Method for purifying polypeptide comprising 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 comprises loading a composition comprising the polypeptide onto an IMAC matrix; eluting the polypeptide from the first IMAC matrix; performing buffer exchange to remove the elution buffer from the first eluate; loading the first eluate from the buffer exchange onto a second IMAC substrate; eluting the polypeptide from the second IMAC matrix; and optionally purifying the eluate from the second IMAC matrix using size exclusion chromatography. In some aspects, the method is fully automated.

Description

Technical Field

[0001] 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, in a high throughput setting using two different IMAC matrices. In some embodiments, the IMAC matrix comprises a chelating moiety that binds nickel. Background Art

[0002] Drug discovery studies and basic research involving peptides often require reliable, high-quality protein reagents, and often require reagents that can be purified rapidly, such as in a high-throughput setting. Purification of secreted His-tagged proteins typically involves concentration of the culture medium containing the His-tagged protein, ultrafiltration to exchange the buffer, a round of immobilized metal affinity chromatography (IMAC) (usually a nickel-containing Ni NTA matrix) to selectively bind the protein to the His tag, and elution of the His-tagged protein from the IMAC column ( Figure 1 ). However, this method involves a time-consuming ultrafiltration step to exchange the buffer for a buffer compatible with the NiNTA 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 standards of modern drug discovery and peptide research. Summary of the invention

[0003] The present disclosure relates to a novel purification method, which involves two different IMAC matrices for purifying polypeptides with His tags or other polypeptides selectively bound to metal ion chelating matrices. Specific embodiments herein include, for example, a method for purifying a polypeptide, the method comprising: (a) loading a composition comprising a 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. In some embodiments, the method further comprises performing size exclusion chromatography (SEC) on the second eluate. In some cases, the method further comprises optionally detecting the polypeptide in the first eluate or the second eluate or after SEC 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 when exposed to a 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 cell 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 a 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, the controller guides the 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 an elution time window for the first eluate and the second eluate.The disclosure herein also relates to a polypeptide that has been purified according to the methods herein.

[0004] The present disclosure also includes a system for purifying polypeptides. In some embodiments, the system includes: (a) an injection valve for injecting a composition containing a polypeptide into the system, wherein the injection valve is 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 path of the equilibration buffer, the elution buffer, and the SEC buffer; (c) a desalting column configured to receive a flow-through or eluate from the first IMAC matrix; (d) a second IMAC matrix configured to receive a flow-through or eluate from a desalting tower; (e) a size exclusion chromatography (SEC) column configured to receive a flow-through or eluate from a second IMAC matrix; and (f) a detection device for detecting polypeptides 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 spectrophotometer or a mass spectrometer.

[0005] In particular, the present disclosure further includes a method for purifying a His-tagged polypeptide, the method comprising: (a) loading a composition comprising a 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 a buffer exchange using a desalting column to remove the elution buffer from the first eluate; (d) loading the first eluate from step (c) onto a second IMAC matrix, wherein the second IMAC matrix comprises 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 comprises detecting the purified polypeptide collected from SEC, optionally by electrophoresis or mass spectrometry. In some cases, the first IMAC matrix comprises agarose beads and a chelating ligand bound to nickel. In some cases, the first IMAC matrix retains nickel when exposed to a cell culture medium. In some cases, the second IMAC matrix comprises agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel. The disclosure also includes a polypeptide that has been 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 following description and will be understood in part from the description or may be learned through practice. These objects and advantages will be realized and obtained through the elements and combinations particularly pointed out in the appended claims. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and explanatory and are not limitations of the claims.

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

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

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

[0010] Figure 2B Shown are gel electrophoresis images of major fractions from tests at 0 mM imidazole, 10 mM imidazole, and 20 mM imidazole.

[0011] Figure 3A Shown is the elution profile of the His-tagged polypeptide from the Ni Excel column.

[0012] Figure 3B Shown from Figure 3A Gel electropherogram of the main Ni Excel peak elution fractions tested.

[0013] Figure 4 Shown is the elution profile of the two-stage purification method of a His-tagged polypeptide and a zoomed-in view of a portion of the profile as the His-tagged polypeptide and impurities elute, as depicted in the bottom panel.

[0014] Figure 5A A flow chart showing a portion of an exemplary two-stage purification method described herein, comprising purification of a polypeptide from BEVS culture medium using a Ni Excel column, followed by purification in Further purification was performed on a SEC column and a Ni Excel column was regenerated.

[0015] Figure 5B Shown are the elution profiles of multiple runs of the same polypeptide and an overlay of the gel electrophoresis results from Figure 5A Purification of the indicated His-tagged polypeptides highly expressed in BEVS medium.

[0016] Fig. 6A Shown is the Ni Excel elution profile of a low expressed His-tagged polypeptide in BEVS medium.

[0017] Figure 6B Shows Fig. 6A Gel electrophoresis of a low-expressing His-tagged polypeptide and the low purity of the Ni-Excel elution indicated that, although compatible with BEVS medium, The SEC column was not adequate to remove all contaminants in the Ni-Excel eluted material.

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

[0019] Figure 7B shows that from loading Fig. 7A The gel electrophoresis results of the eluate from the Ni Excel column of a His-tagged polypeptide highly expressed in CHO culture medium are shown, and show that the Ni Excel column removes several impurities as well as the desired His-tagged polypeptide.

[0020] Fig. 8A Shows Fig. 7A and Figure 7B Elution profile of a His-tagged peptide after SEC purification.

[0021] Figure 8B Shown from Fig. 8A Gel electrophoresis results of the SEC-purified eluate showed that impurities were not effectively removed by SEC.

[0022] Fig. 9 Shown is an example of a system for purifying His-tagged polypeptides via a two-stage process using two IMACs, in this case Ni Excel followed by NiNTA followed by SEC.

[0023] Fig.10 It shows that Fig. 9 Elution profiles and gel electrophoresis results for the two-stage purification process and the four-column system are shown. A His-tagged peptide that was lowly expressed in BEVS culture medium was purified.

[0024] Figures 11A-11B The results show that proteins expressed in insect cells in baculovirus expression system (BEVS) buffer were expressed on four columns, namely NiExcel, desalting, NiNTA and Purification in 75 system. Fig.11A A flow chart of the purification process is provided, and Fig. 11B The elution profile and electrophoresis gel of the fractions eluted from the column are shown.

[0025] Figures 12A-12B The results show the protein expressed in CHO cells on four columns, namely NiExcel, Desalting, NiNTA and 200 system for purification. Fig. 12A A flow chart of the purification process is provided, and Fig. 12B The elution profile and electrophoresis gel of the fractions eluted from the column are shown.

[0026] Detailed Description of Certain Embodiments

[0027] 1. Definition

[0028] Unless otherwise defined, scientific and technical terms related to the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0029] In this application, the use of "or" means "and / or" unless otherwise stated. In the context of multiple dependent claims, the use of "or" only refers back to more than one preceding independent or dependent claim in an alternative manner. In addition, unless specifically stated otherwise, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising more than one subunit.

[0030] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0031] Units, prefixes and symbols are expressed in the form accepted by its Système International de Unites (SI). Numerical ranges include the numbers defining the ranges. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0032] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0033] " compositions " comprising polypeptide to be purified herein broadly comprises at least one polypeptide of interest and one or more pollutants or impurities, such as other polypeptides and / or other non-polypeptide molecules. In certain embodiments, compositions are derived from cell culture (that is, derived from cells for expressing polypeptides), such as cell lysates or clarified cell lysates, or provide polypeptides expressed in cells and are intended to other types of samples of purification. In other cases, compositions can be derived from polypeptide synthesis reactions. In other cases again, compositions can be derived from living organisms, such as plants or animals for expressing polypeptides of interest, such as samples taken from the organism. Compositions can be " partially purified " (that is, one or more previous purification steps, such as chromatography), or can be directly obtained from host cells or organisms producing polypeptides (for example, compositions are cell culture fluids or lysates of homogenate or harvest).

[0034] "Contaminants" or "impurities" are materials other than the desired polypeptide product. Contaminants can be, but are not limited to, variants, fragments, aggregates or derivatives of the desired polypeptide (e.g., variants without a His tag where the desired polypeptide is His-tagged), another polypeptide, nucleic acid, endotoxin, nucleic acid, lipid and membrane components, cell debris, small molecule chemicals, and other materials that may be found in cells or when preparing polypeptides synthetically.

[0035] "Affinity chromatography" or "affinity-based capture" refers to a separation method based on a specific interaction between an affinity column molecule and a specific polypeptide to be purified, such as the binding of a polypeptide to a ligand, the binding of a His-tag or other peptide tag to a metal ion or a specific antibody placed in a chromatography matrix.

[0036] As used herein, "immobilized metal affinity chromatography (IMAC)" refers to a type of affinity-based capture in which polypeptides are separated from contaminants based on their affinity for metal ions coordinated to ligands on an insoluble matrix. In some embodiments, the IMAC matrix is ​​disposed in a column, i.e., an "IMAC column." In some embodiments, the metal ions are nickel ions.

[0037] The term "matrix" is used herein to refer to chromatographic materials, such as affinity chromatography materials. In certain embodiments, the matrix may include beads or particles, and the beads or particles include materials that the polypeptide can selectively bind, such as comprising a chelating ligand combined with nickel. In certain embodiments, the matrix of the affinity chromatography material can be placed in a column, and the material to be purified can flow through the column. In other cases, it can be placed in a spin column, or placed on a plate or chip or other device. In some cases, the matrix may include beads or particles (such as magnetic particles), which can be separated from a solution by, for example, introducing a magnet.

[0038] "Size Exclusion Chromatography (SEC)" is a chromatographic method that separates molecules by size.

[0039] "Eluate" as used herein refers to material eluted from a chromatography matrix or column by application of an elution buffer.

[0040] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain natural and / or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. These terms also include amino acid polymers that have modifications (such as, for example, glycosylation, sialylation, etc.) or are complexed with other molecules.

[0041] In some embodiments, the polypeptide or protein to be purified is an antibody. The term "antibody" herein is used in the broadest sense and encompasses 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, (Fab')2, etc., as long as they exhibit antibody-antigen binding activity.

[0042] The term "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and 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 (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0043] The term "isolated" or "purified" polypeptide or protein means a polypeptide that has been at least partially separated from one or more contaminants. In some embodiments, the polypeptide is purified to a purity greater than 80%, 90%, 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0044] As used herein, the term "unpurified polypeptide" or "unpurified protein" in connection with the methods herein is a polypeptide or protein that has not been subjected to any chromatography or other method intended to at least partially separate the polypeptide from contaminants. For example, unpurified polypeptides can be found in solutions after cell lysates or peptide synthesis, etc.

[0045] The term "buffer exchange" refers to the process of changing the buffer in which the polypeptide of interest to be purified is found, such as to reduce the concentration of salts in the buffer or to remove components that may interfere with one or more chromatographic processes. In some embodiments, buffer exchange can be performed on a filter or column (such as a "desalting column").

[0046] 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 can have a molecular weight cutoff of, for example, between 17,000 Da and 90,000 Da, depending on the polypeptide to be purified.

[0047] As used herein, an "equilibrium buffer" is an aqueous buffer that is compatible with the polypeptide to be purified and that facilitates the interaction and binding between metal ions and the polypeptide in a chromatography column (such as an IMAC column).

[0048] "Elution buffer" is used to elute (ie, remove) polypeptides bound to a column or matrix (such as an IMAC or SEC matrix). The elution buffer can elute polypeptides from the matrix based on, for example, conductivity, pH, charge, ionic strength, etc.

[0049] A "regeneration buffer" may be used to regenerate a chromatography matrix (such as an IMAC matrix) so that it can be reused.

[0050] As used herein, an "automated" or "automatically controlled" process is a process that can be run, for example, by a computerized control system with appropriate software (as opposed to a system that requires active, manual intervention during or between at least one step), such as to move a sample containing an analyte 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 buffer and eluate through the system.

[0051] 2. Methods

[0052] The present disclosure relates to methods and systems for purifying polypeptides, for example. As described in more detail in the examples below, His tags and His tag affinity resins are often used to produce very pure protein reagents. However, secreted proteins with His tags in cell culture media cannot be directly loaded onto many nickel-containing IMAC matrices because the culture media detaches nickel from the resin chelation, thereby limiting the options for how such proteins can be purified. A traditional low-throughput approach to address this problem is to perform buffer exchange before loading the conditioned medium onto the IMAC matrix. Buffer exchange for each expression is a time-consuming, hands-on process that makes the purification flux of secreted His-tagged proteins very low. Certain IMAC matrices (such as Ni-excel) are not stripped by conditioned medium strips, making them suitable for direct loading without buffer exchange. However, we have observed that such matrices non-specifically bind to contaminated proteins in cell culture media compositions, and these impurities cannot be fully removed by further purification steps. For example, direct loading of mammalian or insect cell culture media (such as baculovirus (BEVS) and CHO culture media) containing low expression levels of His-tagged proteins on the Ni-Excel results in material with very low purity, even with a further size exclusion (SEC) step. For polypeptides expressed in CHO culture media, both high and low expressers exhibit significant non-specific binding that cannot be completely removed by SEC. We tested whether to add low concentrations of imidazole to the conditioned media before loading and observed that this did not result in an increase in purity and also resulted in a significant loss of protein yield.

[0053] The methods disclosed herein solve these many problems of purifying His-tagged polypeptides from cell culture media such as mammalian and insect cell culture media (such as BEVS and CHO culture media). In some embodiments, the method comprises (a) loading a composition comprising a 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.

[0054] In some embodiments, the composition is derived from a cell culture, i.e., a cell culture of a host cell expressing a polypeptide to be purified. Therefore, the composition from which the polypeptide is to be purified can be a cell lysate or a clarified cell lysate, etc. Therefore, in some embodiments, the composition comprises a cell culture medium. In some embodiments, the cell culture medium can be an artificial culture medium, a serum-containing culture medium, a serum-free culture medium, a chemically defined culture medium, or a protein-free culture medium. Examples of cell culture media include, but are not limited to, culture media for mammalian and insect cells. Examples of such culture media include baculovirus expression vector system (BEVS) culture media, Chinese hamster ovary (CHO) culture media, N293 cell culture media, RPMI1640 culture media, and Dulbecco modified Eagle medium (DMEM). In some embodiments, the cell culture medium can be a conditioned cell culture medium. The conditioned cell culture medium includes proteins and / or cytokines secreted by the cells. In some embodiments, the composition can also include non-protein contaminants, such as nucleic acids, lipids, etc. In other embodiments, the composition can be from a protein synthesis chemical reaction, and may contain contaminants, such as incompletely reacted polypeptide components, etc.

[0055] In some embodiments, the polypeptide comprises a polyhistidine tag (His tag) or other suitable tags intended to specifically bind to an IMAC matrix (such as a matrix comprising a chelating ligand bound to nickel). The polypeptide with the His tag can be produced by any suitable cell or can be added during the peptide synthesis reaction. Examples of cells that can express polypeptides with His tags include, but are not limited to, bacteria, insect cells, Escherichia coli, or mammalian cells. In some embodiments, BEVS medium is used to produce polypeptides with His tags in insect cells or CHO medium is used to produce polypeptides in CHO cells. CHO medium typically comprises Dulbecco's modified Eagle 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), 2mM L-glutamine, 100U / mL penicillin, and 100μg / mL streptomycin. BEVS medium generally comprises RPMI1640 medium supplemented with FBS or equivalent serum (e.g., in some cases supplemented with 10% FBS), and may further contain a surfactant such as F68, L-glutamine, penicillin and streptomycin. In some cases, BEVS medium also contains 100 U / mL penicillin and 100 μg / mL streptomycin. Culture media such as BEVS and CHO medium also contain essential amino acids and buffers to closely mimic the composition of mammalian cells.

[0056] The polypeptide to be purified can be any His-tagged polypeptide that can be bound to the IMAC matrix used herein. Examples of polypeptides include, but are not limited to, antibodies, cell receptors, intracellular proteins, secreted proteins, membrane proteins, etc. In some cases, the polypeptide can be an antigen or target for antibody binding. In some embodiments, the polypeptide is secreted from cells of a cell culture, i.e., is a secreted polypeptide.

[0057] The composition containing the polypeptide is loaded onto the first IMAC matrix and can be carried out by any known means available to those of ordinary skill in the art. For example, the composition can be loaded in an automated manner, wherein a pump extracts the composition from a reservoir containing the composition and pumps the fluid to a post containing the first IMAC matrix. In certain embodiments, the composition can be loaded into a tube (i.e., a loop), and the pump guides the buffer from the reservoir through the tube containing the composition and arrives on the post containing the first IMAC matrix. Alternatively, the composition can be manually, or by other methods that do not utilize a pump, or usually loaded (i.e., poured) into the first IMAC matrix by gravity flow.

[0058] In some embodiments, the first IMAC matrix comprises a chelating ligand that binds to nickel ions. A polypeptide comprising a His tag or similar tag that binds to a nickel-containing matrix is ​​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 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 protein ligand that binds to polyhistidine. In some embodiments, the chelating ligand comprises a sulfopropyl, sulfoethyl, carboxymethyl, trimethylammonium, or diethylaminoethyl group.

[0059] In some embodiments, the first IMAC matrix retains nickel when exposed to BEVS or CHO culture medium. In some cases, the cell culture medium may contain a stripping agent that, when exposed to an IMAC matrix comprising nickel, will cause the nickel to be washed or leached from the matrix. In some embodiments, the first IMAC matrix is ​​any matrix described in EP 1276716 (incorporated by reference in its entirety). In some embodiments, the first IMAC matrix is ​​available under the brand name NiSepharose TM Excel or HisTrap TM Excel (Sigma-Aldrich Co.) (ie, NiExcel herein) or an equivalent of such a matrix.

[0060] In some embodiments, the first IMAC matrix is ​​contained in a column. In some embodiments, the first IMAC column comprises a bed volume of 2 ml to 20 ml. In some embodiments, the first IMAC column comprises 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 in a spin column and may comprise, for example, a bed volume of 0.5, 0.75, 1 or 2 ml.

[0061] 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 be in the range of 0 to 20mM. In some embodiments, the amount of imidazole in the wash buffer can be in the range of 5mM to 15mM. In some embodiments, the amount of imidazole in the wash buffer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15mM. In some embodiments, the wash buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the wash buffer can be in the range of 20mM to 70mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be in the range of 40mM to 60mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be 45, 50 or 55mM. In some embodiments, the wash buffer comprises sodium chloride (NaCl). The amount of NaCl in the wash buffer can be in the range of 5mM to 35mM. In some embodiments, the amount of NaCl in the wash buffer may be in the range of 15mM to 25mM. In some embodiments, the wash buffer may include a surfactant. In some embodiments, the amount of NaCl in the wash buffer may be 15, 20 or 25mM. In some embodiments, the wash buffer comprises 50mM Na3PO4, 20mM NaCl and 10mM imidazole. In some embodiments, the wash buffer comprises 50mM Na3PO4, 20mM NaCl, 10mM imidazole and 0.1% Triton X-114. In some embodiments, the amount of wash buffer for washing the first IMAC column may be in the range of one to five times the bed volume of the column. In some embodiments, the IMAC matrix may be first washed with a buffer without a surfactant, and then washed with a buffer containing a surfactant.

[0062] Elution buffer can be used to elute polypeptide from the first IMAC matrix. In certain embodiments, the elution buffer comprises imidazoles. The amount of imidazoles in the elution buffer can be in the range of 0 to 1000mM. In certain embodiments, the amount of imidazoles in the elution buffer can be in the range of 100mM to 1000mM, 100mM to 800mM, 200mM to 600mM or 300mM to 500mM. In certain embodiments, the amount of imidazoles in the elution buffer can be 100, 150, 200, 250, 300, 350, 400, 450, 500 or 550mM. In certain embodiments, the elution buffer comprises trisodium phosphate (Na3PO4). Na3PO4 in the elution buffer can be in the range of 20mM to 70mM. In certain embodiments, Na3PO4 in the elution buffer can be in the range of 40mM to 60mM. In some embodiments, the amount of Na3PO4 in the elution buffer can be 45, 50 or 55mM. In some embodiments, the elution buffer comprises sodium chloride (NaCl). The amount of NaCl in the elution buffer can be in the range of 5mM to 35mM. In some embodiments, the amount of NaCl in the elution buffer can be in the range of 15mM to 25mM. In some embodiments, the amount of NaCl in the elution buffer can be 15, 20 or 25mM. In some embodiments, the elution buffer comprises 50mM Na3PO4, 20mM NaCl and 400mM imidazole. In some embodiments, the amount of the elution buffer for eluting polypeptides from the first IMAC column can be in the range of one to five times the bed volume of the column.

[0063] In certain 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 high salt concentration is used in the elution buffer, such imidazoles or salts can be substantially removed when the first eluate is prepared to be loaded onto the second IMAC matrix. In certain embodiments, buffer exchange includes passing the first eluate through a desalting column. Polypeptide passes through a desalting column faster than molecules of lower molecular weight, thereby allowing polypeptides to be collected while retaining substances of lower molecular weight, such as imidazoles. In certain embodiments, the desalting column comprises a bed volume of 2ml to 20ml. In certain embodiments, the desalting column comprises a bed volume of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20ml. Other means for performing buffer exchange include using dialysis or ultrafiltration, using a membrane or filter that captures polypeptides but allows buffer components and small molecules to pass through.

[0064] After the buffer exchange step, the first eluate is loaded onto the second IMAC matrix. The first eluate is loaded onto the second IMAC matrix and can be carried out similarly to loading the composition onto the first IMAC matrix. In certain embodiments, the eluate from the desalting column is collected in a pipe (i.e., loop) sequentially connected to the second IMAC matrix. Once the eluate is collected in the pipe, a pump can direct the fluid onto the second IMAC matrix.

[0065] In some embodiments, the second IMAC matrix comprises nickel. In some embodiments, the second IMAC matrix comprises agarose beads and a nitrilotriacetic acid chelating portion (Ni NTA) bound to nickel. The nitrilotriacetic acid (NTA) chelating portion is attached to the agarose beads. In other embodiments, the first IMAC and the 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 in a column. In some embodiments, the second IMAC column comprises a bed volume of 2ml to 20ml. In some embodiments, the second IMAC column comprises a bed volume of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20ml. In other embodiments, the second IMAC matrix is ​​contained in a spin column and can comprise, for example, a bed volume of 0.5, 0.75, 1 or 2ml.

[0066] In some embodiments, after the polypeptide has been loaded onto 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 be in the range of 0 to 20mM. In some embodiments, the amount of imidazole in the wash buffer can be in the range of 5mM to 15mM. In some embodiments, the amount of imidazole in the wash buffer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15mM. In some embodiments, the wash buffer comprises trisodium phosphate (Na3PO4). The amount of Na3PO4 in the wash buffer can be in the range of 20mM to 70mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be in the range of 40mM to 60mM. In some embodiments, the amount of Na3PO4 in the wash buffer can be 45, 50 or 55mM. In some embodiments, the wash buffer comprises sodium chloride (NaCl). The amount of NaCl in the wash buffer can be in the range of 5mM to 35mM. In some embodiments, the amount of NaCl in the wash buffer may be in the range of 15mM to 25mM. In some embodiments, the amount of NaCl in the wash buffer may be 15, 20 or 25mM. In some embodiments, the wash buffer comprises 50mM Na3PO4, 20mM NaCl, 10mM imidazole and 0.1% Triton X-114. In some embodiments, the wash buffer may include a surfactant. In some embodiments, the wash buffer comprises 50mM Na3PO4, 20mM NaCl and 10mM imidazole. In some embodiments, the amount of wash buffer for washing the second IMAC column may be in the range of one to five times the bed volume of the column. In some embodiments, the IMAC matrix may be first washed with a buffer without a surfactant, and then washed with a buffer containing a surfactant.

[0067] Elution buffer can be used to elute polypeptide from the second IMAC matrix. In certain embodiments, the elution buffer comprises imidazole. The amount of imidazole in the elution buffer can be in the range of 0 to 1000mM. In certain embodiments, the amount of imidazole in the elution buffer can be in the range of 100mM to 1000mM, 100mM to 800mM, 200mM to 600mM or 300mM to 500mM. In certain embodiments, the amount of imidazole in the elution buffer can be 100, 150, 200, 250, 300, 350, 400, 450, 500 or 550mM. In certain embodiments, the elution buffer comprises trisodium phosphate (Na3PO4). Na3PO4 in the elution buffer can be in the range of 20mM to 70mM. In certain embodiments, Na3PO4 in the elution buffer can be in the range of 40mM to 60mM. In some embodiments, the amount of Na3PO4 in the elution buffer can be 45, 50 or 55mM. In some embodiments, the elution buffer comprises sodium chloride (NaCl). The amount of NaCl in the elution buffer can be in the range of 5mM to 35mM. In some embodiments, the amount of NaCl in the elution buffer can be in the range of 15mM to 25mM. In some embodiments, the amount of NaCl in the elution buffer can be 15, 20 or 25mM. In some embodiments, the elution buffer comprises 50mM Na3PO4, 20mM NaCl and 400mM imidazole. In some embodiments, the amount of elution buffer for eluting polypeptides from the second IMAC column can be in the range of one to five times the volume of the column bed. In some embodiments, the elution buffer for the first IMAC matrix and the second IMAC matrix is ​​the same.

[0068] In certain embodiments, the time of polypeptide elution from the first IMAC column and the second IMAC column can be determined experimentally using the UV absorbance of the eluate fraction, such as at a wavelength suitable for observing the polypeptide, such as 280nm, etc. The elution time window can be determined and the correct fraction can be collected. For example, during a specific elution time window, the eluate can be collected in a tube (i.e., loop) to prepare for processing by the next post. Outside the elution time window, the eluate can be directed to a waste collector. In certain embodiments, the eluate can be further analyzed by, for example, gel electrophoresis or mass spectrometry or other detection techniques to assess its purity and productivity.

[0069] If desired, for example, the second eluate can be further purified using size exclusion chromatography (SEC) or other chromatographic methods (such as anion or cation exchange). In some embodiments, the method includes the step of performing SEC on the second eluate. The step of performing SEC can include loading the second eluate onto a 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, 75 or ELISA® 200 or equivalent can be used as the SEC matrix. In some embodiments, the eluate from the SEC matrix can be collected in different fractions for later analysis, such as on a 96-well plate.

[0070] In some embodiments, the method comprises detecting the purified polypeptide collected from either the first IMAC eluate or the second IMAC eluate, or if SEC is performed, detecting the purified polypeptide collected from the SEC eluate. A variety of methods can be used to detect the elution of the polypeptide from the SEC column. In some embodiments, UV spectroscopy can be used to detect the elution of the polypeptide from the SEC column. Other methods include, but are not limited to, electrophoresis or mass spectrometry.

[0071] In some embodiments, the method comprises regenerating the first IMAC matrix and / or the second IMAC matrix by washing with a regeneration buffer, and if used, an additional chromatography matrix such as SEC. In some embodiments, the regeneration buffer comprises sodium hydroxide. In some embodiments, the regeneration buffer comprises 0.1M to 1M 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 1M sodium hydroxide. In some embodiments, an equilibration buffer is then run through the matrix to remove such sodium hydroxide and prepare the matrix for a new polypeptide purification process.

[0072] In some embodiments, the method for purifying a His-tagged polypeptide herein comprises (a) loading a composition comprising a 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 a buffer exchange using a desalting column to remove the elution buffer from the first eluate; (d) loading the first eluate from step (c) onto a second IMAC matrix, wherein the second IMAC matrix comprises 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 such embodiments, the first IMAC matrix comprises a chelating ligand that binds to nickel ions. The polypeptide comprising a His-tagged or similar tag bound to a nickel-containing matrix is ​​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 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 protein 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 culture medium. For example, in some cases, the cell culture medium may contain a stripping agent that, when exposed to an IMAC matrix comprising nickel, will cause nickel to be washed or leached from the matrix. In some embodiments, the first IMAC matrix is ​​any matrix described in EP 1276716 (the entire contents of which are incorporated by reference). In some embodiments, the first IMAC matrix is ​​a NiSepharose® matrix under the brand name NiSepharose®. TM Excel or HisTrap TM Excel (Sigma-Aldrich Co.) (ie, NiExcel herein) or an equivalent of such a matrix.

[0073] In some embodiments, the first IMAC matrix and the second IMAC matrix are contained in a column. In some embodiments, the first IMAC matrix and the second IMAC matrix are columns comprising a bed volume of 2 ml to 20 ml. In some embodiments, the first IMAC column and the second IMAC column comprise 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 SEC step after chromatography with two IMAC matrices.

[0074] In some embodiments, when the IMAC and / or SEC matrix is ​​disposed on a column, the controller directs flow through a column containing a first IMAC matrix and a second IMAC matrix, and the method is automated. In some embodiments, the controller directs flow through a column containing a first IMAC matrix, a desalting column, a second IMAC matrix, and a SEC column, and the method is automated.

[0075] The flow rate of the composition, buffer, eluate through the column can be selected based on the size of the column. The flow rate through the column (IMAC, desalination and SEC) can be the same or different. In certain embodiments, the flow rate through the first IMAC column and the second IMAC column can be 2ml / min to 8ml / min. In certain embodiments, the flow rate through the first IMAC column and the second IMAC column can be 5ml / min. In certain embodiments, the flow rate through the desalting column can be 5ml / min to 15ml / min. In certain embodiments, the flow rate through the first IMAC column and the second IMAC column can be 10ml / min.

[0076] In some embodiments, the method herein is superior to the existing method using a single IMAC column because before the composition is loaded onto an IMAC matrix (such as a column), there is no need to first exchange the buffer of the starting composition containing a cell culture medium (such as BEVS or CHO culture medium). Such a buffer exchange process for removing the cell culture medium may be relatively time-consuming. At the same time, the process herein allows better purification of His-tagged polypeptides than the traditional one-IMAC method, as can be seen in the examples and drawings of the present disclosure. The method herein is also compatible with both high-expression and low-expression polypeptides from specific cell culture host cells. The method herein can also be partially or fully automated. In particular, the automated method can also allow high-throughput protein preparation, otherwise if the starting composition such as cell lysate must be loaded onto the IMAC matrix in batches, or if the cell lysate must first be buffer exchanged before the chromatography to be compatible with the IMAC composition, then this is impossible. In addition, as described in more detail in the following section, the system can be designed to perform the method herein so that several large-scale polypeptide expressions can be purified in series with minimal hands-on requirements.

[0077] 3. System

[0078] The present disclosure also relates to systems capable of implementing the methods described herein, including those described in the above sections. In some cases, the system includes (a) an injection valve for injecting a composition containing a polypeptide into the system, wherein the injection valve is connected to a first immobilized metal affinity chromatography (IMAC) matrix and a pump to control sample flow through the first IMAC matrix; (b) a column valve for controlling the flow path of an equilibrium buffer, an elution buffer, and a SEC buffer; (c) a desalting column or a similar device for buffer exchange, which is configured to receive a flow-through or eluate from the first IMAC matrix; and (d) a second IMAC matrix, which is configured to receive a flow-through or eluate from a desalting column; and (f) a detection device, such as a UV spectrophotometer or a mass spectrometer, for detecting polypeptides in the system. In some cases, the system further includes an additional chromatographic column for further purification of the polypeptide, such as (e) a size exclusion chromatography (SEC) column, which is configured to receive a flow-through or eluate from a second IMAC matrix. In some cases, the injection valve and the column valve can be automatically controlled.

[0079] For example, Fig. 9 An embodiment of an exemplary system 10 for purifying polypeptides according to the methods of the present invention is shown. For example, the system 10 includes four separate columns, two IMAC columns (a) an injection valve 11 for injecting a composition 12 containing polypeptides into the system 10, wherein the injection valve 11 is connected to a first immobilized metal affinity chromatography (IMAC) column 13 and a pump for controlling the flow of the sample through the first IMAC column 13; (b) a column valve 14 for controlling the flow path of the equilibration buffer 15, the elution buffer 16, and the SEC buffer; (c) a desalting column 17, which is configured to receive the effluent or eluate from the first IMAC column 13; and (d) a second IMAC column 18, which is configured to receive the effluent or eluate from the desalting column 17; (e) a size exclusion chromatography (SEC) column 19, which is configured to receive the effluent or eluate from the second IMAC column 18; and (f) a detection device 20 for detecting the polypeptides in the system. Optionally, the system may also include a mixer ( Fig. 9 The mixer mixes the mobile phase buffer gradient if such a gradient is used in the chromatography method.

[0080] In certain embodiments, the system incorporates a controller, pump and interface of a commercial multi-column chromatography system such as an AKTA chromatography system (Cytiva). Each post can be connected in series to the pipeline of such a chromatography system. In certain embodiments, an injection valve and a column valve can be automatically controlled to guide buffer to pass through a suitable column at the appropriate time.

[0081] As described above, the systems disclosed herein can have certain advantages over systems that utilize only one IMAC matrix. For example, the entire starting composition can be loaded onto the IMAC matrix without breaking it down into batches. In some embodiments, there is no need to first exchange the buffer of the starting composition comprising a cell culture medium (such as BEVS or CHO medium) before loading the composition onto the IMAC matrix, while allowing for greater purification of His-tagged polypeptides compared to traditional one-IMAC methods, as can be seen in the examples and figures of the present disclosure. The systems herein can be used with both highly expressed and lowly expressed polypeptides from specific cell culture host cells. In particular, automated systems can also allow for high-throughput protein preparation, which would otherwise be impossible if starting compositions such as cell lysates had to be loaded onto IMAC columns or other matrices in batches, or if the cell lysates had to be first buffer exchanged prior to chromatography to be compatible with the IMAC composition. In addition, systems such as Fig. 9 The system shown in can perform the methods herein, allowing several large-scale polypeptide expressions to be purified in series with minimal hands-on requirements. For example, a 5 mL Ni Excel column used in the methods and systems herein has been used in approximately 30 1 L runs using compositions in CHO or BEVS medium over a 3 month period with no color change or capacity loss.

[0082] Examples

[0083] Example 1. Effect of Imidazole on Protein Yield from Ni Excel

[0084] The effect of adding up to 20 mM imidazole on protein purity and yield from Ni Excel columns (Cytiva catalog number 17371201) was tested by adding the polypeptide composition to be purified in CHO culture medium to a column containing 0, 10 or 20 mM imidazole. Typically, imidazole can be added when performing metal ion affinity chromatography to purify a specific polypeptide because it can help remove impurities caused by nonspecific binding to the chromatographic matrix. The filtered culture medium (through a 0.2 micron filter) is directly loaded on the column; no additives are added. The column is pre-equilibrated with 50 mM sodium phosphate, 200 mM NaCl and 10 mM imidazole (pH 8), 2 mM NaN3. The same buffer is then used to wash the column. The second wash is performed with 50 mM Tris Na3PO4, 200 mM NaCl, 10 mM imidazole, 2 mM NaN3, 0.1% Triton-X (TX) 114. The bound polypeptides were eluted in a buffer containing 50 mM Tris Na3PO4, 200 mM NaCl, 400 mM imidazole and 2 mM NaN3. Figure 2A and Figure 2BThe results in show that increasing imidazole concentration leads to a loss of protein yield, as indicated by Figure 2A The size of the elution trace shown in FIG. 1 is reduced, while the purity is not significantly improved, as depicted in FIG. Figure 2B As indicated in Figure 2A Gel electrophoresis of the fractions collected in .

[0085] The effect of washing Ni-Excel column (Cytiva) with different concentrations of imidazoles on protein purity and yield was further tested with the starting composition of 1L high expressor, which secreted the His-tagged protein produced in baculovirus (BEVS) culture medium and was loaded on the 5mL Ni-Excel column in the buffer without imidazoles. Washing buffer containing 10, 20, 30, 40 or 50mM imidazoles was tested. Each washing buffer had a pH of 8 and contained 50mM sodium phosphate and 200mM NaCl. The His-tagged protein was eluted from the Ni Excel column using an elution buffer containing 50mM sodium phosphate, 200mM NaCl and 300-400mM imidazoles (pH 8). 0.5M NaOH was used to regenerate the Ni Excel column. The buffer containing 50mM sodium phosphate, pH 8, 200mM NaCl and 10mM imidazoles was selected as washing buffer.

[0086] Figure 3A The UV trace of the solution leaving the Ni Excel column is shown. The peak at about 240 min corresponds to the eluted protein from the Ni Excel column. No loss in yield was observed up to 50 mM imidazole concentration in the wash buffer. Figure 3B The elution profile of the His-tagged polypeptide fraction corresponding to elution at about 220 minutes to about 260 minutes is shown. Figure 3A The trace of . Elution was tested using both 300 mM and 400 mM imidazole in the elution buffer. The buffer containing 50 mM sodium phosphate, pH 8, 200 mM NaCl and 400 mM imidazole produced a sharper peak in the gel and was selected as the elution buffer.

[0087] Example 2. Two-step automated purification using Ni Excel and SEC (BEVS medium)

[0088] A two-step automated purification system was tested, which included a Ni Excel column (Cytiva, Cat. No. 17371201) and a Superdex 200 (SEC) column. Figure 5AFlowchart showing the steps and some features of the two-step procedure. High expressors, 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 (pH 8), and the elution buffer contained 50 mM sodium phosphate, 200 mM NaCl, and 400 mM imidazole (pH 8). Figure 4 Separation of the His-tagged protein of interest from early eluting impurities is shown. The total run time for nickel and SEC column purification was 7 hours with minimal hands-on time required.

[0089] For BEVS medium, when the expression level of the His-tagged protein is high, the protein eluted from the column is pure enough that a size exclusion (SEC) step reduces the purity to more than 95% (see Figure 5B ). This purification process was also performed using low-expressing, secreted proteins in BEVS medium, such as FIG. 6A to FIG. 6B As shown in these figures, the peptide could not be purified to a high enough purity using the Ni-Excel column that SEC was sufficient to remove the remaining residue (see Fig. 6A and Figure 6B This may be because Ni Excel shows more non-specific binding to non-His-tagged proteins than other IMAC matrices such as Ni NTA.

[0090] Example 3. Two-step automated purification (CHO culture medium)

[0091] In this example, the protein with His tag was directly loaded in CHO culture medium. Specifically, the filtered culture medium (filtered by 0.2 micron filter) containing the heterodimeric polypeptide with His tag to be purified was directly loaded onto a Ni Excel column (Cytiva, catalog number 17371201), which had been pre-balanced in a buffer containing 40mM sodium phosphate, 200mM NaCl and 10mM imidazole (pH 8) and 2mM sodium azide. The same buffer was used for the first wash of the column, followed by a second wash in 50mM Tris sodium phosphate, 200mM NaCl, 10mM imidazole, 2mM sodium azide and 0.1% TX 114. The polypeptide was eluted in 50mM Tris sodium phosphate, 200mM NaCl, 400mM imidazole and 2mM sodium azide. Fig. 7A shows the UV trace at 280 nm of the solution eluted from the Ni Excel column, and Figure 7B The protein content of the fractions collected from the Ni Excel column analyzed by gel electrophoresis is shown. Figure 7BAs can be seen in the figure, there are a lot of impurities in the fractions containing the protein of interest.

[0092] Further purification is then performed by SEC chromatography. However, the SEC procedure cannot completely remove impurities from His-tagged peptides, such as Fig. 8A and Figure 8B shown in . Specifically, Fig. 8A The UV trace of the eluate from the SEC column at 280 nm is shown, where each peak represents each subunit of the heterodimeric polypeptide, and Figure 8B An electrophoretic run of the eluted polypeptide species is shown, with arrows indicating the polypeptide subunits of interest. Fig. 7A The white box at about 180 minutes in FIG. 4 depicts impurities that can be removed by subsequent SEC chromatography steps, while Figure 8B Indicates other impurities remaining.

[0093] Thus, in CHO medium, both high and low expressers exhibited substantial nonspecific binding that could not be completely removed by SEC and required additional purification steps. Addition of 10 mM or 20 mM imidazole to the conditioned medium prior to loading was tested; however, this did not result in an increase in Ni eluate purity but rather resulted in a significant loss in protein yield.

[0094] Example 4. Four-column purification using Ni Excel, Ni NTA, and SEC

[0095] Next tested the system of using two nickel columns in series, to determine whether such settings can improve the purity of high expressors and low expressors from BEVS and CHO culture medium. Tested four-column automatic purification system, it comprises 5mL Ni Excel column (Cytiva, catalog number 17371201), desalting column, 5mL Ni NTA column (Qiagen, catalog number 30761) and Superdex200 (SEC) column. These columns are connected to UNICORN 7.3.0AKTA purification system. Conditioned medium is filtered by 0.2 micron filter, and is loaded on the 5ml Ni Excel resin with 50mM Na PO , 20mM NaCl and 10mM imidazole pre-equilibration. Once loaded, just use the washing buffer containing 50mM Na PO , 20mM NaCl and 10mM imidazoles and the second washing buffer containing 50mM Na PO , 20mM NaCl, 10mM imidazoles and 0.1% Triton X-114 to wash the column, to remove endotoxin. Once the wash was complete, the His-tagged protein was eluted with 14.5 ml of 50 mM Na3PO4, 20 mM NaCl, and 400 mM imidazole. The flow rate of conditioned medium, wash buffer, and elution buffer through the Ni Excel column was set to 5 ml / min.

[0096] Desalting columns (Cytiva HiPrep TM 26 / 10 with a bed volume of 53 ml and a molecular weight cutoff of 1000-5000) was used for buffer exchange, and the His-tagged protein eluted from the Ni Excel column was then loaded onto a Ni NTA column. Due to the high salt content of the NiExcel eluate, it could not be loaded directly onto the Ni NTA column. The desalting column was a HiPrep TM 26 / 10 desalting column (Cytiva, catalog number 17-5087-01). The 50 ml desalting column used in this method is capable of buffer exchange up to 15 ml of protein solution. The 10 ml loop captures the eluate from the Ni Excel column, and the eluate is loaded onto the desalting column from the loop. The flow rate of the desalting column is set to 10 ml / min.

[0097] The buffer-exchanged protein from the desalting column was collected in a 20 ml loop and loaded from the loop onto a 5 ml Ni NTA Superflow pre-equilibrated with 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole. TMThe column was loaded. Once loading was complete, the column was washed with 50 mM Na3PO4, 20 mM NaCl, and 10 mM imidazole to remove endotoxins and 50 mM Na3PO4, 20 mM NaCl, 10 mM imidazole, and 0.1% Triton X-114 to remove endotoxins. Once washing was complete, 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 to 5 ml / min. The Ni NTA Superflow TM The eluate was collected in a 7 ml loop and from there loaded onto a Superdex (SEC) column.

[0098] Both nickel columns were regenerated with 0.5 M NaOH after each run and the sample pump was also washed after each run.The system can queue purifications of several expressions simultaneously in a hands-free, fully automated mode, regardless of expression level.

[0099] Fig. 9 A schematic diagram of the system is shown. Fig.10 The examples using the methods described in this application are shown to purify a His-tagged polypeptide in high yield. The polypeptide was purified to greater than 95% purity despite low expression levels in cell culture. Without the Ni-Excel + Ni-NTA column tandem purification method used herein, it would have been impossible to purify such a low-expressing polypeptide to such a high purity in a high-throughput manner. The same method as above was performed except that the polypeptide purified from CHO medium instead of BEVS medium was used.

[0100] Example 5: Four-column purification of proteins expressed in insect cells

[0101] In another example, a 5 mL Ni Excel column (Cytiva, catalog number 17371201), a desalting column, a 5 mL Ni NTA column (Qiagen, catalog number 30761), and a 120 mL 75 (SEC) column to purify proteins in BEVS culture medium. 10 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. Before eluting the protein, the column was washed with 50 mM sodium phosphate pH 8.0, 200 mM NaCl, 10 mM imidazole and 0.1% Triton X114. 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 of 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, washing and elution buffers as above. Then, in 120 mL The NiNTA column eluate was further purified by size exclusion chromatography (SEC) on a 75 column in 25 mM Tris pH 7.5, 150 mM NaCl and 2 mM EDTA. (See Fig.11A . ) Fig. 11B The results of the batch purification process are shown. The protein was purified to a concentration of 2.9 mg / mL (specifically 31.9 mg in a volume of 11 mL) and had an endotoxin concentration of 0.08 U / mg. Fig. 11B As shown in the gel in, the protein is highly pure after this process.

[0102] Example 6: Four-column purification of proteins expressed in CHO cells

[0103] In additional experiments, additional proteins were expressed in CHO cells. A 35-liter volume of protein in CHO buffer was concentrated to 3 liters and loaded in batches onto a column containing a 5 mL NiExcel column, a 50 mL desalting column, a 5 mL NiNTA column, and The concentrated protein in CHO buffer was loaded in batches onto a 5 mL NiExcel column pre-equilibrated in 50 mM sodium phosphate pH 8.0, 200 mM NaCl, and 10 mM imidazole. Before eluting the protein, the column was washed with 50 mM sodium phosphate pH 8.0, 200 mM NaCl, 10 mM imidazole, and 0.1% Triton X114. The protein was eluted in 50 mM sodium phosphate pH 8.0, 200 mM NaCl, and 400 mM imidazole. After elution, the protein was subjected to desalting in a 50 mL volume. The desalted solution was then loaded onto a 5 mL NiNTA column using the same equilibration, washing, and elution buffers. Then, the column was washed with 120 mL PBS and 400 mM elution buffers. The NiNTA column eluate was further purified by size exclusion chromatography (SEC) on a 200 column in 25 mM Tris pH 7.5, 150 mM NaCl and 1 mM EDTA. (See Fig. 12A . ) Fig. 12B The results of the purification are shown, resulting in a protein concentration of 0.67 mg / mL (16 mL, containing 10.7 mg protein).

Claims

1. A method for purifying a polypeptide, comprising: (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.

2. The method of claim 1, further comprising subjecting the second eluate to size exclusion chromatography (SEC).

3. The method according to 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. The method according to any one of claims 1 to 3, wherein the first IMAC matrix comprises agarose beads and a chelating ligand bound to nickel.

5. The method of any one of claims 1 to 4, wherein the first IMAC matrix retains nickel when exposed to cell culture medium.

6. The method according to any one of claims 1 to 5, wherein the second IMAC matrix comprises agarose beads and a nitrilotriacetic acid chelating moiety bound to nickel.

7. The method according to any one of claims 1 to 6, further comprising regenerating the first IMAC matrix and / or the second IMAC matrix by washing with a regeneration buffer.

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. The method of any one of claims 1 to 9, wherein the polypeptide is an antibody, a cell receptor, an intracellular protein, a secreted protein or a membrane protein.

11. The method of any one of claims 1 to 10, wherein the buffer exchange comprises passing the first eluate through a desalting column.

12. The method according to any one of claims 1 to 11, wherein the first elution buffer and the second elution buffer are the same.

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

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

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

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

20. A system for purifying a polypeptide, wherein the system comprises: (a) an injection valve for injecting a composition containing the polypeptide into the system, wherein the injection valve is connected to a first immobilized metal affinity chromatography (IMAC) matrix and a pump to control the flow of a sample through the first IMAC matrix; (b) a column valve for controlling the flow paths of the equilibration buffer, the elution buffer, and the 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.

21. The system of claim 20, wherein the injection valve and the column valve are capable of being automatically controlled.

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

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

24. A method for purifying a His-tagged polypeptide, the method comprising: (a) loading the 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 a buffer exchange using a desalting column to remove the elution buffer from the first eluate; (d) loading the first eluate from step (c) onto a second IMAC matrix, wherein the second IMAC matrix comprises 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.

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

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

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

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

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

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