Methods for isolating polypeptides

Abstract

The present disclosure is directed to methods of isolating and / or purifying a protein species comprising contacting a mixture comprising the species and one or more impurities with two or more chromatography columns in a sequential operation mode. In some embodiments, the protein species is a charge variant.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 321,531, filed March 18, 2022, which is incorporated by reference in its entirety.

[0002] The present application relates to the field of protein separation and purification using two or more chromatography columns. [Background technology]

[0003] Therapeutic proteins, especially monoclonal antibodies (mAbs), undergo a variety of post-translational modifications (PTMs), including oxidation, deamidation, glycosylation, and lysine truncation. Some of these modifications give rise to charge variants of the protein. Characterization and analysis of charge variants of therapeutic proteins is required to ensure that they do not affect the quality of the formulation. However, large-scale protein purification and isolation can be costly and time-consuming. Traditional methods for separating specific protein species rely on HPLC or FPLC, which can take weeks to months to generate substantial product for further analysis and / or use. Traditional methods typically force a choice between high productivity or high purity, but it is usually not possible to achieve both simultaneously.

[0004] Thus, there remains a need in the field of protein purification for methods that maximize yield and purity while reducing the time required to generate a purified sample. Summary of the Invention

[0005] Some embodiments of the present disclosure are directed to a method of isolating a protein species from a mixture that contains the protein species and one or more impurities, the method comprising contacting the mixture with two or more chromatography columns in a continuous operation mode.

[0006] Some embodiments of the present disclosure are directed to a method of increasing the purity and / or yield of a protein species from a mixture that contains the protein species and one or more impurities, the method comprising contacting the mixture with two or more chromatography columns in a sequential operation mode.

[0007] Some aspects of the present disclosure include: 1. A method for concentrating a protein species for analytical characterization, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous mode of operation of a chromatographic separation system; and (b) subjecting the species from (a) to analytical characterization. The present invention relates to a method comprising:

[0008] Some aspects of the present disclosure provide a method for performing analytical characterization of a protein species, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode of a chromatographic separation system; and (b) performing analytical characterization of the chemical species from (a); The present invention relates to a method comprising:

[0009] In some embodiments, analytical characterization is performed by HPLC systems, capillary isoelectric focusing (cIEF) gel electrophoresis, imaging capillary isoelectric focusing (iCIEF), cation exchange chromatography (CEX), anion exchange chromatography (AEX), MFI, SEC-MALS, SEC, or mass spectrometry. In some embodiments, the method results in increased purity and / or increased yield of protein species compared to HPLC or FPLC.

[0010] In some embodiments, two or more chromatographic columns concentrate the species.

[0011] In some embodiments, the method further comprises loading the mixture onto a first chromatographic column. In some embodiments, the loaded mixture is passed through the first chromatographic column and separated into enriched species comprising said species and waste species ("enrichment stage I"). In some embodiments, the enriched species is passed through a second column and the waste species is discarded after the first chromatographic column ("enrichment stage II").

[0012] In some embodiments, the method further comprises re-equilibrating the first chromatography column.

[0013] In some embodiments, the method further comprises contacting the enriched species with a first chromatographic column. In some embodiments, the method further comprises loading an additional mixture onto the first column, where the additional mixture comprises the species and one or more impurities. In some embodiments, the additional mixture is added at the same time that the enriched species is added to the first chromatographic column. In some embodiments, the additional mixture is added after the enriched species is added to the first chromatographic column and before the enriched species passes through the first chromatographic column.

[0014] In some embodiments, enrichment steps I and II are repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.

[0015] In some embodiments, the method further comprises a removal step. In some embodiments, the removal step comprises contacting the enriched species with a first chromatographic column in the absence of the additional mixture. In some embodiments, the removal step further comprises passing the enriched species through the first chromatographic column and separating the species from one or more impurities. In some embodiments, the removal step further comprises passing the enriched species through a second chromatographic column and separating the species from one or more impurities.

[0016] In some embodiments, the method further comprises eluting the species, hi some embodiments, the method produces a protein species that is at least 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0017] In some embodiments, the concentration of the eluted species is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.5, at least about 3.0, at least about 3.5, at least about 4.0, at least about 4.5, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, or at least about 10.0 times higher than the concentration of the species in the mixture.

[0018] In some embodiments, one or more chromatography columns comprises a salt gradient, a pH gradient, or both. In some embodiments, the salt gradient comprises a sodium chloride gradient. In some embodiments, the salt gradient comprises the presence or absence of salt. In some embodiments, the concentration of the salt is between about 50 mM and about 600 mM, between about 100 mM and about 550 mM, between about 150 mM and about 500 mM, between about 200 mM and about 450 mM, between about 250 mM and about 400 mM, between about 100 mM and about 400 mM, between about 100 mM and about 350 mM, between about 100 mM and about 300 mM, between about 100 mM and about 250 mM, between about 300 mM and about 600 mM, between about 350 mM and about 550 mM, between about 400 mM and about 500 mM, or between about 350 mM and about 450 mM. In some embodiments, the concentration of salt is at least 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 260 mM, at least about 270 mM, at least about 280 mM, at least about 290 mM, at least about 300 mM, at least about 310 mM, at least about 320 mM, at least about 330 mM, at least about 340 mM, at least about 350 mM, at least about 360 mM, at least about 370 mM, at least about 380 mM, at least about 390 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM, or at least about 600 mM.

[0019] In some embodiments, the pH of the pH gradient is between about pH 3 and about pH 11. In some embodiments, the mixture is in a buffer. In some embodiments, the buffer is MES, phosphate buffer, Tris, or any combination thereof.

[0020] In some embodiments, the method further comprises measuring a post-translational modification. In some embodiments, the post-translational modification is N-terminal glutamine pyroglutamylation, C-terminal lysine truncation, C-terminal proline amidation, glycosylation, sialylation, deamidation, aspartic acid isomerization, general truncation, or any combination thereof.

[0021] In some embodiments, the protein comprises a fusion protein or an antibody or antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof binds to an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), MICA / B PSCA, IL-8, EGFR, HER3, HER4, and any combination thereof.

[0022] In some embodiments, the fusion protein comprises an immunoglobulin component and a growth factor. In some embodiments, the fusion protein comprises an Fc fusion protein. In some embodiments, the fusion protein comprises Fc fused to CTLA-4. In some embodiments, the fusion protein comprises abatacept or belatacept.

[0023] In some embodiments, the fusion protein comprises an Fc fused to an interleukin.

[0024] In some embodiments, the species of the fusion protein or antibody is an acidic species, a basic species, or a predominant species. In some embodiments, the fusion protein or antibody is partially purified by Protein A affinity chromatography.

[0025] In some embodiments, the chemical species is concentrated by a countercurrent purification system. In some embodiments, the countercurrent purification system is a multi-column countercurrent solvent gradient purification (MCSGP) system.

[0026] Some aspects of the present disclosure are directed to protein species prepared by the methods disclosed herein. In some aspects, the protein species is a charge variant.

[0027] Some aspects of the present disclosure are directed to methods of treating a disease or condition in a subject in need of treatment comprising administering to the subject a protein species disclosed herein. [Brief description of the drawings]

[0028] [Figure 1] Schematic diagram of the enrichment method described herein using MCSGP twin-column sequential chromatography. The load material is represented by a mixture of three species (red ("R"), green ("G"), and blue ("B")), with the green species being the species of interest. Enrichment of the green species is achieved via a three-step operation, including enrichment of the green species, removal of the red and blue species, and elution of the green species. Specifically, Figure 1A shows the enrichment step, where the mixture is loaded onto the first column, the species that first elutes from the column (blue) is discarded, and then the species to be enriched (green) is recycled to the second column. After all the green components have been recycled, the species that later elutes (red) is discarded from the first column via a strip process. An additional load (mixture) is injected into column 2, and the same separation as column 1 (discard blue, recycle green, discard red) is performed on column 2, resulting in enrichment of the green over an increasing number of cycles. Figure 1B shows the stripping step, where the species of interest (green) remains on the column and the undesired species (blue and red) are removed. An elution step follows stripping to recover the green species from the system. Figure 1C shows a simulation of the UV trace of the entire operation described above. In the left panel, the load material contains equal amounts of the three species: blue, green, and red. The middle panel is the product after 10 cycles of enrichment of the green species. Finally, the right panel shows that after one cycle of stripping, virtually none of the blue and red species remain in the system, while the green species remains unchanged. This product is eluted in the final step of the process and collected for subsequent analysis. The duration of each step for one of the molecules featured in this paper was approximately 45 minutes per cycle. Considering that there were 10 cycles of enrichment, two cycles of stripping, and one cycle of elution, the total processing time was approximately 10 hours.

[0029] [Diagram 2]Figures 2A, 2B, 2C, and 2D show charge variant separation of mAb1 antibody using one of the following chromatography systems: (i) high performance liquid chromatography (HPLC), (ii) fast protein liquid chromatography (FPLC), and (iii) sequential chromatography as described herein (CUBE). Figure 2A shows charge variant separation using an HPLC system. The fraction in front of the arrow is defined as the acidic fraction. Figure 2B shows charge variant separation using an FPLC system. The fraction in front of the arrow is defined as the acidic fraction. Figure 2C shows concentration of the acidic fraction using CUBE. A comparison of sequential chromatographic profiles of cycle 2 (black; 1) and cycle 10 (red; 2) from column 1 (left) and column 2 (right) is provided. Figure 2D provides a comparison of processing time (black (1) bars) and purity of acidic fraction (red (2) bars) by the methods of separation or concentration using HPLC, FPLC, and sequential chromatography methods. The time was calculated to generate 10 mg of acidic fraction from a load material of 17% acidic variant. Purity was determined by the iCIEF method.

[0030] [Diagram 3] Provides images of capillary electrophoresis profiles of charge variants of mAb1 antibody isolated using sequential chromatography. Panel (1) shows the load material before separation or enrichment. Both acidic and basic regions are identified. Panel (2) shows enrichment of acidic region 1 shown in panel (1). Panel (3) shows enrichment of acidic region 2 shown in panel (1). Panel (4) shows enrichment of basic region 1 shown in panel (1). And panel (5) shows enrichment of basic region 2 shown in panel (1). Y-axis is normalized based on peak maximum.

[0031] [Figure 4]Figure 1 provides an imaged capillary electrophoresis profile of mAb2 antibody charge variants isolated by sequential chromatography. The top panel (a) is of the load material before fractionation or concentration. The second panel (b) is of the load material after concentration. Arrows indicate acidic species present in the sample. The Y-axis is normalized based on the peak maximum.

[0032] [Figure 5A] Intact masses of deglycosylated samples of mAb3 are shown (from top to bottom: standard reference material, unenriched material, enriched acidic species, and enriched basic species). The peak around 144673 m / z is assigned to the deglycosylated species. The peaks around 144835 m / z and 144999 m / z are assigned to species with one and two glycosylations, respectively. The peak around 144558 m / z from the basic sample, which is barely visible in the unenriched material, is assigned to the C-terminal proline amidated species.

[0033] [Figure 5B] The presence of truncation species in the deglycosylated samples is shown in Figure 4A (top to bottom panels are standard reference material, load material, enriched acidic species, and enriched basic species). The peak around 121385 m / z is assigned to a species missing one light chain. The peak around 131538 m / z is assigned to a heavy chain truncation species with one heavy chain missing residues beyond 329.

[0034] [Figure 6]Figure 1 provides imaged capillary electrophoresis profiles of charge variants of mAb3 antibody. Acidic species of mAb3 were fractionated (FPLC) or enriched (continuous chromatography) using Mono S CEX or Mono Q AEX with salt or pH gradients. Panel (1) shows the load material before fractionation or enrichment. Panel (2) shows acidic species fractionated using Mono S with salt gradient and FPLC. Panel (3) shows acidic species enriched using Mono S with salt gradient and continuous chromatography. Panel (4) shows acidic species fractionated using Mono Q with pH gradient and FPLC. Panel (5) shows enriched acidic species using Mono Q with pH gradient in continuous mode. Y-axis is normalized based on peak maximum.

[0035] [Figure 7]Figures 7A, 7B, 7C, 7D, and 7E show peptide mapping mass spectrometry results of mAb3 antibody samples separated using cation exchange chromatography (CEX) (Mono S) and anion exchange chromatography (AEX) (Mono Q). Samples labeled as "fractionated" were prepared using the conventional FPLC method, and samples labeled as "enriched" were prepared using the continuous chromatography method. Figure 7A shows the degree of sialylation of samples separated using Mono S with a salt gradient (upper panel) or Mono Q with a pH gradient (lower panel). The bars indicate the different sialylated glycans detected: G1FS (white), G2FS (light gray), and G2FS2 (dark gray). G1FS is a monosialylated glycan of G1F glycan. G2FS and G2FS2 are mono- and di-sialylated glycans of G2F glycan, respectively. Figure 7B shows the deamidation of samples isolated using Mono S with a salt gradient (top panel) or Mono Q with a pH gradient (bottom panel). The bars represent the different amino acid residues N84 (white), N325 (light grey), N384 (grey), and N389 (dark grey). Figure 7C shows the glycation of samples isolated using Mono S with a salt gradient (top panel) or Mono Q with a pH gradient (bottom panel). The bars represent the different peptides: peptide 1 (white) and peptide 2 (grey). Figure 7D shows the percentage of N-terminal glutamine detected in samples isolated using Mono S with a salt gradient (top panel) or Mono Q with a pH gradient (bottom panel). Figure 7E shows the percentage of C-terminal proline amidation observed in samples isolated using Mono S with a salt gradient (top panel) or Mono Q with a pH gradient (bottom panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Large-scale protein purification and isolation is costly and time-consuming. Traditional methods for separating specific protein species rely on HPLC and / or FPLC. However, obtaining high yields, such as more than 10 mg of a desired protein species, can take weeks or more. Some embodiments of the present disclosure are directed to a method for isolating a protein species from a mixture containing the protein species and one or more impurities, comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode. Some embodiments of the present disclosure are directed to a method for increasing the purity and / or yield of a protein species from a mixture containing the protein species and one or more impurities, comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode.

[0037] Some aspects of the present disclosure provide a method for concentrating a protein species for analytical characterization, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous mode of operation of a chromatographic separation system; and (b) subjecting the species from (a) to analytical characterization. The present invention relates to a method comprising:

[0038] Some aspects of the present disclosure provide a method for performing analytical characterization of a protein species, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode of a chromatographic separation system; and (b) performing analytical characterization of the chemical species from (a); The present invention relates to a method comprising:

[0039] In some embodiments, the protein species is a charge variant. In some embodiments, the two or more chromatography columns comprise at least two ion exchange columns. In some embodiments, the two or more chromatography columns comprise a pH gradient. In some embodiments, the two or more chromatography columns comprise a salt gradient. In some embodiments, the two or more chromatography columns comprise a pH gradient and a salt gradient.

[0040] I. Terminology In order that this disclosure may be more readily understood, certain terms are defined first. As used in this application, each of the following terms shall have the meaning set forth below, unless expressly stated otherwise herein. Additional definitions are set forth throughout the application.

[0041] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "a" (or "an"), as well as the terms "one or more," and "at least one," can be used interchangeably herein. In some embodiments, the term "a" or "an" means "single." In other embodiments, the term "a" or "an" includes "two or more" or "multiple."

[0042] The term "and / or" as used herein is deemed to specifically disclose each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0043] The term "about" or "comprising essentially of" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "substantially comprising" can mean within one standard deviation or more than one standard deviation, according to the practice of the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude, or up to 5 times the value. When a particular value or composition is described in the present specification and claims, unless otherwise indicated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value or composition.

[0044] Where an embodiment is described herein using the term "comprising," it is understood that analogous embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0045] As used herein, the term "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that is 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or clear from the context (except where such value exceeds 100% of the possible values).

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

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the technical field to which this disclosure pertains.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0048] Units, prefixes, and symbols are written in the format accepted by the International System of Units (SI). The headings provided herein are not intended to limit the various aspects of the present disclosure, but may be understood by reference to the entire specification. Thus, defined terms are more fully defined by reference to the entire specification.

[0049] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in further detail in the following paragraphs.

[0050] The terms "purification", "separation" or "isolation" as used interchangeably herein refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition. In some embodiments, the protein of interest is a first charge variant of a protein, e.g., a charge variant of an antibody, and the one or more impurities include a second charge variant of the same protein.

[0051] As used herein, the term "chromatography" refers to a dynamic separation technique by which a target molecule, such as a target protein (e.g., a charge variant of a protein, e.g., an antibody), can be separated and isolated from other molecules in a mixture (e.g., other charge variants). Typically, in a chromatographic method, a liquid mobile phase transports a sample containing the target molecule of interest across or through a stationary phase (usually solid) medium. Due to differences in partitioning or affinity for the stationary phase, selected molecules temporarily bind to the stationary phase, while the mobile phase carries away different molecules at different times.

[0052] The term "continuous operation mode" or "continuous chromatography" refers to a chromatography process in which a sample is passed through at least two tandem chromatography columns (i.e., the eluate from the first column is loaded directly onto the second column). In some embodiments, the sample is loaded onto the first column, the eluate from the first column is applied directly to the second column, and the eluate from the second column is collected. In some embodiments, the sample is loaded onto the first column, the eluate from the first column is loaded directly onto the second column, and the eluate from the second column is loaded back onto the first column, and this process is repeated at least once, at least twice, at least three times, at least four times, or at least five times before the eluate from the second column is collected.

[0053] As used herein, the term "ion exchange chromatography" refers to a mode of chromatography in which target molecules, such as proteins (e.g., charge variants of proteins) to be separated, are separated based on polar interactions with charged molecules (e.g., positively or negatively charged molecules) immobilized on a chromatography resin. Elution from an ion exchange chromatography column can be achieved using a salt gradient or by changing the pH.

[0054] "Anion exchange chromatography" or "AEX" refers to ion exchange chromatography involving a positively charged ion exchange resin that has an affinity for molecules with positive and negative surface charges. To separate the protein of interest from other bound proteins, a salt gradient can be applied to the column, and the proteins are eluted in order according to their net surface charge.

[0055] "Cation exchange chromatography" or "CEX" refers to ion exchange chromatography involving a negatively charged ion exchange resin that has an affinity for molecules that have a net surface charge. To separate the protein of interest from other bound proteins, a salt gradient can be applied to the column and the proteins are eluted in order according to their net surface charge.

[0056] The term "affinity chromatography" as used herein refers to a mode of chromatography in which target molecules, such as protein molecules to be separated (e.g., charge variants of a protein), are separated by a "lock-and-key" interaction with a molecule immobilized on a chromatography resin (e.g., a Protein A-based ligand). This specific interaction causes the target molecule to bind to the molecule immobilized on the resin, while undesired molecules pass through. Altering the temperature, pH, or ionic strength of the mobile phase releases the target molecule in high purity. In various embodiments described herein, affinity chromatography involves the addition of a sample containing the target molecule (e.g., an immunoglobulin or other Fc-containing protein) to a solid support carrying a ligand based on the C domain of Protein A (referred to as Protein A affinity chromatography media or resin). Other ligands used in affinity chromatography can include, for example, Protein G from Streptococci, which binds to the Fc region of immunoglobulins.

[0057] As used herein, the term "high performance liquid chromatography," or "HPLC," or "high pressure liquid chromatography" refers to a chromatography system that relies on pumps to pass pressurized liquids and sample mixtures through columns filled with sorbent, leading to separation of the sample components. The components of the sample mixture are separated from one another due to differing degrees of interaction with the sorbent particles.

[0058] The term "capillary isoelectric focusing gel electrophoresis" or "cIEF gel electrophoresis" as used herein refers to a high-resolution analytical technique that can separate protein / peptide mixtures, protein glycosylation, and other charge variants based on their isoelectric point (pI).

[0059] As used herein, the term "imaged capillary isoelectric focusing" or "iCIEF" refers to an analytical technique in which amphoteric components of biomolecules are separated according to their isoelectric points in an electric field.

[0060] The term "contacting" as used herein refers to applying a solution, e.g., a mixture containing a protein product and contaminants, to a chromatography matrix, as described herein. In some embodiments, the term "contacting" is synonymous with "loading" a solution onto a chromatography column. As used herein, "column packing" or "chromatography matrix" refers to an adsorbent solid material contained within a chromatography column. In some aspects, the column packing comprises SuperQ. In some aspects, the column packing comprises GigaCap. In some aspects, the column packing comprises FRACTOGEL® SO3 - Includes.

[0061] When used in the context of a gradient being applied to a chromatography matrix, the term "applied" broadly means that the gradient is formed, directly or indirectly, in and / or around the chromatography matrix. In some embodiments, the chromatography matrix is ​​present in a column and the gradient is formed within the column. In some embodiments, the gradient applied to the chromatography matrix is ​​formed within the column, as opposed to a gradient that is formed externally and then added to the column. In certain embodiments, the gradient applied to the chromatography matrix is ​​formed within the column as a result of the addition of multiple buffers to the chromatography matrix. In other embodiments, the gradient applied to the chromatography matrix is ​​formed externally and then added to the column.

[0062] As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of cells, either surface-attached or in suspension, maintained or grown in a medium (see definition of "medium" below) under conditions suitable for the survival and / or growth of the population of cells. As will be apparent to one of skill in the art, these terms, as used herein, can refer to a combination that includes a population of cells and the medium in which the population is suspended.

[0063] As used herein, the term "expression" or "expressing" refers to the transcription and translation that occurs in a cell. The expression level of a product gene in a host cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced in the cell, or both.

[0064] The term "antibody" refers in some embodiments to a protein consisting of at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region comprises a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and regions that are more conserved called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The term "antibody" can include bispecific or multispecific antibodies.

[0065] As used herein, "IgG antibodies", e.g., human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments have the structure of a naturally occurring IgG antibody, i.e., have the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an IgG1, IgG2, IgG3, or IgG4 antibody may consist of two heavy chains (HC) and two light chains (LC), with the two HCs and LCs having the same number and positions of disulfide bridges that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bridges).

[0066] Immunoglobulins may be of any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species, IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. There are several allotypes of immunoglobulins, such as IgG1, that differ from each other by at most a few amino acids. "Antibody" includes naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, and fully synthetic antibodies.

[0067] The term "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody is exerted by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (papain cleavage fragment) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (ii) a F(ab')2 fragment (pepsin cleavage fragment) or a similar bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be recombinantly linked by a synthetic linker that allows the VL and VH regions to be made into a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art and screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0068] The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, including, for example, (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by other means involving splicing of human immunoglobulin gene sequences with other DNA sequences.

[0069] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) that is encoded by heavy chain constant region genes.

[0070] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by their commonly accepted one-letter codes.

[0071] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The terms "polypeptide" or "protein" or "product" or "product protein" or "amino acid residue sequence" are used interchangeably. The term "polypeptide" refers to a chain containing two or more amino acids and does not refer to a specific length. As used herein, the term "protein" is intended to encompass molecules containing one or more polypeptides, which may in some cases be linked by bonds other than amide bonds. Alternatively, a protein may be a single polypeptide chain. In this latter example, a single polypeptide chain may be formed by fusing two or more polypeptide subunits to form a protein. The terms "polypeptide" and "protein" also refer to the product of post-expression modifications, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide or protein may be derived from a natural biological source or produced by recombinant technology.

[0072] The term "polynucleotide" or "nucleotide" as used herein is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). The term "nucleic acid" refers to one or more nucleic acid segments present in a polynucleotide, e.g., DNA, cDNA, RNA fragments. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment, e.g., a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or polynucleotides that have been purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules that have been produced synthetically. In addition, a polynucleotide or nucleic acid can contain regulatory elements, such as a promoter, an enhancer, ribosome binding sites, transcription termination signals, and the like.

[0073] As used herein, the term "impurity" or "impurities" refers to one or more molecules, e.g., polypeptides, nucleic acid molecules, small molecules, or any combination thereof, that are present in a mixture with a target molecule, e.g., a target species of a polypeptide, e.g., a target charge variant of a polypeptide. In some embodiments, the impurities are different polypeptides, e.g., polypeptides that have a different structure, sequence, or function than the target polypeptide. In some embodiments, the impurities are different species, e.g., charge variants or HMW species, of the target polypeptide.

[0074] As used herein, the term "purity" refers to the degree to which a composition, e.g., a solution containing a target polypeptide, contains one or more impurities. For example, a solution containing a target polypeptide in which 98% of the target polypeptide in the solution is charge variant A and 2% of the target polypeptide contains one or more charge variants other than charge variant A has a purity of 98%.

[0075] II. Methods of the Disclosure Some embodiments of the present disclosure are directed to a method of isolating a protein species from a mixture comprising the protein species and one or more impurities, the method comprising contacting the mixture with two or more chromatography columns in a continuous operation mode. Some embodiments of the present disclosure are directed to a method of increasing the purity and / or yield of a protein species from a mixture comprising the protein species and one or more impurities, the method comprising contacting the mixture with two or more chromatography columns in a continuous operation mode. In some embodiments, the chromatography comprises a countercurrent purification system. In some embodiments, the countercurrent purification system is a multi-column countercurrent solvent gradient purification (MCSGP) system.

[0076] The term "species" or "variant" of a protein refers to different forms that are encoded by the same nucleotide sequence but differ in protein chain length, protein mass, and / or post-translational modifications. This includes, but is not limited to, species with different degrees of glycosylation, monomers, oligomers or multimers (also called high molecular weight (HMW) species, truncated forms, charged forms, etc. For example, monoclonal antibodies (mAbs) are heterogeneous in their biochemical and biophysical properties due to multiple post-translational modifications and degradation events. The charge heterogeneity of mAbs is affected by such modifications, which alter the net charge and local charge distribution. Charge variants of mAbs are identified as acidic, basic and major species. As used herein, the term "major species", "major peak" or "major variant" of a mAb refers to a species that is characterized by a neutral isoelectric point (p I) refers to the mAb that elutes as the main peak. As used herein, the term "acidic species" or "acidic variant" of a mAb refers to a variant that has a lower pI than the main species. As used herein, the term "basic species" or "basic variant" of a mAb refers to a variant that has a higher pI than the main species. The C-terminal lysine residue of fmAb provides an additional positive charge, increasing the basic species of the mAb. Inefficient cleavage of C-terminal lysine residues by endogenous carboxypeptidases during antibody production is one of the main reasons that result in mAbs with 0, 1 or 2 C-terminal lysines (Zhang et al., 2015).

[0077] In some embodiments, the methods disclosed herein result in an increased purity of a chemical species compared to conventional methods. In some embodiments, the methods disclosed herein result in an increased purity of a chemical species compared to HPLC or FPLC methods. In some embodiments, the purity of a sample is increased by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to HPLC or FPLC methods.

[0078] In some embodiments, the methods disclosed herein reduce the total time required to obtain a sufficient amount of a chemical species compared to conventional methods. In some embodiments, the methods disclosed herein reduce the total time required to obtain a sufficient amount of a chemical species compared to HPLC or FPLC methods. In some embodiments, a sufficient amount of a chemical species is at least about 5 mg, at least about 6 mg, at least about 7 mg, at least about 8 mg, at least about 9 mg, at least about 10 mg, at least about 11 mg, at least about 12 mg, at least about 13 mg, at least about 14 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 75 mg, or at least about 100 mg of a chemical species. In some embodiments, a sufficient amount of a chemical species is at least about 10 mg. In some embodiments, the time required to obtain a sufficient amount of the chemical species is reduced by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, as compared to conventional methods (e.g., HPLC or FPLC). In some embodiments, the time required to obtain a sufficient amount of the chemical species is less than about 90%, less than about 80%, less than about 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the time required to obtain the same or comparable amount of the chemical species using conventional methods, e.g., HPLC or FPLC. In some embodiments, the methods disclosed herein obtain at least about 10 mg of the target species in less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 48 hours, less than about 36 hours, less than about 30 hours, or less than about 24 hours. In some embodiments, the methods disclosed herein obtain at least about 10 mg of the target species in less than about 48 hours.In some embodiments, the methods disclosed herein obtain at least about 10 mg of the target species in less than about 36 hours. In some embodiments, the methods disclosed herein obtain at least about 10 mg of the target species in less than about 30 hours. In some embodiments, the methods disclosed herein obtain at least about 10 mg of the target species in less than about 24 hours.

[0079] In some embodiments, the methods disclosed herein have increased productivity (measured by normalizing yield (e.g., grams of chemical species) to time period) compared to conventional methods, e.g., HPLC or FPLC. In some embodiments, the productivity is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold compared to conventional methods (e.g., HPLC or FPLC).

[0080] In some embodiments, the method further comprises subjecting the isolated species to one or more analytical characterizations. Thus, some embodiments of the present disclosure provide a method of concentrating a protein species for analytical characterization, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous mode of operation of a chromatographic separation system; and (b) subjecting the species from (a) to analytical characterization. Some aspects of the present disclosure are directed to a method of performing analytical characterization of a protein species, comprising: (a) separating a protein species from a mixture containing the protein species and one or more impurities, the separation comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode of a chromatographic separation system; and (b) performing analytical characterization of the chemical species from (a); The present invention relates to a method comprising:

[0081] In some embodiments, the analytical characterization comprises HPLC system, capillary isoelectric focusing (cIEF) gel electrophoresis, imaged capillary isoelectric focusing (iCIEF), cation exchange chromatography (CEX), anion exchange chromatography (AEX), MFI, SEC MALS, SEC, mass spectrometry, or any combination thereof. In some embodiments, the analytical characterization comprises subjecting the chemical species, e.g., charge variants of a protein (e.g., an antibody), to an HPLC system. In some embodiments, the analytical characterization comprises subjecting the chemical species, e.g., charge variants of a protein (e.g., an antibody), to capillary isoelectric focusing (cIEF) gel electrophoresis. In some embodiments, the analytical characterization comprises subjecting the chemical species, e.g., charge variants of a protein (e.g., an antibody), to imaged capillary isoelectric focusing (iCIEF). In some embodiments, the analytical characterization comprises subjecting the chemical species, e.g., charge variants of a protein (e.g., an antibody), to cation exchange chromatography (CEX). In some embodiments, analytical characterization involves subjecting a species, e.g., a charge variant of a protein (e.g., an antibody), to anion exchange chromatography (AEX).

[0082] A. Column Chromatography Some embodiments of the disclosure include contacting a mixture comprising a protein species and one or more impurities with two or more chromatography columns in a continuous operation mode. In some embodiments, the two or more chromatography columns concentrate species, e.g., charge variants. In some embodiments, the method includes loading a mixture comprising a protein species and one or more impurities onto a first chromatography column. The first chromatography column can include any chromatography matrix. In some embodiments, the chromatography matrix of the first column is an AEX matrix. In some embodiments, the chromatography matrix of the first column is a CEX matrix. In some embodiments, the chromatography matrix of the first column is a mixed-mode chromatography matrix. In some embodiments, the chromatography matrix of the first column is an affinity chromatography matrix. In some embodiments, the chromatography matrix of the first column is a size-exclusion matrix.

[0083] In some embodiments, the load mixture passes through a first chromatographic column and is separated into (i) an enriched species containing the species and (ii) one or more waste species containing one or more impurities. This stage is referred to herein as "enrichment stage I." In enrichment stage I, the waste species are eluted from the column and discarded. The enriched species leaving the column are then loaded onto a second column. In some embodiments, the second column is positioned such that the enriched species is eluted directly from the first column to the second column. In some embodiments, the enriched species is collected from the first column and applied to the second column. In some embodiments, after the enriched species has passed through the column, a second waste species is eluted from the first column and discarded, i.e., a second waste species that passes through the column more slowly than the enriched species is eluted from the first column and discarded.

[0084] In some embodiments, the enriched species is applied to a second column. The enriched species passes through the second column and is further separated into (i) an enriched species comprising the species and (ii) one or more additional waste species comprising one or more additional impurities. This stage is referred to herein as "enrichment stage II." In enrichment stage II, an additional waste species is eluted from the column and discarded. In some embodiments, the additional waste species elutes from the second column before the enriched species. In some embodiments, the additional waste species elutes from the second column after the enriched species. In some embodiments, the additional waste species elutes from the second column before the enriched species and the additional waste species elutes from the second column after the enriched species.

[0085] Once the enriched species has passed through the second column, it is loaded onto the first column. In some embodiments, an additional mixture (comprising the species and one or more impurities) is added to the first column simultaneously with the enriched species. In some embodiments, the enriched species is combined with the additional mixture prior to loading onto the first column. In some embodiments, the enriched species is loaded onto the first column and then the additional mixture is loaded onto the same first column. In some embodiments, the additional mixture is loaded onto the first column and then the enriched species is loaded onto the same first column. In some embodiments, the additional mixture is added after the enriched species has been added to the first chromatographic column and prior to passing the enriched species through the first chromatographic column. In some embodiments, the first column is re-equilibrated prior to loading. In some embodiments, the second column is re-equilibrated prior to loading.

[0086] In some embodiments, enrichment steps I and II are repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, enrichment steps I and II are repeated until all of the starting mixture has been applied to the first column.

[0087] In some embodiments, the method further comprises a "depletion step". That is, after enrichment steps I and II have been repeated n times, the enriched species proceeds to a depletion step. In some embodiments, n is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some embodiments, the depletion step comprises contacting the enriched species with a first chromatographic column in the absence of additional mixture. The enriched species is then passed through the first chromatographic column to separate the charged species from one or more remaining impurities. In some embodiments, the first remaining impurity exits the first column before the enriched species, and the first remaining impurity is discarded. In some embodiments, the enriched species exits the first column and is applied to a second column. The enriched species is then passed through a second chromatographic column to separate the enriched species from one or more remaining impurities. Following the depletion step, the species is eluted from the second column.

[0088] In some embodiments, the eluted species has a purity of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 100%. In some embodiments, the eluted species has a purity of at least about 90%. In some embodiments, the eluted species has a purity of at least about 95%. In some embodiments, the eluted species has a purity of at least about 96%. In some embodiments, the eluted species has a purity of at least about 97%. In some embodiments, the eluted species has a purity of at least about 98%. In some embodiments, the eluted species has a purity of at least about 99%.

[0089] In some embodiments, the species is eluted at a concentration that is at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, or at least about 10.0 times greater than in the mixture.

[0090] In some embodiments, the method further comprises measuring a post-translational modification of the protein species. In some embodiments, the modification comprises N-glutamic acid pyroglutamylation, C-terminal lysine truncation, C-terminal proline amidation, glycosylation, sialylation, deamidation, aspartic acid isomerization, general truncation, or any combination thereof.

[0091] In some embodiments, one or more chromatography columns comprises a salt gradient. In some embodiments, one or more chromatography columns comprises a pH gradient. In some embodiments, one or more chromatography columns comprises a salt gradient and a pH gradient. In some embodiments, the salt gradient comprises sodium chloride (NaCl gradient). In some embodiments, the salt gradient comprises a gradient from no salt, e.g., no NaCl, to a high salt concentration.

[0092] In some embodiments, the salt (e.g., NaCl) concentration is 0 mM to at least about 500 mM, 0 mM to at least about 450 mM, 0 mM to at least about 400 mM, 0 mM to at least about 350 mM, 0 mM to at least about 300 mM, 0 mM to at least about 290 mM, 0 mM to at least about 280 mM, 0 mM to at least about 270 mM, 0 mM to at least about 260 mM, 0 mM to at least about 250 mM, about 50 mM to at least about 500 mM, about 50 mM to at least about 450 mM, about 50 mM to at least about 400 mM, about 50 mM to at least about 350 mM, about 50 mM to at least about 300 mM, about 50 mM to at least about 290 mM, about 50 mM to at least about 280 mM, about 50 from about 270 mM, from about 50 mM to at least about 260 mM, from about 50 mM to at least about 250 mM, from about 100 mM to at least about 500 mM, from about 100 mM to at least about 450 mM, from about 100 mM to at least about 400 mM, from about 100 mM to at least about 350 mM, from about 100 mM to at least about 300 mM, from about 100 mM to at least about 290 mM, from about 100 mM to at least about 280 mM, from about 100 mM to at least about 270 mM, from about 100 mM to at least about 260 mM, from about 100 mM to at least about 250 mM, from about 150 mM to at least about 350 mM, from about 1200 mM to at least about 300 mM, or from about 225 mM to at least about 375 mM of salt (e.g., NaCl).

[0093] In some embodiments, the concentration of salt (e.g., NaCl) is at least 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 260 mM, at least about 270 mM, at least about 280 mM, at least about 290 mM, at least about 300 mM, at least about 310 mM, at least about 320 mM, at least about 330 mM, at least about 340 mM, at least about 350 mM, at least about 360 mM, at least about 370 mM, at least about 380 mM, at least about 390 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM, or at least about 600 mM salt (e.g., NaCl).

[0094] In some embodiments, the salt gradient is a linear gradient. In some embodiments, the salt gradient is a step gradient.

[0095] In some embodiments, the salt gradient mobile phase further comprises a buffer. In some embodiments, the salt gradient mobile phase comprises MES. In some embodiments, the salt gradient mobile phase comprises at least about 10 mM MES, at least about 15 mM MES, at least about 20 mM MES, at least about 25 mM MES, or at least about 30 mM MES. In some embodiments, the salt gradient mobile phase comprises at least about 20 mM MES. In some embodiments, the pH of the salt gradient mobile phase is at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, or at least about 6.5. In some embodiments, the salt gradient mobile phase comprises 20 mM MES with and without 250 mM sodium chloride at pH 6.0. In some embodiments, the salt gradient mobile phase comprises 20 mM MES, pH 5.8, with and without 400 mM sodium chloride.

[0096] In some embodiments, one or more chromatographic columns comprise a pH gradient. In some embodiments, the pH of the pH gradient mobile phase is between about pH 3 and about pH 11, about pH 3 and about pH 10, about pH 3 and about pH 9, about pH 3 and about pH 8, about pH 3 and about pH 7, about pH 4 and about pH 11, about pH 5 and about pH 11, about pH 6 and about pH 11, or about pH 7 and about pH 11. In some embodiments, the pH of the pH gradient mobile phase is between about pH 3 and about pH 11.

[0097] In some embodiments, the pH gradient mobile phase further comprises a buffer, which in some embodiments comprises MES, phosphate buffer, Tris, bis-Tris, 1,3 diaminopropane, diethanolamine, piperazine, imidazole, acetic acid, malonic acid, formic acid, MOPSO, HEPES, BICINE, CHES, CAPS, or any combination thereof.

[0098] In some embodiments, the buffer comprises at least about 10 mM MES, at least about 15 mM MES, at least about 20 mM MES, at least about 25 mM MES, or at least about 30 mM MES, hi some embodiments, the buffer comprises at least about 20 mM MES.

[0099] In some embodiments, the buffer comprises at least about 1 mM Tris, at least about 2 mM Tris, at least about 3 mM Tris, at least about 4 mM Tris, at least about 5 mM Tris, at least about 6 mM Tris, at least about 7 mM Tris, at least about 8 mM Tris, at least about 9 mM Tris, and at least about 10 mM Tris. In some embodiments, the buffer comprises at least about 5 mM Tris.

[0100] In some embodiments, the buffer comprises at least about 1 mM bis-Tris, at least about 2 mM bis-Tris, at least about 3 mM bis-Tris, at least about 4 mM bis-Tris, at least about 5 mM bis-Tris, at least about 6 mM bis-Tris, at least about 7 mM bis-Tris, at least about 8 mM bis-Tris, at least about 9 mM bis-Tris, and at least about 10 mM bis-Tris. In some embodiments, the buffer comprises at least about 5 mM bis-Tris.

[0101] In some embodiments, the buffer is at least about 1 mM 1,3 diaminopropane, at least about 2 mM 1,3 diaminopropane, at least about 3 mM 1,3 diaminopropane, at least about 4 mM 1,3 diaminopropane, at least about 5 mM 1,3 diaminopropane, at least about 6 mM 1,3 diaminopropane, at least about 7 mM 1,3 diaminopropane, at least about 8 mM 1,3 diaminopropane, at least about 9 mM 1,3 diaminopropane, and at least about 10 mM 1,3 diaminopropane. In some embodiments, the buffer comprises at least about 5 mM 1,3 diaminopropane.

[0102] In some embodiments, the buffer comprises at least about 1 mM diethanolamine, at least about 2 mM diethanolamine, at least about 3 mM diethanolamine, at least about 4 mM diethanolamine, at least about 5 mM diethanolamine, at least about 6 mM diethanolamine, at least about 7 mM diethanolamine, at least about 8 mM diethanolamine, at least about 9 mM diethanolamine, and at least about 10 mM diethanolamine. In some embodiments, the buffer comprises at least about 5 mM diethanolamine.

[0103] In some embodiments, the buffer comprises at least about 1 mM piperazine, at least about 2 mM piperazine, at least about 3 mM piperazine, at least about 4 mM piperazine, at least about 5 mM piperazine, at least about 6 mM piperazine, at least about 7 mM piperazine, at least about 8 mM piperazine, at least about 9 mM piperazine, and at least about 10 mM piperazine. In some embodiments, the buffer comprises at least about 5 mM piperazine.

[0104] In some embodiments, the buffer comprises at least about 1 mM imidazole, at least about 2 mM imidazole, at least about 3 mM imidazole, at least about 4 mM imidazole, at least about 5 mM imidazole, at least about 6 mM imidazole, at least about 7 mM imidazole, at least about 8 mM imidazole, at least about 9 mM imidazole, and at least about 10 mM imidazole. In some embodiments, the buffer comprises at least about 5 mM imidazole.

[0105] In some embodiments, the buffer comprises at least about 1 mM acetate, at least about 2 mM acetate, at least about 3 mM acetate, at least about 4 mM acetate, at least about 5 mM acetate, at least about 6 mM acetate, at least about 7 mM acetate, at least about 8 mM acetate, at least about 9 mM acetate, and at least about 10 mM acetate. In some embodiments, the buffer comprises at least about 5 mM acetate.

[0106] In some embodiments, the buffer comprises at least about 1 mM malonic acid, at least about 2 mM malonic acid, at least about 3 mM malonic acid, at least about 4 mM malonic acid, at least about 5 mM malonic acid, at least about 6 mM malonic acid, at least about 7 mM malonic acid, at least about 8 mM malonic acid, at least about 9 mM malonic acid, and at least about 10 mM malonic acid. In some embodiments, the buffer comprises at least about 5 mM malonic acid.

[0107] In some embodiments, the buffer comprises at least about 1 mM formic acid, at least about 2 mM formic acid, at least about 3 mM formic acid, at least about 4 mM formic acid, at least about 5 mM formic acid, at least about 6 mM formic acid, at least about 7 mM formic acid, at least about 8 mM formic acid, at least about 9 mM formic acid, and at least about 10 mM formic acid. In some embodiments, the buffer comprises at least about 5 mM formic acid.

[0108] In some embodiments, the buffer comprises at least about 1 mM MOPSO, at least about 2 mM MOPSO, at least about 3 mM MOPSO, at least about 4 mM MOPSO, at least about 5 mM MOPSO, at least about 6 mM MOPSO, at least about 7 mM MOPSO, at least about 8 mM MOPSO, at least about 9 mM MOPSO, and at least about 10 mM MOPSO. In some embodiments, the buffer comprises at least about 5 mM MOPSO.

[0109] In some embodiments, the buffer comprises at least about 1 mM HEPES, at least about 2 mM HEPES, at least about 3 mM HEPES, at least about 4 mM HEPES, at least about 5 mM HEPES, at least about 6 mM HEPES, at least about 7 mM HEPES, at least about 8 mM HEPES, at least about 9 mM HEPES, and at least about 10 mM HEPES. In some embodiments, the buffer comprises at least about 5 mM HEPES.

[0110] In some embodiments, the buffer contains at least about 1 mM BICINE, at least about 2 mM BICINE, at least about 3 mM BICINE, at least about 4 mM BICINE, at least about 5 mM BICINE, at least about 6 mM BICINE, at least about 7 mM BICINE, at least about 8 mM BICINE, including at least about 9 mM BICINE, and at least about 10 mM BICINE. In some embodiments, the buffer contains at least about 5 mM BICINE.

[0111] In some embodiments, the buffer comprises at least about 1 mM CHES, at least about 2 mM CHES, at least about 3 mM CHES, at least about 4 mM CHES, at least about 5 mM CHES, at least about 6 mM CHES, at least about 7 mM CHES, at least about 8 mM CHES, at least about 9 mM CHES, and at least about 10 mM CHES. In some embodiments, the buffer comprises at least about 5 mM CHES.

[0112] In some embodiments, the buffer comprises at least about 1 mM CAPS, at least about 2 mM CAPS, at least about 3 mM CAPS, at least about 4 mM CAPS, at least about 5 mM CAPS, at least about 6 mM CAPS, at least about 7 mM CAPS, at least about 8 mM CAPS, at least about 9 mM CAPS, and at least about 10 mM CAPS. In some embodiments, the buffer comprises at least about 5 mM CAPS.

[0113] In some embodiments, the chromatography column comprises an AEX matrix and the pH gradient mobile phase comprises about 5 mM 1,3 diaminopropane, about 5 mM diethanolamine, about 5 mM Tris, about 5 mM imidazole, about 5 mM bis-Tris, and about 5 mM piperazine, and the pH is about 11.1. In some embodiments, the chromatography column comprises an AEX matrix and the pH gradient mobile phase comprises about 5 mM 1,3 diaminopropane, about 5 mM diethanolamine, about 5 mM Tris, about 5 mM imidazole, about 5 mM bis-Tris, about 5 mM piperazine, and about 5 mM acetic acid, and the pH is about 3.5.

[0114] In some embodiments, the chromatography column comprises a CEX matrix and the pH gradient mobile phase comprises about 5 mM malonic acid, about 5 mM formic acid, about 5 mM acetic acid, about 5 mM MES, about 5 mM MOPSO, about 5 mM HEPES, about 5 mM BICINE, about 5 mM CHES, and about 5 mM CAPS, and the pH is about 4.0. In some embodiments, the chromatography column comprises a CEX matrix and the pH gradient mobile phase comprises about 5 mM malonic acid, about 5 mM formic acid, about 5 mM acetic acid, about 5 mM MES, about 5 mM MOPSO, about 5 mM HEPES, about 5 mM BICINE, about 5 mM CHES, and about 5 mM CAPS, and the pH is about 11.0.

[0115] B. Polypeptides The methods disclosed herein can be used to isolate and / or purify any polypeptide species. In some embodiments, the polypeptide is a protein. In some embodiments, the species is a charge variant of a protein. In some embodiments, the species is an acidic species. In some embodiments, the species is a basic species. In some embodiments, the species is a predominant species.

[0116] In some embodiments, the protein is subjected to a purification process before being subjected to the method disclosed herein.In some embodiments, the protein is subjected to affinity chromatography beforehand, for example, partially purified.In some embodiments, the affinity chromatography beforehand comprises Protein A affinity chromatography.

[0117] In some embodiments, the protein comprises a fusion protein. In some embodiments, the protein comprises an immunoglobulin component fused to a biologically active polypeptide. In some embodiments, the immunoglobulin component comprises a fragment of an antibody. In some embodiments, the immunoglobulin component comprises a fragment of a constant region of an antibody. In some embodiments, the immunoglobulin component comprises an Fc.

[0118] In some embodiments, the protein comprises an immunoglobulin fused to a growth factor, a clotting factor, a cytokine, a chemokine, an enzyme, a hormone, or any combination thereof. In some embodiments, the protein comprises an Fc fused to a CTLA-4 polypeptide. In some embodiments, the protein comprises an abatacept. In some embodiments, the protein comprises a belatacept. In some embodiments, the protein comprises an Fc fused to an interleukin.

[0119] In some embodiments, the protein comprises an antibody or antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof binds to a tumor antigen. In some embodiments, the antibody or antigen-binding portion thereof binds to a checkpoint inhibitor. In some embodiments, the antibody or antigen-binding portion thereof binds to an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD1 la, tissue factor (TF), MICA / B PSCA, IL-8, EGFR, HER3, HER4, and any combination thereof.

[0120] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-1. A variety of human monoclonal antibodies that specifically bind to PD-1 with high affinity are described in U.S. Patent Nos. 8,008,449, 6,808,710, 7,488,802, 8,168,757, 8,354,509, U.S. Patent Application Publication No. 2016 / 0272708, and PCT International Publication No. WO2012 / 145. 493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206 107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 0208 58, WO2016 / 197367, 2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 10615 9, WO2014 / 194302, 2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, No. 6,393,991, each of which is incorporated by reference in its entirety.

[0121] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), semipilimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS00I (TAIZHOU JUNSHI PHARMA; also known as toripalimab; Si Yang Liu et al., J Hematol. Oneal. 10:136 (2017)), BGB-A317 (Beigene; also known as tislelizumab; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847; Si-Yang Liu et al., J Hematol. Oneal. JO:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al., J Hematol. Oneal.JO:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics, see WO2017 / 19846), BCD-100 (Biocad; Kaplon et al., mAbs 10(2):183-203 (2018)), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, WO2017 / 133540).

[0122] In some embodiments, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab.

[0123] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1. Examples of anti-PD-L1 antibodies include, but are not limited to, the antibodies disclosed in U.S. Patent No. 9,580,507. In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see U.S. Patent No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol 3 l(suppl): 3000), durvalumab (AstraZeneca; IMFINZ Immune System Inflammatory Bowel Disease (IMID)). (商標), also known as MEDI-4736; WO2011 / 066389), avelumab (Pfizer; also known as BAVENC10®, MSB-0010718C; see WO2013 / 079174), STI- 1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D MED / alfumab; see Zhang et al., Cell Diseov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO2017 / 034916), BGB-A333 (BeiGene; Desai et al., JCO 36 (15supp():TPS3 113 (2018)), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

[0124] In one embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). In one embodiment, the PD-L1 antibody is durvalumab (IMFINZ®). (商標) In one embodiment, the PD-L1 antibody is avelumab (BAVENC10®).

[0125] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to CTLA-4. A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described in, for example, U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121, and International Publication Nos. WO2012 / 122444, WO2007 / 113648, WO2016 / 196237, and WO2000 / 037504, each of which is incorporated herein by reference in its entirety. In certain embodiments, the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc., see WO 2016 / 196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206, see WO 2000 / 037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In certain embodiments, the anti-CTLA-4 antibody is ipilimumab. In certain embodiments, the CTLA-4 antibody is tremelimumab. In certain embodiments, the CTLA-4 antibody is MK-1308. In a particular embodiment, the CTLA-4 antibody is AGEN-1884.

[0126] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to LAG-3. Antibodies that bind to LAG-3 include those described in International Publication WO / 2015 / 042246 and US Patent Application Publication Nos. 2014 / 0093511 and 2011 / 0150892, each of which is incorporated herein by reference in its entirety. Non-limiting examples of anti-LAG-3 antibodies include, but are not limited to, 25F7 (described in US Patent Application Publication No. 2011 / 0150892), BMS-986016, IMP731 (H5L7BW), MK-4280 (28G-10), REGN3767, humanized BAP050, IMP-701 (LAG-5250), TSR-033, BI754111, MGD013, or FS-118. These and other anti-LAG-3 antibodies useful in the claimed invention are described in, e.g., WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220569 , WO2018 / 071500, WO2017 / 015560, WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227, and WO2014 / 140180, each of which is incorporated by reference in its entirety.

[0127] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to CD137. Antibodies that bind to CD137 are described in U.S. Patent Publication No. 2005 / 0095244 and U.S. Patent Nos. 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863, and 6,210,669, each of which is incorporated herein by reference in its entirety. In some embodiments, the anti-CD137 antibody is urelumab (BMS-663513), described in U.S. Patent No. 7,288,638 (20H4.9-IgG4[10C7 or BMS-663513). In some embodiments, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1) described in U.S. Patent No. 7,288,638. In some embodiments, the anti-CD137 antibody is 4E9 or BMS-554271 described in U.S. Patent No. 6,887,673. In some embodiments, the anti-CD137 antibody is an antibody disclosed in U.S. Patent No. 7,214,493; No. 6,303,121; No. 6,569,997; No. 6,905,685; or No. 6,355,476. In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492; No. 3H3 or BMS-469497; or No. 3E1 described in U.S. Patent No. 6,362,325. In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued U.S. Patent No. 6,974,863 (such as 53A2). In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued U.S. Patent No. 6,210,669 (such as 1D8, 3B8, 3E1). In some embodiments, the antibody is Pfizer's PF-05082566 (PF-2566).

[0128] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to KIR. Examples of anti-KIR antibodies are disclosed in International Publication Nos. WO / 2014 / 055648, WO2005 / 003168, WO2005 / 009465, WO2006 / 072625, WO2006 / 072626, WO2007 / 042573, WO2008 / 084106, WO2010 / 065939, WO2012 / 071411, and WO / 2012 / 160448, each of which is incorporated herein by reference in its entirety. One anti-KIR antibody useful in the present disclosure is lirilumab (also known as BMS-986015, IPH2102, or the S241P variant of 1-7F9), first described in International Publication WO2008 / 084106. An additional anti-KIR antibody useful in the present disclosure is 1-7F9 (also known as IPH2101), described in International Publication WO2006 / 003179.

[0129] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to GITR. Examples of anti-GITR antibodies are described in International Publications WO / 2015 / 031667, WO2015 / 184,099, WO2015 / 026,684, WO11 / 028683, and WO / 2006 / 105021, U.S. Patent Nos. 7,812,135, and 8,388,967, and U.S. Publication Nos. 2009 / 0136494, 2014 / 0220002, 2013 / 0183321, and 2014 / 0348841, each of which is incorporated herein by reference in its entirety. In one embodiment, the anti-GITR antibody useful in the present disclosure is TRX518 (e.g., as described in Schaer et al., Curr Opin Immunol. (2012) Apr; 24(2): 217-224, and WO / 2006 / 105021). In another embodiment, the anti-GITR antibody is selected from MK4166, MK1248, and the antibodies described in WO11 / 028683 and US8,709,424. In an embodiment, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015 / 031667. In an embodiment, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015 / 184099, such as antibody Hum231#1 or Hum231#2, or CDRs thereof, or derivatives thereof (e.g., pab1967, pab1975, or pab1979). In some embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in JP2008278814, WO09 / 009116, WO2013 / 039954, US20140072566, US20140072565, US20140065152, or WO2015 / 026684, or is INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (Medimmune). In some embodiments, the anti-GITR antibody is an anti-GITR antibody described in PCT / US2015 / 033991 (e.g., an antibody comprising the variable regions of 28F3, 18E10, or 19D3).

[0130] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to TIM3. In some embodiments, the anti-TIM3 antibody is disclosed in International Publication WO2018013818, WO / 2015 / 117002 (e.g., MGB453, Novartis), WO / 2016 / 161270 (e.g., TSR-022, Tesaro / AnaptysBio), WO2011155607, WO2016 / 144803 (e.g., STI-600, Sorrento Therapeutics), WO2016 / 071448, WO17055399; WO17055404, WO17178493, WO18036561, WO18039020 (e.g., Ly-3221367, Eli Lilly), WO2017205721, WO17079112; WO17079115; WO17079116, WO11159877, WO13006490, WO2016068802, WO2016068803, WO2016 / 111947, and WO / 2017 / 031242, each of which is incorporated by reference in its entirety.

[0131] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L). In some embodiments, the anti-OX40 antibody is BMS-986178 (Bristol-Myers Squibb Company), described in International Publication WO20160196228. In some embodiments, the anti-OX40 antibody is selected from the anti-OX40 antibodies described in International Publications WO95012673, WO199942585, WO14148895, WO15153513, WO15153514, WO13038191, WO16057667, WO03106498, WO12027328, WO13028231, WO16200836, WO17063162, WO17134292, WO17096179, WO17096281, and WO17096182, each of which is incorporated by reference in its entirety.

[0132] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to NKG2A. In some embodiments, the anti-NKG2A antibody is BMS-986315. In some embodiments, the anti-NKG2A antibodies are described in, e.g., WO2006 / 070286 (Innate Pharma SA; University of Genova); U.S. Patent No. 8,993,319 (Innate Pharma SA; University of Genova); WO2007 / 042573 (Innate Pharma SA; Novo Nordisk A / S; University of Genova); U.S. Patent No. 9,447,185 (Innate Pharma S / A; Novo Nordisk A / S; University of Genova); WO2008 / 009545 (Novo Nordisk A / S); U.S. Patent Nos. 8,206,709; 8,901,283; 9,683,041 (Novo Nordisk A / S); WO2009 / 092805 (Novo Nordisk A / S); Nos. 8,796,427, and 9,422,368 (Novo Nordisk A / S); WO2016 / 134371 (Ohio State Innovation Foundation); WO2016 / 032334 (Janssen); WO2016 / 041947 (Innate); WO2016 / 041945 (Academisch Ziekenhuis Leiden HODN LUMC); WO2016 / 041947 (Innate Pharma); and WO2016 / 041945 (Innate Pharma), each of which is incorporated herein by reference in its entirety.

[0133] In some embodiments, the antibody or antigen-binding portion thereof specifically binds ICOS. In some embodiments, the anti-ICOS antibody is BMS-986226. In some embodiments, the anti-ICOS antibody is any of the antibodies described in, e.g., WO2016 / 154177 (Jounce Therapeutics, Inc), WO2008 / 137915 (Medimmune), WO2012 / 131004 (INSERM, French National Institute of Health and Medical Research), EP3147297 (INSERM, French National Institute of Health and Medical Research), WO2011 / 041613 (Memorial Sloan Kettering Cancer Center), EP2482849 (Memorial Sloan Kettering Cancer Center), WO1999 / 15553 (Robert Koch Institute), U.S. Pat. Nos. 7,259,247 and 7,722,872 (Robert Koch Institute), U.S. Pat. Nos. 7,045,615; 7,112,655, and 8,389,690 (Japan Tobacco Inc.), U.S. Pat. Nos. 9,738,718 and 9,771,424 (GlaxoSmithKline), and WO2017 / 220988 (Kymab Limited), each of which is incorporated by reference in its entirety.

[0134] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to TIGIT. In some embodiments, the anti-TIGIT antibody is BMS-986207. In some embodiments, the anti-TIGIT antibody is clone 22G2 as described in WO2016 / 106302. In some embodiments, the anti-TIGIT antibody is MTIG7192A / RG6058 / RO7092284, or clone 4.1D3 as described in WO2017 / 053748. In some embodiments, the anti-TIGIT antibody is selected from the anti-TIGIT antibodies described in, for example, WO2016 / 106302 (Bristol-Myers Squibb Company) and WO2017 / 053748 (Genentech).

[0135] In some embodiments, the antibody or antigen-binding portion thereof specifically binds to CSF1R. In some embodiments, the anti-CSF1R antibody is an antibody species disclosed in any of International Publications WO2013 / 132044, WO2009 / 026303, WO2011 / 140249, or WO2009 / 112245, such as cavilizumab, RG7155 (emactuzumab), AMG820, SNDX 6352 (UCB 6352), CXIIG6, IMC-CS4, JNJ-40346527, MCS110, or the anti-CSF1R antibody in the method is replaced with an anti-CSF1R inhibitor or an anti-CSF1 inhibitor, such as BLZ-945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486, ARRY-382, or PLX-73086.

[0136] C. Treatment Method Some embodiments of the present disclosure are directed to a method of treating a subject, comprising administering a protein species isolated and / or purified according to the methods disclosed herein. In some embodiments, the subject has a tumor. In some embodiments, the tumor is selected from tumors derived from hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer (e.g., NSCLC or SCLC), kidney cancer, renal cell carcinoma, head and neck cancer (e.g., small cell carcinoma of the head and neck), colon cancer, prostate cancer, breast cancer, and any combination thereof. In some embodiments, the tumor is recurrent or refractory. In some embodiments, the tumor is locally advanced or metastatic.

[0137] III. Compositions of the Present Disclosure Some embodiments of the present disclosure are directed to a species of a polypeptide, such as a fusion protein and / or an antibody, isolated and / or purified according to any of the methods disclosed herein. In some embodiments, the polypeptide is a protein. In some embodiments, the species is a charge variant of a protein. In some embodiments, the species is an acidic species of a protein. In some embodiments, the species is a basic species of a protein. In some embodiments, the species is a primary species of a protein.

[0138] In some embodiments, the protein comprises a fusion protein. In some embodiments, the protein comprises an immunoglobulin component fused to a biologically active polypeptide. In some embodiments, the immunoglobulin component comprises a fragment of an antibody. In some embodiments, the immunoglobulin component comprises a fragment of a constant region of an antibody. In some embodiments, the immunoglobulin component comprises an Fc.

[0139] In some embodiments, the protein comprises an immunoglobulin fused to a growth factor, a clotting factor, a cytokine, a chemokine, an enzyme, a hormone, or any combination thereof. In some embodiments, the protein comprises an Fc fused to a CTLA-4 polypeptide. In some embodiments, the protein comprises an abatacept. In some embodiments, the protein comprises a belatacept. In some embodiments, the protein comprises an Fc fused to an interleukin.

[0140] In some embodiments, the protein comprises an antibody or antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof binds to a tumor antigen. In some embodiments, the antibody or antigen-binding portion thereof binds to a checkpoint inhibitor. In some embodiments, the antibody or antigen-binding portion thereof binds to an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD1 la, tissue factor (TF), MICA / B, PSCA, IL-8, EGFR, HER3, HER4, and any combination thereof.

[0141] Various aspects of the disclosure are described in further detail in the following subsections. The disclosure is further illustrated by the following examples, which should not be construed as further limiting. EXAMPLES

[0142] Example 1: Materials and Methods In the examples described below, one or more of the following materials and methods are used.

[0143] Device For chromatographic separation, we used AKTA (Cytiva, formerly GE Healthcare) (商標)The following chromatography systems were used: Avant 25 (FPLC), ALLIANCE E2695 (HPLC) from Waters Corporation coupled with Fraction Collector III from Waters Corporation, and CONTICHROM® CUBE 30 (MCC) from ChromaCon. HPLC systems are designed for analytical separations and when coupled with a fraction collector provide high resolution separations but with very low throughput. FPLC systems are designed for preparative chromatography and provide low resolution separations but with much higher throughput than HPLC systems. Finally, the MCC process provides separations comparable to the FPLC system but can utilize multiple (2) columns and has a built-in method (N-Rich) that allows for sample enrichment.

[0144] Where appropriate, the following analyzers were also used: iCE3 from Protein Simple (商標) System and Alcott720Autosampler, DropSense96 from Unchained Labs (formerly Trinean), Maxis II from Bruker Daltonics (商標) ACQUITY UPLC Coupled with a Quadrupole Time-of-Flight Mass Spectrometer (商標) System (Waters).

[0145] Chemicals and Materials Four recombinant human monoclonal IgG antibodies (mAb1-3) were expressed in Chinese Hamster Ovary (CHO) cells and purified by affinity chromatography. Trypsin was purchased from Promega (Madison, WI, USA). Peptide N-glycosidase F enzyme (PNGaseF) was obtained from New England Biolabs (Ipswich, MA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

[0146] Charge variant fractionation and enrichment using liquid chromatography Charge variant fractionation using HPLC and FPLC:

[0147] Monoclonal antibody samples were injected onto MabPac SCX-10 (4 mm × 250 mm) and MabPac SCX-10 (9 mm × 250 mm) columns (Thermo Fisher Scientific, Wilmington, DE, USA) on a Waters Alliance e2695 HPLC system and a GE Avant 25 FPLC system, respectively. To maximize the productivity of the separation, the maximum injection volume by mass based on the vendor's recommendations was used. The salt gradient mobile phases were 20 mM MES with and without 250 mM sodium chloride at pH 6.0, and 20 mM MES with and without 400 mM sodium chloride at pH 5.8. The flow rate and elution gradient time were optimized to achieve the desired separation. Fractions from the HPLC system were collected using a Waters Fraction Collector III. Fractions from the FPLC were collected on an Avant built-in fraction collector. The fractions were further pooled and characterized.

[0148] Charge variant enrichment using sequential chromatography:

[0149] Two Mono S CEX columns (10 mm × 100 mm) and two Mono Q AEX columns (10 mm × 100 mm) were purchased from Cytiva (Chicago, IL, USA). The column length of 100 mm was chosen to fit the pressure limit of the continuous chromatography system (<50 bar) when two columns were connected in series in continuous operation mode. Salt gradient elution was performed using 20 mM MES with and without 250 mM sodium chloride as described above for the CEX columns and 50 mM Tris with and without 250 mM sodium chloride at pH 9.0 for the AEX columns. The AEX pH gradient mobile phases were 5 mM 1,3 diaminopropane, 5 mM diethanolamine, 5 mM Tris, 5 mM imidazole, 5 mM bis-tris, 5 mM piperazine, pH 11.1, and 5 mM 1,3 diaminopropane, 5 mM diethanolamine, 5 mM Tris, 5 mM imidazole, 5 mM bis-Tris, 5 mM piperazine, 5 mM acetic acid, pH 3.5. The CEX pH gradient mobile phases were 5 mM malonic acid, 5 mM formic acid, 5 mM acetic acid, 5 mM MES, 5 mM MOPSO, 5 mM HEPES, 5 mM BICINE, 5 mM CHES, 5 mM CAPS, pH 4, and 5 mM malonic acid, 5 mM formic acid, 5 mM acetic acid, 5 mM MES, 5 mM MOPSO, 5 mM HEPES, 5 mM BICINE, 5 mM CHES, 5 mM CAPS, pH 11.0. Charge variant separation conditions for these columns were optimized using an FPLC system before transfer to a Contichrom CUBE (ChromaCon) continuous chromatography system. Single runs using the transfer method were performed using ChromIQ software. Regions containing the charge variants of interest were defined using the chromatographic profiles of the single runs. After 10 cycles of enrichment and 2 cycles of depletion in the CUBE system, fractions from the CUBE system were collected using an external fraction collector for further pooling and analytical characterization.

[0150] Characterization of charge variant samples Imaged Capillary Isoelectric Focusing (iCIEF):

[0151] To confirm the separation, iCIEF was performed using an iCE31M system and an Alcott 720 Autosampler (Protein Simple) to quantify the relative amounts of charge variants (acidic, main, and basic). Separation cartridges and capillaries were purchased from Convergent Bioscience. The capillaries were immobilized on a glass substrate and separated from catholytes and anolytes by two hollow fiber membranes. Samples were prepared by mixing 2 g / L of protein with a stock master mix solution containing relevant pI markers (Protein Simple), 1% methylcellulose solution (Protein Simple), PHARMAL YTE® 3-10 (Cytiva), and urea, and diluting to 0.25 g / L with deionized water. After injection of the prepared samples into the cartridge, a prefocus period of 1–1.5 min at 1500 V and a focus period of 8–12 min at 3000 V were applied to achieve optimal resolution. Final images of the IEF traces were captured with a deuterium lamp detector at 280 nm. Results were processed by pI calibration using Protein Simple iCE software, and integration and quantification were calculated using Waters Empower3 software.

[0152] Mass spectrometric characterization of mAB fractions: To identify the root cause of the charge variants, samples were analyzed intact after deglycosylation and proteolytic cleavage with trypsin. The molecular weights of the intact and deglycosylated mAbs were analyzed by liquid chromatography-mass spectrometry (LC-MS) analysis:

[0153] Deglycosylated samples were prepared by mixing the samples with PNGaseF (New England Biolabs, Ipswich, Mass.) at 12.5 Units / μg of protein at 37° C. for 1 hour.

[0154] The molecular weight of mAb samples was measured using Maxis II (商標) Quadrupole time-of-flight mass spectrometer (Bruker, Daltonics Inc., Billerica, MA) and POROS (商標) R2 / 10 PERFUSION CHROMATOGRAPHY (商標) ACQUITY UPLC coupled with a column (2.1 mm × 100 mm, Thermo Scientific, Waltham, MA) (商標) A HPLC-MS system (Waters Corporation, Milford, MA) was used. The flow rate was 0.2 mL / min and the column temperature was set at 65 °C during the analysis. Samples were injected onto the column in 90% mobile phase A (0.1% formic acid in LC-MS grade water) and 10% mobile phase B (0.1% formic acid in LC-MS grade acetonitrile). A linear gradient from 10% to 90% mobile phase B was used to elute the mAbs in 10 min.

[0155] Maxis II mass spectrometer COMPASS HYSTAR (商標) The software was controlled and run in positive mode with the following settings: scan range m / z 500-4000, gas temperature 220°C, drying gas 6 L / min, nebulizer 2.5 Bar, and capillary voltage 4500 V. COMPASS DATAANALYSIS was used for deconvolution of mass spectra. (商標) , version 4.4 was used.

[0156] Peptide Mapping using LC / MS / MS:

[0157] One hundred microliters of concentrated sample was denatured with 8 M guanidine hydrochloride (pH 8), reduced with 10 mM DTT for 20 min at 37°C, and alkylated with 15 mM IAA for 20 min at 37°C in the dark. The alkylated sample was buffer exchanged into digestion buffer (2 M urea, 50 mM TRIS, 10 mM CaCl2, pH 7.6) by passing through Micro Bio-Spin 6 columns (Bio-Rad, Hercules, California). The eluate was enzymatically digested with trypsin at a ratio of 1:25 (w / w, enzyme / protein) for 3 h at 37°C. After digestion, the digested sample was acidified by adding 1 N hydrochloric acid.

[0158] Tryptic digests were analyzed using a Waters ACQUITY UPLC (商標) After chromatographic separation using a Thermo Scientific ORBITRAP Q EXACTIVE system (Milford, MA, USA), (商標) The peptides were analyzed on a Q EXACTIVE PLUS mass spectrometer (Thermo Scientific, Bremen, Germany). Separation was performed on a Waters Acquity BEH C18 column (1.7 μm, 2.1 × 150 mm) with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B at 45 °C. Peptides were eluted with a linear gradient of mobile phase B from 1% to 80% over 105 min at a flow rate of 0.2 mL / min. (商標) The mass spectrometer was operated in data-dependent mode to switch between MS and MS / MS acquisition. Ions were generated using a sheath gas flow rate of 40 units, auxiliary gas flow rate of 10 units, spray voltage of 3 kV, capillary temperature of 275 °C, and S-lens RF level of 60 units. Resolution was set to 70,000 (AGC target 3e6) for survey scans and 17,500 (AGC target 1e5) for MS / MS events. A dynamic exclusion time of 10 seconds was used with one repeat count. Mass spectrometry data analysis was performed using the THERMO PROTEOME DISCOVER (商標)The software package, version 1.4 (Thermo Scientific, Bremen, Germany) was used.

[0159] Example 2: Concentration using a multi-column sequential chromatography method As described herein, the enrichment methods provided herein generally involve the use of two identical (twin) columns and MCSGP techniques. A schematic of the overall process is shown in Figures 1A-1B. As shown, enrichment is achieved through a three-step operation including enrichment (green - i.e., of species of interest), removal (red and blue - i.e., of species not of interest), and elution (green). First, a switch refers to the run of a single column, whereas a cycle refers to the run of each of the two columns, corresponding to two switches. The feed material is represented by a mixture of three components (species) shown in red, green, and blue, with the green species representing the species of interest for enrichment.

[0160] In the enrichment step (see Figure 1A), the feed material is loaded into the first column. After discarding the species that first elute from the column (blue), the species to be enriched (green) are recycled to the second column by connecting the inlet of column 2 to the outlet of column 1. After all species for enrichment (green) are recycled from the first column to the second column, the species that elute later (red) are discarded from the first column through a strip step, after which column 1 is re-equilibrated. During the re-equilibration of column 1, an additional load (mixture) is injected into column 2 in addition to the green species recycled from column 1. The same separation operation as column 1 (discard blue, recycle green, discard red) is performed on column 2. As the number of cycles increases, the desired species (green) is enriched. In the removal step (see Figure 1B), the separation cycling pattern performed in the enrichment step is performed again, but no additional load material is added to the column after the recycle is completed. This stage consists of one cycle, meaning one separation on each of the two columns. The desired species (green) remains in the column, while the undesired species (blue and red) are removed from the system as pruning occurs. Pruning is followed by a third stage called elution, to recover the green species from the system. The elution stage is an elongated version of the separation gradient used to concentrate the green species, where fractionation of the remaining product can be performed.

[0161] FIG. 1C provides simulated UV trace results using the concentration method described above. The left panel shows the presence of three species (red, blue, and green) in the initial feed material. The center panel shows the product resulting from 10 cycles of concentration of the green species. The right panel shows that after one cycle of removal, virtually no non-target species (i.e., blue and red species) remain in the system, while the target species (i.e., green) remains unchanged. As demonstrated herein, the target species was eluted at the final stage of processing and collected for subsequent analysis.

[0162] Example 3: Comparison of batch and multicolumn continuous chromatography methods for charge variant enrichment To evaluate the capabilities of the enrichment methods provided herein (e.g., continuous chromatography MCSGP), HPLC, FPLC, and continuous chromatography (CUBE) were used to separate acidic variants of a recombinant human IgG antibody (mAb1). The specific methods used are provided in Example 1.

[0163] As shown in Figures 2A and 2B, the separation patterns using HPLC (Figure 2A) and FPLC (Figure 2B) were largely similar with the acidic variants present in the early elution fractions collected (before the arrow). For HPLC and FPLC, improved resolution was observed with HPLC. However, the advantage of FPLC over HPLC was that a larger amount of initial material could be processed (injection volume was more than 10 times larger). Figure 2C provides the separation profile with the CUBE system. As shown, the chromatograms only show the separation of the acidic species, since the enrichment method discards any material that is not the target of enrichment. Similar to FPLC, the CUBE system allowed the processing of a larger amount of material (i.e., more material could be loaded onto the CUBE system in one injection). In terms of resolution, it appears to be intermediate between the values ​​achieved with the HPLC and FPLC systems. As shown in Figure 2C, the main advantage of the enrichment method using the CUBE system is that the acidic peak after 10 enrichment cycles (red) was significantly larger than the peak observed after 2 cycles (black), demonstrating the ability of the CUBE system to highly enrich the species of interest (acidic variants). The implementation of fractionation after depletion was an option that allowed flexible pooling of the product at the elution stage.

[0164] From the data generated by these three different methodologies, a prediction of the time required to produce 10 mg of acidic variants from a load sample containing 17% acidic variants was extrapolated. As shown in Figure 2D, the estimated times were 300 h, 48 h, and 10 h, and the resulting sample purity (or acidity rate) measured using the iCIEF method was 87%, 78%, and 95% for the HPLC, FPLC, and CUBE systems, respectively. These results confirmed that the continuous chromatographic concentration method provided herein can achieve the highest purity in the shortest time.

[0165] Another important property of the enrichment method provided herein is the ability to separate subspecies variants in the enriched acidic or basic peak regions using elution fractions. To further evaluate this aspect, two sequential chromatographic enrichment runs, one focused on the acidic peak region and the other on the basic peak region, were performed to separate different variants of the mAb1 antibody.

[0166] As shown in Figure 3 (panel 1), two peaks were fractionated in each run, region 1 and region 2. As shown in panels 2 and 3, acidic variants were enriched in both acidic regions 1 and 2, with acidic region 1 containing more acidic variants. In addition, the distribution of acidic variants differed between acidic regions 1 and 2. Acidic region 1 also contained more acidic variants containing two extra negative charges (resulting in pIs of -7.2 and -7.4, respectively) that were barely detected in the load material (see panel 1 in Figure 3). Similar separation efficiency and patterns were observed for enrichment in the basic region, as shown in panels 4 and 5. These results indicate that the enrichment method provided herein, by combining enrichment of specific peak regions with further fractionation of the elution peak, can identify specific charge variants present in a given sample.

[0167] Example 4: Comparison of separation conditions for charge variant enrichment To further evaluate different separation / enrichment conditions, both CEX and AEX columns were used with salt and pH gradients to separate acidic and basic charge variants of the mAb3 antibody.

[0168] As shown in Figure 6, the best separation results were observed when CEX (Mono S) was used with a salt gradient. For AEX (Mono Q), the best separation results were observed with a pH gradient. Using these optimized separation conditions, samples from the FPLC fractionation runs and concentrated samples from the continuous chromatography runs were collected and further analyzed using iCIEF and MS. As shown in the iCIEF data provided in Figure 6, much improved enrichment was observed with the continuous chromatography method (panels 3 and 5) compared to the FPLC fractionation method with batch mode separation (panels 2 and 4).

[0169] Comparing only the iCIEF profiles using CEX with a salt gradient (panel 3) and AEX with a pH gradient (panel 5), the differences in separation efficiency of these methods could not be visually distinguished. However, several acidic variants including sialylated, deamidated, and glycosylated glycans were observed in the LC / MS peptide mapping (see Figures 7A, 7B, and 7C). Interestingly, the two different methods mentioned above resulted in enrichment of different acidic variants to different degrees based on MS analysis. Without being bound by theory, this may be due to the fact that the separation during ion exchange chromatography did not rely solely on charge interactions.

[0170] As shown in Figures 7B and 7C, the use of AEX with a pH gradient resulted in more effective separation of deamidated and glycosylated (acidic) species compared to the use of CEX with a salt gradient. The deamidation levels at different Asn sites (including N84, N325, N384, and N389) showed very little difference in the acidic, main, and basic fractions from the CEX column with a salt gradient (Figure 7B, upper panel). The deamidation levels observed at N384 and N389 in the PENNY loop were less than 7% even in the acidic fractions. However, the deamidated species were significantly enriched in the acidic fractions of the AEX column with a pH gradient. The deamidation levels of N384 and N389 were approximately 20% in the acidic samples recovered from the FPLC and approximately 9% in the acidic samples recovered from the CUBE (Figure 7B, lower panel).

[0171] As shown in Figure 7C, a similar trend was observed in the glycation levels of the two identified peptides, suggesting that the AEX column (Mono Q) with a pH gradient was more effective in enriching glycated species. In contrast, CEX with a salt gradient (Mono S) was more effective in enriching basic species, including noncyclized N-terminal glutamine and C-terminal proline amidation, as shown in Figures 7D and 7E. N-terminal glutamine and C-terminal lysine in all fractions of AEX were present at similar levels. However, N-terminal glutamine and C-terminal lysine were detected at much higher levels in the basic fractions, as shown in the upper panels of Figures 7D and 7E. In general, compared with samples fractionated from the FPLC system, enriched samples recovered from the CUBE system contained the same or higher levels of target species, except for deamidated species.

[0172] Taken together, these results demonstrate that sample generation using the enrichment method provided herein (MCSGP method) is highly effective and efficient, facilitating studies to establish structure-function relationships.

[0173] Example 5: Impact of increasing sample purity on analytical characterization As demonstrated in the above examples, the enrichment methods provided herein allowed for the identification of species that were not clearly detected in either the initial load sample or the samples generated using FPLC. Further advantages of the enrichment methods provided herein are further highlighted below.

[0174] iCIEF An image comparison of the capillary electrophoresis profile of the mAb2 antibody sample before and after the acidic region enrichment is provided in Figure 4. After enrichment, the two shoulder signals that existed before enrichment became clearer peak signals after enrichment (see the arrows in the upper and lower panels of Figure 4). In addition, two acidic peaks that were not detected in the load sample before enrichment were observed (see * in the lower panel of Figure 4).

[0175] mass spectrometry As described herein, the enrichment method provided herein was able to enrich not only the charge variants observed in the initial sample, but also to identify additional variants that were previously undetectable. As shown in Figure 5A, the glycation signal from the deglycosylation LC-MS assay was stronger in the enriched acidic sample (third panel from the top). Furthermore, in the acidic variant separation of mAb1, a new peak was detected in the enriched acidic region 1 sample (leftmost peak in panel 2 of Figure 3). A similar new peak was also observed in the acidic variant separation of mAb2 (see * in the bottom panel of Figure 4).

[0176] Based on MS analysis, a new peak (around 144557.8 m / z) was also observed in the enriched basic variant sample (left peak in the lower panel of Figure 5A). This peak is attributed to a proline amidated species after removal of the C-terminal glycine. As shown in Figure 5B (lower panel), a peak around 131538 m / z was clearly observed only in the enriched acidic variant sample. This peak was assigned to a heavy chain truncation form with deletion of residues 330 and beyond. Without the use of the continuous chromatographic enrichment method provided herein, detection of such a peak would not have been possible, especially in such a short period of time.

[0177] In addition to the above, as described below, the enrichment methods provided herein also resulted in improved detection of specific post-translational modifications (PTMs). Results of all PTMs identified in fractionated and enriched samples of different recombinant human monoclonal IgG antibodies are provided, for example, in Figure 3 (mAb1), Figure 4 (mAb2), and Figures 5A-5B and 6 (mAb3).

[0178] Sialic acid As shown in Figure 5A, MS analysis demonstrated that the Fc-sialylation level was increased in the acidic fractions, especially in the enriched acidic fraction of mAb3, using the enrichment method provided herein. Furthermore, MS analysis suggested that the detected sialylation occurred at the termini of N-glycans (G1F and G2F) (Figure 7A).

[0179] Deamidation and Aspartic Acid Isomerization Asn deamidation was observed at residues in both the CDRs and constant regions of the antibody. The two most frequently deamidated residues were N384 and N389. N384 was followed by a Gly residue, and N389 was in the PENNY motif NG, which was previously reported as a deamidation hotspot. The other two deamidation sites detected were N84 and N325, followed by Ser and Lys, respectively (Figure 7B).

[0180] Saccharification Using the enrichment method provided herein, glycation of peptides located in the variable region near the N-terminus of the mAb was observed. As shown in Figure 7C, the identification of glycation in fractions generated from the CEX column with salt gradient was different from samples generated using the AEX column with pH gradient. There was more variability in samples using the AEX column with pH gradient, indicating that this separation method is superior in resolving this charge variant species.

[0181] N-terminal glutamine modification N-glutamine was also detected in high proportions in the basic fractions (Figure 7D). Continuous chromatographic enrichment showed better separation compared to batch mode fractionation. The use of Mono S (CEX) with a salt gradient improved the detection of pyroglutamic acid. This PTM was not expected to affect the activity of the product for two reasons: (1) the first residue is not present in the antigen-binding interface, and (2) Due to the presence of glutaminyl cyclase, the non-pyrolyzed N-terminus is converted to a pyrolyzed N-terminus when injected into the human body.

[0182] C-Terminal Lysine Basic variants of the mAb1 antibody were enriched using a Mono Q (AEX) column and a pH gradient (Figure 3, panels 4 and 5). Analysis of these enriched basic species after treatment with carboxypeptidase B (CpB) confirmed the presence of a C-terminal Lys. Differences in antigen affinity for these variants were minimal because the complementarity determining region (CDR) was on the opposite end of the mAb from where the Lys variants were found.

[0183] Amidation of C-terminal proline From the LC-MS data, a C-terminal amidation modification in the basic species of mAb3 was observed using a Mono S (CEX) column with a salt gradient. When this sample was enriched using the method provided herein (MCSGP), the relative content of this modification in the basic species sample increased 6-fold over the load material and 20% over the FPLC fractionated sample (Figure 7E).

[0184] Truncation species As shown in FIG. 5B, using the enrichment method provided herein, a deglycosylated truncation species of 131,538 Da was observed in the MS analysis. This species was due to a truncation of one of the heavy chains with the loss of residues 330 to 446. The cleavage site (P329) was in the loop region before the last β-strand of the CH2 domain. In addition, a truncation species of 97.5 kDa was also observed in the acidic fraction. This truncation species was due to a heavy chain fragment from the hinge region to the end.

[0185] Taken together, the results provided herein demonstrate that the disclosed continuous chromatographic concentration method (MCSGP) allows for the concentration of target charge species with both high productivity and high purity compared to traditional batch mode methods. The use of continuous chromatographically concentrated samples offers significant advantages for analytical characterization.

[0186] It is understood that it is intended that the Detailed Description section be used to interpret the claims, and not the Summary or Abstract sections. The Summary and Abstract sections set forth one or more exemplary embodiments of the invention contemplated by the inventor, but are not exhaustive, and therefore are not intended to limit the scope of the invention and the appended claims.

[0187] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specific functions and their relationships. The boundaries of these functional building blocks are arbitrarily defined herein for the convenience of description. Alternative boundaries can be defined as long as the specific functions and their relationships are appropriately performed.

[0188] The above description of specific embodiments fully reveals the general nature of the present invention, so that those skilled in the art can easily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept of the present invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the terms or phrases used herein are for the purpose of description and not for the purpose of limitation, as they should be interpreted by those skilled in the art in light of the teachings and guidance.

[0189] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0190] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0191] All publications, patent applications, patents and other literature mentioned herein are incorporated by reference in their entirety. Database entries and electronic publications disclosed in this disclosure are incorporated by reference in their entirety. The version of a database entry or electronic publication incorporated by reference in this application is the latest version of the database entry or electronic publication that was publicly available at the time this application was filed. Database entries corresponding to gene or protein identifiers disclosed in this application (e.g., genes or proteins identified by accession numbers or database identifiers in public databases such as Genbank, Refseq, or Uniprot) are incorporated by reference in their entirety. The incorporation information associated with a gene or protein is not limited to the sequence data contained in the database entry. The information incorporated by reference includes the entire contents of the database entry in the latest version of the database that was publicly available at the time this application was filed. In the event of any conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. A method for isolating a protein species from a mixture containing a protein species and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode.

2. A method for increasing the purity and / or yield of a protein species from a mixture containing a protein species and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode.

3. A method for enriching protein chemical species for analytical characterization, (a) Separating a protein species from a mixture containing a protein species and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode of a chromatography separation system; and (b) To subject the chemical species from (a) to analytical characterization. Methods that include...

4. A method for performing analytical characterization of protein chemical species, (a) Separating a protein species from a mixture containing a protein species and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode of a chromatography separation system; and (b) Analyze the chemical species from (a) Methods that include...

5. The method according to claim 3 or 4, wherein analytical characterization is performed by an HPLC system, capillary isoelectric focusing (cIEF) gel electrophoresis, imaging capillary isoelectric focusing (iCIEF), cation exchange chromatography (CEX), anion exchange chromatography (AEX), MFI, SEC-MALS, SEC, or mass spectrometry.

6. The method according to any one of claims 1 to 4, which results in increased purity and / or increased yield of protein chemical species compared to HPLC or FPLC.

7. The method according to any one of claims 1 to 4, wherein two or more chromatography columns concentrate chemical species.

8. The method according to claim 7, further comprising loading a mixture onto a first chromatography column.

9. The method according to claim 8, wherein the loaded mixture passes through a first chromatography column to separate the concentrated species containing the chemical species from the waste chemical species ("concentration step I").

10. The method according to claim 9, wherein the concentrated species passes through a second column and the waste species are discarded after the first chromatography column ("concentration step II").

11. The method according to claim 10, further comprising re-equilibriumating the first chromatography column.

12. The method according to claim 10, further comprising contacting a concentrated chemical species with a first chromatography column.

13. The method according to claim 8, further comprising loading an additional mixture into a first column, wherein the additional mixture comprises the chemical species and one or more impurities.

14. The method according to claim 13, wherein the additional mixture is added at the same time that the concentrated chemical species is added to the first chromatography column.

15. The method according to claim 13, wherein the additional mixture is added after the concentrated species has been added to the first chromatography column, but before the concentrated species has passed through the first chromatography column.

16. The method according to claim 13, wherein the concentration steps I and II are repeated at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times.

17. The method according to claim 16, further comprising a removal step.

18. The method according to claim 17, wherein the removal step includes contacting the concentrated chemical species with a first chromatography column in the absence of additional mixtures.

19. The method according to claim 18, wherein the removal step further comprises passing the concentrated chemical species through a first chromatographic column and separating the chemical species from one or more impurities.

20. The method according to claim 19, wherein the removal step further comprises passing the concentrated species through a second chromatographic column and separating the species from one or more impurities.

21. The method according to any one of claims 1 to 4, further comprising eluting a chemical species.

22. The method according to any one of claims 1 to 4, which produces a protein chemical species with a purity of at least 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%.

23. The method according to claim 21, wherein the concentration of the eluted chemical species is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.5, at least about 3.0, at least about 3.5, at least about 4.0, at least about 4.5, at least about 5.0, at least about 5.5, at least about 6.0, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, or at least about 10.0 times higher than the concentration of the chemical species in the mixture.

24. The method according to any one of claims 1 to 4, wherein one or more chromatography columns include a salt gradient, a pH gradient, or both.

25. The method according to claim 24, wherein the salt gradient includes a sodium chloride gradient.

26. The method according to claim 24, wherein the salt gradient includes the presence or absence of salt.

27. The method according to claim 24, wherein the salt concentration is between approximately 50 mM and approximately 600 mM, between approximately 100 mM and approximately 550 mM, between approximately 150 mM and approximately 500 mM, between approximately 200 mM and approximately 450 mM, between approximately 250 mM and approximately 400 mM, between approximately 100 mM and approximately 400 mM, between approximately 100 mM and approximately 350 mM, between approximately 100 mM and approximately 300 mM, between approximately 100 mM and approximately 250 mM, between approximately 300 mM and approximately 600 mM, between approximately 350 mM and approximately 550 mM, between approximately 400 mM and approximately 500 mM, or between approximately 350 mM and approximately 450 mM.

28. The method according to claim 24, wherein the concentration of the salt is at least 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 260 mM, at least about 270 mM, at least about 280 mM, at least about 290 mM, at least about 300 mM, at least about 310 mM, at least about 320 mM, at least about 330 mM, at least about 340 mM, at least about 350 mM, at least about 360 mM, at least about 370 mM, at least about 380 mM, at least about 390 mM, at least about 400 mM, at least about 450 mM, at least about 500 mM, at least about 550 mM, or at least about 600 mM.

29. The method according to claim 24, wherein the pH of the pH gradient is between approximately pH 3 and approximately pH 11.

30. The method according to any one of claims 1 to 4, wherein the mixture is present in the buffer solution.

31. The method according to claim 30, wherein the buffer is MES, phosphate buffer, Tris, or a combination thereof.

32. The method according to any one of claims 1 to 4, further comprising measuring post-translational modifications.

33. The method according to claim 32, wherein the post-translational modification is N-glutamine pyroglutamylation, C-terminal lysine truncation, C-terminal proline amidation, glycation, sialylation, deamidation, aspartate isomerization, general truncation, or any combination thereof.

34. The method according to any one of claims 1 to 4, wherein the protein comprises a fusion protein or an antibody or its antigen-binding portion.

35. The method according to claim 34, wherein the antibody or its antigen-binding portion binds to an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), MICA / B PSCA, IL-8, EGFR, HER3, HER4, and any combination thereof.

36. The method according to claim 34, wherein the fusion protein comprises an immunoglobulin component and a growth factor.

37. The method according to claim 36, wherein the fusion protein comprises an Fc fusion protein.

38. The method according to claim 36, wherein the fusion protein comprises Fc fused to CTLA-4.

39. The method according to claim 36, wherein the fusion protein comprises abatacept or beratacept.

40. The method according to claim 36, wherein the fusion protein comprises Fc fused to an interleukin.

41. The method according to claim 34, wherein the chemical species of the fusion protein or antibody is an acidic chemical species, a basic chemical species, or a dominant chemical species.

42. The method according to claim 34, wherein the fusion protein or antibody is partially purified by protein A affinity chromatography.

43. The method according to any one of claims 1 to 4, wherein the chemical species are concentrated by a countercurrent purification system.

44. The method according to claim 43, wherein the countercurrent purification system is a multi-column countercurrent solvent gradient purification (MCSGP) system.

45. A chemical species of protein prepared by the method described in any one of claims 1 to 4.

46. A chemical species of the protein according to claim 45, which is a charge variant.

47. A pharmaceutical composition for treating a target disease or condition, comprising the chemical species of protein described in claim 45.

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