Excel column (Cytiva catalog number 17371201) pre-equilibrated in a buffer containing 40 mM sodium phosphate, 200 mM NaCl, and 10 mM imidazole at pH 8 with 2 mM sodium azide. This same buffer was used for the first wash of the column, followed by a second wash in 50 mM Tris-sodium phosphate, 200 mM NaCl, 10 mM imidazole, 2 mM sodium azide, and 0.1% TX 114. The polypeptide was eluted in 50 mM Tris-sodium phosphate, 200 mM NaCl, 400 mM imidazole, and 2 mM sodium azide. Figure 7A shows the UV trace at 280 nm of the solution eluted from the Ni Excel column, and Figure 7B shows the protein content in the fractions collected from the Ni Excel column analyzed by gel electrophoresis. As can be seen in Figure 7B, significant amounts of impurities were present in the fractions containing the protein of interest.

[0085] Further purification by SEC chromatography was then performed. However, the SEC procedure failed to completely remove impurities from the His-tagged polypeptide, as shown in Figures 8A and 8B. Specifically, Figure 8A shows the UV trace at 280 nm of the eluate from the SEC column, with each peak representing a subunit of the heterodimeric polypeptide, and Figure 8B shows an electrophoresis run of the eluted polypeptide species with arrows indicating the polypeptide subunit of interest. Furthermore, the white box in Figure 7A at approximately 180 minutes indicates an impurity that was able to be removed by the subsequent SEC chromatography step, while Figure 8B shows that other impurities remained.

[0086] Therefore, in CHO medium, both high and low expressers exhibit significant nonspecific binding that cannot be completely removed by SEC and requires additional purification steps. The addition of 10 or 20 mM imidazole to the conditioned medium before uptake was tested. However, this did not result in an increase in the purity of the Ni eluate, but did result in a significant decrease in protein yield.

[0087] Example 4. Four-column purification using Ni Excel, Ni NTA, and SEC Next, a system using two nickel columns in parallel was tested to determine whether such a setup would improve the purity of high and low expressers from BEVS and CHO cultures. A four-column automated purification system was tested, including a 5 mL Ni Excel column (Cytiva, catalog no. 17371201), a desalting column, a 5 mL Ni NTA column (Qiagen, catalog no. 30761), and a Superdex 200 (SEC) column. The columns were connected to a UNICORN 7.3.0 AKTA purification system. Conditioned medium was filtered over a 0.2 micron filter and loaded onto 5 mL of Ni Excel resin pre-equilibrated with 50 mM NaPO, 20 mM NaCl, and 10 mM imidazole. Once loading was complete, the column was washed with a wash buffer containing 50 mM NaPO, 20 mM NaCl, and 10 mM imidazole, followed by a second wash buffer containing 50 mM NaPO, 20 mM NaCl, 10 mM imidazole, and 0.1% Triton X-114 to remove endotoxin. Once washing was complete, the His-tagged protein was eluted with 14.5 ml of 50 mM NaPO, 20 mM NaCl, and 400 mM imidazole. The flow rate of the conditioned medium, wash buffer, and elution buffer through the Ni Excel column was set at 5 ml / min.

[0088] Before loading the His-tagged protein eluted from the Ni Excel column onto the Ni NTA column, a desalting column (Cytiva HiPrep™ 26 / 10, 53 ml bed volume, 1000–5000 molecular weight cutoff) was used for buffer exchange. Due to the high salt content of the Ni Excel eluate, the eluate could not be directly loaded onto the Ni NTA column. The desalting column was a HiPrep™ 26 / 10 desalting column (Cytiva, catalog number #17-5087-01). The 50 ml desalting column used in this method has a capacity to buffer exchange up to 15 ml of protein solution. A 10 ml loop captured the eluate from the Ni Excel column and then loaded it onto the desalting column. The set flow rate of the desalting column was 10 ml / min.

[0089] The buffer-exchanged protein from the desalting column was collected in a 20 ml loop and then loaded onto a 5 ml Ni NTA Superflow™ column pre-equilibrated with 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole. After loading, the column was washed with 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole to remove endotoxin, and then with 50 mM Na3PO4, 20 mM NaCl, 10 mM imidazole, and 0.1% Triton X-114 to remove endotoxin. After washing, the His-tagged protein was eluted with 15 ml of 50 mM Na3PO4, 20 mM NaCl, and 400 mM imidazole. The flow rate of the conditioned medium, wash buffer, and elution buffer through the Ni NTA column was set at 5 ml / min. The Ni NTA Superflow™ eluate was collected in a 7 ml loop and then loaded onto a Superdex (SEC) column.

[0090] Both nickel columns were regenerated after each run using 0.5 M NaOH, and the sample pump was also washed after each run. The system can now wait for several expressions to be purified in a hands-free, fully automated mode, without considering the expression level.

[0091] A diagram of this system is shown in Figure 9. Figure 10 shows that an embodiment of the method described herein was used to purify a His-tagged polypeptide with high yield. Despite low expression levels in cell culture, the polypeptide was purified to greater than 95% purity. Without the parallel Ni-Excel + Ni-NTA column purification method used here, it may not have been possible to purify this low-expressing polypeptide to such high purity in a high-throughput manner. The same method as above was performed, except that polypeptide purified from CHO medium was used instead of BEVS medium.

[0092] Example 5: Four-column purification of proteins expressed in insect cells In a further example, proteins in BEVS medium were purified using a 5 mL Ni Excel column (Cytiva, catalog no. 17371201), a desalting column, a 5 mL Ni NTA column (Qiagen, catalog no. 30761), and a 120 mL Superdex® 75 (SEC) column. Ten liters of cell lysate in BEVS buffer, pH 7.2, was concentrated to 1 liter. The concentrated protein in BEVS buffer was loaded onto a 5 mL NiExcel column pre-equilibrated in 50 mM sodium phosphate, pH 8.0, 200 mM NaCl, and 10 mM imidazole. The column was washed with 50 mM sodium phosphate, pH 8.0, 200 mM NaCl, 10 mM imidazole, and 0.1% Triton X114, after which the protein was eluted. The protein was eluted in 50 mM sodium phosphate, pH 8.0, 200 mM NaCl, and 400 mM imidazole. After elution, the protein was desalted on a 50 mL desalting column so that it could be loaded onto a NiNTA column. The desalted solution was then loaded onto a 5 mL NiNTA column using the same equilibration, wash, and elution buffers as above. The NiNTA column eluate was then further purified by size exclusion chromatography (SEC) on a 120 mL Superdex® 75 column in 25 mM Tris pH 7.5, 150 mM NaCl, and 2 mM EDTA (see Figure 11A). Figure 11B shows the results of the batch purification process. The protein was purified to a concentration of 2.9 mg / mL (specifically, 31.9 mg in an 11 mL volume) and had an endotoxin concentration of 0.08 U / mg. As shown by the gel in Figure 11B, the protein was highly pure after this process.

[0093] Example 6: Four-column purification of proteins expressed in CHO cells In further experiments, additional proteins were expressed in CHO cells. A 35-liter volume of protein in CHO buffer was concentrated to 3 liters and batch loaded onto a four-column system containing a 5 mL NiExcel column, a 50 mL desalting column, a 5 mL NiNTA column, and a Superdex® 200 column. The concentrated protein in CHO buffer was batch loaded onto a 5 mL NiExcel column pre-equilibrated in 50 mM sodium phosphate pH 8.0, 200 mM NaCl, and 10 mM imidazole. The column was washed with 50 mM sodium phosphate pH 8.0, 200 mM NaCl, 10 mM imidazole, and 0.1% Triton X114 before the protein was eluted. The protein was eluted in 50 mM sodium phosphate pH 8.0, 200 mM NaCl, and 400 mM imidazole. After elution, the protein was desalted in a 50 mL volume. The desalted solution was then loaded onto a 5 mL NiNTA column using the same equilibration, wash, and elution buffers. The NiNTA column eluate was then further purified by size exclusion chromatography (SEC) on a 120 mL Superdex® 200 column in 25 mM Tris pH 7.5, 150 mM NaCl, and 1 mM EDTA. (See Figure 12A.) Figure 12B shows the results of the purification, which resulted in a protein concentration of 0.67 mg / mL (16 mL containing 10.7 mg of protein).

Claims

1. 1. A method for purifying a polypeptide, comprising: (a) incorporating a composition comprising the polypeptide into a first immobilized metal affinity chromatography (IMAC) matrix; (b) eluting the polypeptide from the first IMAC matrix with an elution buffer to form a first eluate comprising the polypeptide; (c) performing a buffer exchange to remove the elution buffer from the first eluate; (d) capturing the first eluate from step (c) on a second IMAC matrix; (e) eluting the polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate comprising the polypeptide; A method comprising:

2. 10. The method of claim 1, further comprising performing size exclusion chromatography (SEC) on the second eluate.

3. 3. The method of claim 1 or 2, further comprising detecting the polypeptide in the first eluate or the second eluate or after SEC, optionally by electrophoresis or mass spectrometry.

4. 4. The method of claim 1, wherein the first IMAC matrix comprises agarose beads and a chelating ligand bound to nickel.

5. 5. The method of claim 1, wherein the first IMAC matrix retains nickel when exposed to cell culture medium.

6. 6. The method of claim 1, wherein the second IMAC matrix comprises agarose beads and nitrilotriacetic acid chelating moieties bound to nickel.

7. 7. The method of claim 1, further comprising regenerating the first IMAC matrix and / or the second IMAC matrix by washing with a regeneration buffer.

8. 8. The method of claim 7, wherein the regeneration buffer comprises sodium hydroxide.

9. The method according to any one of claims 1 to 8, wherein the polypeptide comprises a polyhistidine tag (His-Tag).

10. 10. The method of claim 1, wherein the polypeptide is an antibody, a cellular receptor, an intracellular protein, a secreted protein, or a membrane protein.

11. 11. The method of claim 1, wherein the buffer exchange comprises passing the first eluate through a desalting column.

12. 12. The method of claim 1, wherein the first elution buffer and the second elution buffer are the same.

13. 13. The method of any one of claims 1 to 12, wherein the composition comprises a cell culture medium.

14. 14. The method of claim 13, wherein the cell culture medium is a conditioned cell culture medium.

15. 15. The method of any one of claims 1 to 14, further comprising washing the first IMAC matrix with a wash buffer after step (a) and before step (b).

16. 16. The method of any one of claims 1 to 15, further comprising washing the second IMAC matrix with a wash buffer after step (d) and before step (e).

17. 17. The method of any one of claims 1 to 16, wherein a controller directs flow through columns containing the first IMAC matrix and the second IMAC matrix, and the method is automated.

18. 18. The method of any one of claims 1 to 17, further comprising determining elution time windows for the first eluate and the second eluate.

19. A polypeptide purified according to the method of any one of claims 1 to 18.

20. 1. A system for purifying a polypeptide, comprising: (a) an injection valve for injecting a composition containing the polypeptide into the system, the injection valve being connected to a first immobilized metal affinity chromatography (IMAC) matrix and a pump to control the flow of the sample through the first IMAC matrix; (b) column valves for controlling the flow paths of the equilibration buffer, elution buffer, and SEC buffer; (c) a desalting column configured to receive the flow-through or eluate from the first IMAC matrix; (d) a second IMAC matrix configured to receive the flow-through or eluate from the desalting column; (e) a size exclusion chromatography (SEC) column configured to receive the flow-through or eluate from the second IMAC matrix; (f) a detection device for detecting the polypeptide in the system; A system comprising:

21. 21. The system of claim 20, wherein the injection valve and the column valve can be automatically controlled.

22. 22. The system of claim 20 or 21, wherein the first IMAC matrix and / or the second IMAC matrix is an IMAC column.

23. 23. The system of any one of claims 20 to 22, wherein the detection device is a UV spectrometer or a mass spectrometer.

24. 1. A method for purifying a His-tagged polypeptide, comprising: (a) incorporating a composition comprising the His-tagged polypeptide into a first immobilized metal affinity chromatography (IMAC) matrix; (b) eluting the His-tagged polypeptide from the first IMAC matrix with an elution buffer to form a first eluate comprising the polypeptide; (c) performing a buffer exchange to remove the elution buffer from the first eluate using a desalting column; (d) capturing the first eluate from step (c) onto a second IMAC matrix, the second IMAC matrix comprising agarose beads and nitrilotriacetic acid chelating moieties bound to nickel; (e) eluting the His-tagged polypeptide from the second IMAC matrix with a second elution buffer to form a second eluate comprising the His-tagged polypeptide; A method comprising:

25. 25. The method of claim 24, further comprising performing size exclusion chromatography (SEC) on the second eluate to collect the purified polypeptide.

26. 26. The method of claim 25, further comprising optionally detecting the purified polypeptide collected from the SEC by electrophoresis or mass spectrometry.

27. 27. The method of any one of claims 24 to 26, wherein the first IMAC matrix comprises agarose beads and a chelating ligand bound to nickel.

28. 28. The method of any one of claims 24 to 27, wherein the first IMAC matrix retains nickel when exposed to cell culture medium.

29. 29. The method of any one of claims 24 to 28, wherein the second IMAC matrix comprises agarose beads and nitrilotriacetic acid chelating moieties bound to nickel.

30. 30. A polypeptide purified according to the method of any one of claims 24 to 29.

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