Ceramic hydroxyapatite chromatography flow-through method

Abstract

This disclosure relates to a method for isolating and / or purifying a protein in flow-through mode ceramic hydroxyapatite chromatography, comprising loading a sample containing a protein and one or more impurities onto a CHT column in flow-through mode.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 519,751, filed on 15 August 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listings The electronically submitted sequence listing contents of the ASCII text file (filename: 3338_331PC01_SequenceListing_ST26, size: 50,639 bytes, creation date: July 30, 2024) submitted with this application are incorporated herein by reference in their entirety.

[0003] This application relates to the field of protein isolation and purification using flow-through mode ceramic hydroxyapatite chromatography. [Background technology]

[0004] The increasing popularity of high-potency processes in biopharmaceutical development can amplify certain challenges associated with the design and optimization of purification processes. Such challenges and obstacles include, but are not limited to, increased consumption of raw materials such as buffers or chromatography resins, longer operating times, a greater number of purification cycles, increased costs, and concerns about product stability. Solutions to these challenges must also consider maintaining the productivity of biopharmaceuticals. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, in the field of protein purification, there is still a need for methods that maximize yield and purity while reducing the cost and time required to produce purified samples. [Means for solving the problem]

[0006] Some aspects of the present disclosure relate to a method for isolating molecules in a sample containing a target molecule and impurities in a flow-through (F / T) mode of ceramic hydroxyapatite chromatography (CHT), the method comprising: a) loading the sample onto a CHT column; and b) obtaining an F / T composition containing the molecule and a smaller amount of impurities compared to the sample before loading.

[0007] A particular aspect of the present disclosure relates to a method for isolating molecules in a sample containing a target molecule and impurities using ceramic hydroxyapatite chromatography (CHT), comprising: a) loading the sample onto a CHT column in flow-through (F / T) mode; and b) obtaining an F / T composition containing the molecule and a smaller amount of impurities compared to the sample before loading.

[0008] In some embodiments, CHT is a polishing step. In some embodiments, the method further includes an additional polishing step.

[0009] In some embodiments, the amount of the target molecule in the sample is such that the target molecule does not bind to the column during and / or after loading. In some embodiments, the amount of the target molecule in the sample is such that some molecules bind to the column during and / or after loading.

[0010] In some embodiments, the method further includes affinity chromatography, ion exchange chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, filtration, or any combination thereof before and after CHT.

[0011] In some embodiments, the target molecule includes a protein. In some embodiments, the target molecule includes a nucleic acid. In some embodiments, the target molecule includes a virus.

[0012] In some embodiments, the loaded sample contains at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, or at least about 500 g / L 樹脂 This includes the loading amount. In some embodiments, the loaded samples are approximately 50-500, approximately 50-450, approximately 50-400, approximately 50-350, approximately 50-300, approximately 70-300, approximately 90-300, approximately 100-300, approximately 120-300, approximately 140-300, approximately 150-300, approximately 50- Approximately 250, approximately 70-250, approximately 90-250, approximately 100-250, approximately 120-250, approximately 140-250, approximately 150-250, approximately 50-200, approximately 70-200, approximately 90-200, approximately 100-200, approximately 120-200, approximately 140-200, or approximately 150-200 g / L 樹脂 Includes the loading amount.

[0013] In some embodiments, loading a sample into a CHT column involves adding the sample to a loading buffer containing a phosphate. In some embodiments, the phosphate in the loading buffer includes sodium phosphate, potassium phosphate, or any combination thereof.

[0014] In some embodiments, the amount of phosphate in the loading buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM. In some embodiments, the amount of phosphate in the loading buffer is about 5–50, about 5–45, about 5–40, about 5–35, about 5–30, about 10–30, about 15–30, about 20–30, about 25–30, about 5–20, about 10–20, about 15–20, about 5–15, or about 10–15 mM. In some embodiments, the F / T composition is pooled as the final product. In some embodiments, the final product is formulated.

[0015] In some embodiments, the method further comprises (c) adding Chase buffer to the CHT column. In some embodiments, the Chase buffer comprises a phosphate. In some embodiments, the phosphate in the Chase buffer comprises sodium phosphate, potassium phosphate, or any combination thereof. In some embodiments, the amount of phosphate in the Chase buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM. In some embodiments, the amount of phosphate in the chase buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM.

[0016] In some embodiments, the method further includes recovering the chase composition containing the molecule after (c). In some embodiments, the chase composition and the F / T composition are pooled as the final product. In some embodiments, the final product is formulated.

[0017] In some embodiments, the method further includes (d) adding an elution buffer when a specific amount of the molecule of interest is bound to the column. In some embodiments, the elution buffer contains a phosphate. In some embodiments, the phosphate in the elution buffer contains sodium phosphate, potassium phosphate, or any combination thereof. In some embodiments, the amount of phosphate in the elution buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM. In some embodiments, the amount of phosphate in the elution buffer is approximately 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 20, 10 to 20, 15 to 20, 5 to 15, or 10 to 15 mM.

[0018] In some embodiments, the loading buffer has a pH of 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, or at least about 8.5. In some embodiments, the pH in the loading buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pH of the loading buffer is about pH 6.3 to about pH 7.7. In some embodiments, the pH of the loading buffer is about 7.5. In some embodiments, the pH of the chase buffer has a pH of 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, or at least about 8.5. In some embodiments, the pH of the chase buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pH of the chase buffer is approximately pH 6.3 to approximately pH 7.7. In some embodiments, the pH of the chase buffer is approximately 7.5. In some embodiments, the pH of the elution buffer is at least approximately 6.0, at least approximately 6.5, at least approximately 7.0, at least approximately 7.5, at least approximately 8.0, or at least approximately 8.5. In some embodiments, the pH in the elution buffer is approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, or approximately 8.5. In some embodiments, the pH of the elution buffer is approximately pH 6.3 to approximately pH 7.7. In some embodiments, the pH of the elution buffer is approximately 7.5.

[0019] In some embodiments, the loading buffer comprises chloride at a concentration of less than about 500 mM, less than about 450 mM, less than about 400 mM, less than about 350 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 190 mM, less than about 180 mM, less than about 170 mM, less than about 160 mM, less than about 150 mM, less than about 140 mM, less than about 130 mM, less than about 120 mM, less than about 110 mM, or less than about 100 mM. In some embodiments, the chase buffer comprises chloride at a concentration of less than about 500 mM, less than about 450 mM, less than about 400 mM, less than about 350 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 190 mM, less than about 180 mM, less than about 170 mM, less than about 160 mM, less than about 150 mM, less than about 140 mM, less than about 130 mM, less than about 120 mM, less than about 110 mM, or less than about 100 mM. In some embodiments, the elution buffer comprises chloride at a concentration of less than about 500 mM, less than about 450 mM, less than about 400 mM, less than about 350 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 190 mM, less than about 180 mM, less than about 170 mM, less than about 160 mM, less than about 150 mM, less than about 140 mM, less than about 130 mM, less than about 120 mM, less than about 110 mM, or less than about 100 mM.

[0020] In some embodiments, the conductivity of the chase buffer is equal to or higher than the conductivity of the loading buffer. In some embodiments, the conductivity of the chase buffer is equal to or lower than the conductivity of the loading buffer. In some embodiments, the conductivity of the elution buffer is equal to or higher than that of the loading buffer. In some embodiments, the conductivity of the elution buffer is equal to or lower than that of the loading buffer.

[0021] In some embodiments, the molecule of interest comprises a protein including an antibody or an antigen-binding portion thereof, an antibody-drug conjugate (ADC), a bispecific molecule, a multispecific molecule, a fusion protein, a cytokine, an immunomodulator, a growth factor, a coagulation factor, a chemokine, an enzyme, a hormone, or any combination thereof.

[0022] In some aspects, the fusion protein comprises an Fc fusion protein, an albumin fusion protein, or any combination thereof. In some aspects, the fusion protein comprises a cytokine. In some aspects, the cytokine comprises IL10, IL6, IL18, IL12, IL4, TGF-beta, IL17, IL8, IL1B, IL13, IL15, IL2, IL7, IL11, IL22, IL21, IL9, IL-1 receptor, IL3, TNF, IFN-gamma, granulocyte-macrophage colony-stimulating factor, IL5, or any combination thereof.

[0023] In some aspects, 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.

[0024] In some aspects, the protein is an immune checkpoint inhibitor.

[0025] In some aspects, the bispecific molecule comprises a first binding moiety and a second binding moiety, wherein the first binding moiety comprises a molecule that specifically binds to an antigen presented on a tumor. In some aspects, the bispecific molecule comprises a molecule that specifically binds to BCMA and CD3, a molecule that specifically binds to CD47 and CD20, a molecule that specifically binds to NKG2D and FLT3, or any combination thereof.

[0026] In some aspects, the molecule of interest is a nucleic acid comprising DNA, RNA (e.g., mRNA), a plasmid, a vector, siRNA, shRNA, an antisense oligonucleotide, or any combination thereof.

[0027] In some aspects, the molecule of interest is a virus comprising an adeno-associated virus, a lentivirus, an adenovirus, or any combination thereof.

[0028] In some embodiments, the impurities include viruses, high molecular weight aggregates (HMW), low molecular weight aggregates (LMW), host cell proteins (HCP), residual deoxyribose nucleic acids (rDNA), residual protein A (rProA), or any combination thereof.

[0029] In some embodiments, this method reduces the risk of polysorbate 80 degradation in the final product.

[0030] Some aspects of this disclosure relate to the target molecule isolated by the method described herein. [Brief explanation of the drawing]

[0031] [Figure 1A] Figure 1A shows a schematic diagram of the molecules used in this disclosure. [Figure 1B] Figure 1B shows the molecular structure and information of the bispecific antibody (bsAb) and Fc fusion protein used in this disclosure. This figure includes process-related impurity challenges such as molecular weight in kilodaltons (kDa) (MW), host cell proteins (HCP), and residual deoxyribose nucleic acid (rDNA), as well as product-related impurity challenges such as low molecular weight (LMW) aggregates, high molecular weight (HMW) aggregates, and residual protein A (rProA). [Figure 2] Figure 2 shows a comparison of the loading breakthrough observed for each bsAb and sodium phosphate condition. The columns in the figure represent the CHT F / T column loading where loading breakthrough was observed. The plotted lines represent the percentage difference in loading breakthrough between the 5 mM and 20 mM sodium phosphate conditions for each bsAb. [Figure 3A]Figure 3A shows the HMW impurity data for bsAb D. Figure 3A shows a chromatogram of bsAb D under 20 mM sodium phosphate conditions, with the ultraviolet absorbance at 280 nanometers (UV280) in milliabsorbance units (mAU) on the right axis, representing the concentration of protein eluted from the CHT column. This curve is segmented by fractions individually collected and tested for impurities. The top of the curve and within each section overlay the HMW percentage of the loading material and its respective individual pool fraction based on size exclusion chromatography using ultra-high performance liquid chromatography (SEC-UPLC) results. This corresponds to the left axis. The slope of the dashed line represents the percentage of the linear sodium phosphate elution gradient. [Figure 3B] Figure 3B shows the HMW impurity data for bsAb D. Figure 3B shows the same HMW percentage information as the chromatogram in Figure 3A, but for bsAb D under both 5 mM and 20 mM sodium phosphate conditions. Each column represents one individual sample, first the loading, followed by each fraction, as are the columns overlaid on the chromatogram above. The chases for each phosphate concentration condition are separated to indicate when they occurred, as they do not align with the same fractions collected across both runs. Chases during the 5 mM sodium phosphate run were collected in fractions 7 and 8, and chases during the 20 mM sodium phosphate run were collected in fractions 9 and 10. [Figure 4] Figure 4A shows the HMW impurity data for bsAb B under both 5 mM and 20 mM sodium phosphate conditions, arranged by the collected fraction. Figure 4B shows the HMW impurity data for bsAb C under both 5 mM and 20 mM sodium phosphate conditions, arranged by the collected fraction. In both Figures 4A and 4B, each point corresponds to the HMW percentage measured via SEC-UPLC for each individual sample for the loading and pooling fractions. [Figure 5]Figures 5A, 5B, 5C, and 5D show LMW impurity data for bsAb A (Figure 5A), B (Figure 5B), C (Figure 5C), and D (Figure 5D) under both 5 mM and 20 mM sodium phosphate conditions, respectively, arranged by the collected fractions. Each point corresponds to the LMW percentage measured via non-reducing capillary electrophoresis of each individual sample for the loading and pooling fractions, via sodium dodecyl sulfate (CAL-NR). The chases for each phosphate concentration condition of bsAb D are separated to indicate when they occurred, as they do not align with the same fractions collected across both runs. Chases during the 5 mM sodium phosphate run of bsAb D were collected in fractions 7 and 8, and chases during the 20 mM sodium phosphate run of bsAb D were collected in fractions 9 and 10. [Figure 6] Figure 6 shows rDNA impurity data for bsAb B under both 5 mM (Figure 6A) and 20 mM (Figure 6B) sodium phosphate conditions, arranged according to the collected fractions. Each point corresponds to the rDNA concentration measured by quantitative polymerase chain reaction (qPCR) for each individual sample for the loading and pooling fractions. Both bar graphs contain the same data, but the lower graph has a y-axis adjusted to a logarithmic scale to more clearly show smaller values. [Figure 7A] Figure 7A shows the CHT F / T chromatograms of Fc-fused A at both bench scale (Figure 7A) and 500-liter scale (Figure 7B). Both show a consistent UV280 curve shape regardless of scale. The bench-scale column used had an inner diameter (ID) of 0.66 cm and a bed height (BH) of 20 cm. The 500-liter scale column used had an ID of 30 cm and a BH of 19.1 cm. [Figure 7B]Figure 7B shows the CHT F / T chromatograms of Fc-fused A at both bench scale (Figure 7A) and 500-liter scale (Figure 7B). Both show a consistent UV280 curve shape regardless of scale. The bench-scale column used had an inner diameter (ID) of 0.66 cm and a bed height (BH) of 20 cm. The 500-liter scale column used had an ID of 30 cm and a BH of 19.1 cm. [Modes for carrying out the invention]

[0032] This disclosure provides insights into the methods and advantages of a flow-through (F / T) operating mode approach for mixed-mode ceramic hydroxyapatite (CHT) chromatography using complex biopharmaceuticals in downstream purification processes. Flow-through CHT chromatography enables the purification of a range of complex biopharmaceuticals, including bispecific antibodies (bsAb) and Fc fusion proteins, with significantly higher productivity than conventional binding and elution (B / E) operating modes. Furthermore, these results demonstrate robust viral clearance capabilities by increasing product pool stability through reduced risk in polysorbate 80 (PS80) degradation and showing significant surrogate viral clearance across multiple surrogate models up to 3log decline values ​​(LRV).

[0033] In some embodiments, a greater column-loading approach was applied to the CHT resin by abandoning the conventional B / E operating mode for this resin and applying the F / T operating mode, which is not typical in the context of CHT. The results of this disclosure demonstrate efficient separation and purification of therapeutic proteins from process and product-related impurities equivalent to B / E, while still employing the significant productivity improvements brought about by the F / T operating mode.

[0034] Some aspects of the present disclosure relate to a method for isolating molecules in a sample containing a target molecule and impurities in flow-through mode of ceramic hydroxyapatite chromatography (CHT), the method comprising: a) loading the sample onto a CHT column; and b) obtaining an F / T composition containing the molecule and a smaller amount of impurities compared to the sample before loading.

[0035] Some aspects of the present disclosure relate to a method for isolating molecules in a sample containing a target molecule and impurities using ceramic hydroxyapatite chromatography (CHT), the method comprising: a) loading the sample onto a CHT column in flow-through mode; and b) obtaining an F / T composition containing the molecule and a smaller amount of impurities compared to the sample before loading.

[0036] I. Terminology To make this disclosure more easily understandable, certain terms are defined first. When used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout this application.

[0037] The singular forms “a,” “an,” and “it” include multiple referents unless the context explicitly indicates otherwise. The terms “a” (or “an”) and “one or more,” and “at least one” are interchangeable herein. In certain embodiments, “a” or “an” means “single.” In other embodiments, “a” or “an” includes “two or more” or “plural.”

[0038] When used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. Thus, when used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, when used in phrases such as "A, B and / or C," the term "and / or" is intended to include 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 (alone); B (alone); and C (alone).

[0039] The terms “approximately” or “essentially derived” refer to a value or composition that is within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, such margin of error depending, in part, on how the value or composition is measured or determined, i.e., on the limits of the measurement system. For example, “approximately” or “essentially derived” may mean within or exceeding one standard deviation, according to convention in the art. Alternatively, “approximately” or “essentially containing” may mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, these terms may mean up to one order of magnitude or up to five times a given value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of “approximately” or “essentially derived” should be assumed to be within an acceptable margin of error for that particular value or composition.

[0040] Whenever an aspect is described in this specification with the term "including," it should be understood that other similar aspects are also provided, described in terms of "consisting of" and / or "essentially consisting of."

[0041] As used herein, the term “approximately” means, when applied to the value of one or more subjects, a value that is similar to the stated reference value. In certain embodiments, unless otherwise specified or evident from the context, the term “approximately” means a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value (except where such a number exceeds 100% of the possible value).

[0042] When described herein, any range of concentration, percentage, ratio, or integer should be understood to include any integer value within the range described and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0043] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in the field relating to this disclosure. 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 general dictionaries of many of the terms used herein.

[0044] Units, prefixes, and symbols are expressed in the format recognized by their respective International System of Units (SI). The headings provided herein are not limitations on the various aspects of this disclosure that can be obtained by referring to this specification as a whole. Thus, defined terms are more fully defined by referring to this specification as a whole.

[0045] Abbreviations used herein are defined throughout this disclosure. Various aspects of this disclosure are described in further detail in the following subsections.

[0046] As used interchangeably herein, the terms “purify,” “separate,” or “isolate” refer to increasing the purity of a protein of interest from a composition or sample containing the protein of interest and one or more impurities. Typically, the purity of a 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 one or more impurities contain a second charge variant of the same protein.

[0047] As used herein, the term "chromatography" refers to a dynamic separation technique that enables the separation and isolation of a target molecule, such as a target protein (e.g., a charged variant of a protein, e.g., an antibody), from other molecules in a mixture (e.g., other charged variants). Typically, in chromatography, a liquid mobile phase transports a sample containing the target molecule of interest across or through a stationary phase (usually a solid) medium. Differences in distribution or affinity to the stationary phase cause the transient binding of selected molecules to the stationary phase, while the mobile phase carries different molecules at different times.

[0048] The term "flow-through" or "F / T" refers to a chromatographic process in which the target protein in a sample containing protein passes through a chromatography column and is then eluted from the column, rather than binding to the column. In contrast, the term "binding-elution" or "B / E" means that the protein in the sample is loaded onto a chromatography column and bound to it. The target protein bound to the column can be debound by applying specific conditions and then eluted for collection.

[0049] The term "ceramic hydroxyapatite chromatography" or "CHT" refers to mixed-mode chromatography that utilizes ceramic hydroxyapatite as a chromatography column. The ions present on the surface of hydroxyapatite make it an ideal candidate for biomolecular mixtures, possessing unique selectivity, separation, and purification properties. The combined presence of calcium ions (C-sites) and phosphate sites (P-sites) provides metallophilic and ion-exchange properties, respectively. The C-sites on the resin surface undergo metallophilic interactions with phosphate or carboxyl groups present on proteins. Simultaneously, these positively charged C-sites tend to repel positively charged functional groups (e.g., amino groups) on proteins. The P-sites undergo cation exchange with positively charged functional groups on proteins. They exhibit electrostatic repulsion with negatively charged functional groups on proteins. A buffer containing high concentrations of phosphate and sodium chloride is used for molecular elution. The properties of the differently charged ions on the surface of hydroxyapatite provide a unique framework for protein selectivity and binding, facilitating robust protein separation.

[0050] As used herein, the term “loading” refers to applying a solution, such as a mixture containing the protein products and contaminants described herein, to a chromatography matrix. In some embodiments, the term “loading” is synonymous with “contact” of the solution on the chromatography column.

[0051] The term "applied to" broadly means that, when used in the context of a gradient applied to a chromatography matrix, the gradient is formed directly or indirectly within and / or around the chromatography matrix. In some embodiments, the chromatography matrix resides within a column, and the gradient is formed within the column. In some embodiments, the gradient applied to the chromatography matrix is ​​formed internally within the column, as opposed to a gradient 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 two or more buffers being added to the chromatography matrix. In other embodiments, the gradient applied to the chromatography matrix is ​​formed externally and then added to the column.

[0052] As used herein, the terms “culture,” “cell culture,” and “eukaryotic cell culture” refer to a population of cells attached to a surface or in suspension, maintained or grown in a culture medium (see the definition of “medium” below) under conditions suitable for the survival and / or proliferation of the cell population. As will be apparent to those skilled in the art, as used herein, these terms may refer to a combination of a cell population and a culture medium in which the population is suspended.

[0053] As used herein, the terms “expression” or “to express” are used to refer to transcription and translation that occur within a cell. The expression level of a product gene in a host cell can be determined based on either or both the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the product gene produced by the cell.

[0054] The term "antibody" in some embodiments refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of 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 consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl 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 the antigen. The term "antibody" may include bispecific or multispecific antibodies.

[0055] As used herein, “IgG antibodies,” such as human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments have the structure of naturally occurring IgG antibodies, i.e., they have the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies 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), the two HC and LC linked by the same number and positions of disulfide crosslinks that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has mutated to modify the disulfide crosslinks).

[0056] Immunoglobulins may originate from any of the generally known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses specific to certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as IgG1, exist in several allotypes, differing from each other by at most a few amino acids. "Antibodies" include, by example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0057] As used herein, the term “antigen-binding site” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding site" of an antibody include: (i) Fab fragments (fragments derived from papain cleavage) or similar monovalent fragments consisting of VL, VH, LC, and CH1 domains; (ii) F(ab')2 fragments (fragments derived from pepsin cleavage) or similar bivalent fragments containing two Fab fragments linked by disulfide crosslinking at the hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et at., (1989), Nature, 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs that can optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined together by a synthetic linker using recombination methods, allowing the VL and VH regions to form a single protein chain (known as single-chain Fv (scFv), see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) to be produced. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as undamaged antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0058] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, created or isolated by recombinant means, for example: (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies, for example, transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences.

[0059] As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0060] Amino acids are referred herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are referred to by their commonly accepted single-letter codes.

[0061] As used herein, the terms “protein,” “protein of interest,” or “polypeptide” refer to molecules composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The terms “polypeptide,” “protein,” “protein of interest,” “product,” “produced protein,” or “amino acid residue sequence” are used interchangeably. The term “polypeptide” refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. As used herein, the term “protein” is intended to encompass molecules composed of one or more polypeptides that may, in some cases, be linked by bonds other than amide bonds. A protein, on the other hand, can be a single polypeptide chain. In this latter example, a single polypeptide chain may, in some examples, contain two or more polypeptide subunits that fuse with each other to form a protein. The terms “polypeptide” and “protein” also refer to products of post-expression modifications, including glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with amino acids that do not exist naturally, but are not limited to these. Polypeptides or proteins may originate from natural biological sources or may be produced by recombinant technologies.

[0062] Where used herein, the terms “polynucleotide” or “nucleotide” are intended to encompass single and multiple nucleic acids and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). The term “nucleic acid” refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA, cDNA, or RNA fragments. Where applied to nucleic acids or polynucleotides, the term “isolated” refers to nucleic acid molecules, DNA, or RNA removed from their natural environment; for example, recombinant polynucleotides encoding antigen-binding proteins contained in a vector are considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. Isolated polynucleotides or nucleic acids by this disclosure further include synthetically produced such molecules. Furthermore, polynucleotides or nucleic acids may contain regulatory elements such as promoters, enhancers, ribosome-binding sites, or transcription termination signals.

[0063] As used herein, the terms “impurity” or “contamination” refer to one or more molecules, e.g., polypeptides, nucleic acid molecules, small molecules, or any combination thereof, present in a mixture with the target molecule, e.g., a target species of polypeptide, e.g., a target charge variant of polypeptide. In some embodiments, the contamination is a different polypeptide, e.g., a polypeptide having a different structure, sequence, or function than the target polypeptide. In some embodiments, the contamination is a different species of the target polypeptide, e.g., an LMW species or an HMW species. In some embodiments, the contamination is a host cell protein, a fragment of the protein of interest, a virus, or any other undesirable substance in the sample being purified.

[0064] As used herein, the term "purity" refers to the degree to which a composition, such as a sample or solution containing a target polypeptide, contains one or more impurities. For example, a solution containing a target polypeptide in which 98% of the substance is the protein of interest and 2% of the substance is impurities has a purity of 98%.

[0065] II. Method of Disclosure Some aspects of the present disclosure relate to a method for isolating molecules in a sample containing a target molecule and impurities in a flow-through (F / T) mode of ceramic hydroxyapatite chromatography (CHT), comprising: a) loading the sample onto a CHT column; and b) obtaining an F / T composition containing the molecule. In some aspects, the F / T composition after F / T contains the target molecule and a smaller amount of impurities compared to the sample before loading.

[0066] Some aspects of the present disclosure relate to a method for isolating molecules in a sample containing a target molecule and impurities using ceramic hydroxyapatite chromatography (CHT), comprising: a) loading the sample onto a CHT column in flow-through (F / T) mode; and b) obtaining an F / T composition containing the molecule. In some aspects, the F / T composition contains the molecule and a smaller amount of impurities compared to the sample before loading.

[0067] CHT resins have two different types of moieties distributed throughout the crystalline structure of the matrix: positively charged calcium groups and negatively charged phosphoryl groups. The calcium moieties interact with the negatively charged carboxyl groups by forming covalent bonds that are 15 to 60 times stronger than normal ionic interactions. This is known as metalloaffinity interaction, which primarily affects acidic proteins and can be weakened by adding buffer salts such as phosphates to the mobile phase. Conversely, the phosphoryl groups interact with the positively charged amine groups via the normal ionic attraction of cation exchange. This primarily affects basic proteins, increases the pH, and can be weakened by adding neutral salts such as chlorides or buffer salts such as phosphates to the mobile phase. Because the positive charge of the calcium moieties repels the negative charge of the phosphoryl moieties, as well as the negative charge of the phosphoryl moieties which repels the negatively charged carboxyl groups, and simultaneously repels the amine groups which share the same charge, the metalloaffinity and cation exchange interactions of the calcium and phosphoryl moieties directly compete with each other. Furthermore, increasing the concentration of chloride (or other neutral salts) in the mobile phase only weakens the cation exchange interaction, not the metal affinity interaction. However, it is possible to increase the strength of the metal affinity interaction by ionically shielding the repulsive, negatively charged phosphoryl moieties of the resin.

[0068] In some embodiments, the methods disclosed herein result in an increase in the purity of the target molecule compared to conventional methods. In some embodiments, the methods disclosed herein result in an increase in the purity of the target molecule compared to binding and elution (B / E) methods. In some embodiments, the purity of the target molecule increases 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 the B / E method.

[0069] In some embodiments, the methods disclosed herein result in increased stability of the target molecule compared to conventional methods. In some embodiments, the methods disclosed herein result in increased stability of the target molecule compared to the B / E method. In some embodiments, the stability of the target molecule increases 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 the B / E method. In some embodiments, the method reduces the risk of polysorbate 80 degradation in the sample.

[0070] In some embodiments, the methods disclosed herein increase productivity compared to conventional methods, such as the B / E method. In some embodiments, productivity increases by at least about 2 times, at least about 3 times, at least about 4 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, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 25 times, or at least about 30 times compared to conventional methods (e.g., the B / E method).

[0071] In some embodiments, the methods disclosed herein reduce the amount of impurities more effectively than conventional methods, such as the B / E method, while maintaining the productivity of the target molecule compared to conventional methods, such as the B / E method.

[0072] In some embodiments, CHT is a polishing step. In some embodiments, the polishing step for isolating the target molecule comprises two polishing steps, one being CHT and the other being a different chromatography, i.e., a chromatography other than CHT. In some embodiments, the polishing step for isolating the target molecule comprises two polishing steps, one being CHT and the other being a second CHT, and the two CHTs are identical. In some embodiments, the two CHTs are different. In some embodiments, the final product after the polishing step is formulated as a pharmaceutical product.

[0073] In some embodiments, the method further includes affinity chromatography, ion exchange chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, filtration, or any combination thereof, before or after the CHT of the present disclosure.

[0074] In some embodiments, the method further includes subjecting the isolated molecule of interest to one or more analytical characterizations. Accordingly, some embodiments of the present disclosure relate to a method for concentrating a molecule of interest for analytical characterization, comprising (a) separating the molecule of interest from a mixture containing the molecule of interest and one or more impurities, comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode in a chromatographic separation system, and (b) subjecting the molecule of interest from (a) to analytical characterization. Some embodiments of the present disclosure relate to a method for performing analytical characterization of a molecule of interest, comprising (a) separating the molecule of interest from a mixture containing the molecule of interest and one or more impurities, comprising contacting the mixture with two or more chromatographic columns in a continuous operation mode in a chromatographic separation system, and (b) performing analytical characterization of the molecule of interest from (a).

[0075] In some embodiments, analytical characterization includes 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, mass spectrometry, or any combination thereof.

[0076] In some embodiments, the amount of target molecule loaded onto the column is such that none of the target molecule binds to the CHT column. For example, the amount of target molecule loaded onto the CHT column is so low that a significant amount of the target molecule does not bind to the CHT column. In some embodiments, the amount of target molecule loaded onto the CHT column is such that it does not exceed the dynamic binding capacity (DBC) of the column for the target molecule. As used herein, the terms “dynamic binding capacity” or “DBC” of a chromatography column refer to the maximum amount of target molecule that can be loaded onto the column without causing unwanted loss, as measured under realistic experimental conditions (default flow rate, actual protein sample).

[0077] In some embodiments, the present disclosure provides a method for isolating molecules in a sample containing a target molecule and impurities using ceramic hydroxyapatite chromatography (CHT), (a) Load the sample into the CHT column in flow-through (F / T) mode. (b) To obtain an F / T composition containing molecules, wherein only the F / T composition is pooled as the final product. This includes methods that include the following: In some embodiments, the F / T composition includes molecules and less impurities compared to the sample before loading.

[0078] In some embodiments, the method of the present disclosure includes a chase step. In some embodiments, the chase step is optional. In some embodiments, the composition collected in the chase step can be pooled as the final product. In some embodiments, the present disclosure is a method for isolating molecules in a sample containing the molecule of interest and impurities using ceramic hydroxyapatite chromatography (CHT). (a) Load the sample into the CHT column in flow-through (F / T) mode. (b) To obtain an F / T composition containing the molecule, (c) Add chase buffer to the CHT column. (d) To obtain a chase composition containing molecules, wherein the F / T composition and the chase composition are pooled as the final product. The method includes the following: In some embodiments, the F / T composition and / or chase composition comprises molecules and less of impurities compared to the sample before loading and / or chasing, respectively.

[0079] In some embodiments, the amount of target molecule loaded onto the column is such that a portion of the target molecule can bind to the CHT column. For example, a high amount of target molecule loaded onto the CHT column allows a significant amount of the target molecule to bind to the CHT column. In some embodiments, the amount of target molecule loaded onto the column exceeds the dynamic binding capacity (DBC) of the column for the target molecule.

[0080] Therefore, in some aspects of the present disclosure, particularly when the amount of the target molecule added to the CHT column is high enough to allow any significant amount of the target molecule to bind to the column, the method of the present disclosure includes an elution step to obtain the target molecule bound to the CHT column to be collected in the final product. In some aspects, the present disclosure is a method for isolating molecules in a sample containing the target molecule and impurities in ceramic hydroxyapatite chromatography (CHT), (a) Load the sample into the CHT column in flow-through (F / T) mode. (b) To obtain an F / T composition containing the molecule, (c) Add the elution buffer to the CHT column. (d) To obtain an elution composition containing the molecule, wherein the F / T composition and the elution composition are pooled as the final product. The method includes the addition of (b) and (c) on the CHT column and the recovery of the chase composition containing the molecule.

[0081] In some embodiments, the present disclosure provides a method for isolating molecules in a sample containing a target molecule and impurities using ceramic hydroxyapatite chromatography (CHT), (a) Load the sample into the CHT column in flow-through (F / T) mode. (b) To obtain an F / T composition containing the molecule, (c) Add chase buffer to the CHT column. (d) Obtain a chase composition containing the molecule, (e) Add the elution buffer to the CHT column. (f) To obtain an elution composition containing molecules, wherein the F / T composition, the chase composition, and the elution composition are pooled as the final product. This includes methods that include [specific methods].

[0082] In some embodiments, the loaded sample contains at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, or at least about 500 g / L 樹脂 Includes the loading amount.

[0083] In the loading embodiment, the samples loaded by the method of the present invention are approximately 50 to approximately 500, approximately 50 to approximately 450, approximately 50 to approximately 400, approximately 50 to approximately 350, approximately 50 to approximately 300, approximately 70 to approximately 300, approximately 90 to approximately 300, approximately 100 to approximately 300, approximately 120 to approximately 300, approximately 140 to approximately 300, approximately 150 to approximately 300, and approximately 50-250, 70-250, 90-250, 100-250, 120-250, 140-250, 150-250, 50-200, 70-200, 90-200, 100-200, 120-200, 140-200, or 150-200 g / L 樹脂 Includes the loading amount.

[0084] In some embodiments, the loaded sample in the method has a concentration of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490 or about 500 g / L 樹脂 of loading amount.

[0085] In some embodiments, the loaded sample in the method has a loading amount of about 200 g / L 樹脂 . In some embodiments, the loaded sample in the method has a loading amount of about 150 g / L 樹脂 . In some embodiments, the loaded sample in the method has a loading amount of about 250 g / L 樹脂 . In some embodiments, the loaded sample in the method has a loading amount of about 300 g / L 樹脂 .

[0086] In some embodiments, the method of the present disclosure can further comprise a washing step. In some embodiments, the method of the present disclosure can comprise any other necessary steps.

[0087] A. Buffer The methods disclosed herein utilize various buffers: loading buffer, chase buffer and / or elution buffer as appropriate. In some embodiments, the buffers in different steps (e.g., loading, chase, or elution) may be the same. In some embodiments, the buffers in different steps may be different. In some embodiments, the pH of the various buffers (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) may be the same. In some embodiments, the pH of the various buffers (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) may be different. In some embodiments, the conductivity of the various buffers (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) may be the same. In some embodiments, the conductivity of the various buffers (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) may be different.

[0088] In some embodiments, the buffer contains phosphate. In some embodiments, the amount of phosphate in the buffer (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) is the same. In some embodiments, the buffer contains phosphate. In some embodiments, the amount of phosphate in the buffer (e.g., loading buffer, chase buffer, elution buffer and / or washing buffer) is different.

[0089] Loading buffer: In some embodiments, loading a sample into a CHT column involves adding the sample to a loading buffer containing a phosphate. In some embodiments, the phosphate in the loading buffer includes sodium phosphate, potassium phosphate, or any combination thereof.

[0090] In some embodiments, the amount of phosphate in the loading buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

[0091] In some embodiments, the amount of phosphate in the loading buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM. In some embodiments, the amount of phosphate in the loading buffer is about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the amount of phosphate in the loading buffer is about 20 mM to about 50 mM.

[0092] In some embodiments, the loading buffer has a pH of 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, or at least about 8.5. In some embodiments, the pH in the loading buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pH of the loading buffer is about pH 6.3 to about pH 7.7. In some embodiments, the pH of the loading buffer is about 7.5.

[0093] In some embodiments, the loading buffer contains chloride at concentrations of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM. In some embodiments, the loading buffer contains chloride at concentrations of less than approximately 100 mM, less than approximately 90 mM, less than approximately 80 mM, less than approximately 70 mM, less than approximately 60 mM, or less than approximately 50 mM. In some embodiments, the loading buffer does not contain chloride.

[0094] Chase buffer: In some embodiments, the method of the present disclosure comprises adding a chase buffer to the CHT column after loading, the chase buffer comprising a phosphate. In some embodiments, the phosphate in the chase buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0095] In some embodiments, the amount of phosphate in the Chase buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

[0096] In some embodiments, the amount of phosphate in the chase buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM. In some embodiments, the amount of phosphate in the loading buffer is about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the amount of phosphate in the chase buffer is about 20 mM to about 50 mM.

[0097] In some embodiments, the chase buffer has a pH of 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, or at least about 8.5. In some embodiments, the chase buffer has a pH of about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pH of the chase buffer is about pH 6.3 to about pH 7.7. In some embodiments, the pH in the chase buffer is about 7.5.

[0098] In some embodiments, the Chase buffer contains chloride at concentrations of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM. In some embodiments, the Chase buffer contains chloride at concentrations of less than approximately 100 mM, less than approximately 90 mM, less than approximately 80 mM, less than approximately 70 mM, less than approximately 60 mM, or less than approximately 50 mM. In some embodiments, the Chase buffer does not contain chloride.

[0099] Elution buffer: In some embodiments, the method of the present disclosure comprises adding an elution buffer to the CHT column after loading or chasing, wherein the elution buffer comprises a phosphate. In some embodiments, the phosphate in the elution buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0100] In some embodiments, the amount of phosphate in the elution buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

[0101] In some embodiments, the amount of phosphate in the elution buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM. In some embodiments, the amount of phosphate in the elution buffer is about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the amount of phosphate in the elution buffer is about 20 mM to about 50 mM.

[0102] In some embodiments, the elution buffer has a pH of 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, or at least about 8.5. In some embodiments, the pH in the elution buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pH of the elution buffer is about pH 6.3 to about pH 7.7. In some embodiments, the pH of the elution buffer is about 7.5.

[0103] In some embodiments, the elution buffer contains chloride at concentrations of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM. In some embodiments, the elution buffer contains chloride at concentrations of less than approximately 100 mM, less than approximately 90 mM, less than approximately 80 mM, less than approximately 70 mM, less than approximately 60 mM, or less than approximately 50 mM. In some embodiments, the elution buffer does not contain chloride.

[0104] B. Sample In some embodiments, the sample subjected to the method of the present disclosure contains the molecule to be isolated or purified and one or more impurities. In some embodiments, the impurities in the sample are in traceable amounts requiring a polishing step.

[0105] Target molecule In some embodiments, the molecule of interest to be isolated or purified may be any molecule having biological activity. In some embodiments, the molecule of interest may be a nucleic acid, a protein, a virus, or any combination thereof. In some embodiments, the molecule of interest may be a nucleic acid. In some embodiments, the molecule of interest may include DNA, RNA (e.g., mRNA), plasmids, vectors, siRNA, shRNA, antisense oligonucleotides, or any combination thereof. In some embodiments, the molecule of interest may include a vector.

[0106] In some embodiments, the molecule of interest to be isolated or purified may be a virus or a viral vector. In some embodiments, the molecule of interest may include an adeno-associated virus, a lentivirus, an adenovirus, or any combination thereof. In some embodiments, the molecule of interest may include a retrovirus. In some embodiments, the molecule of interest for this method may include an adeno-associated virus (AAV). In some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, and AAV12.

[0107] In some embodiments, the target molecule for this method includes a lentivirus.

[0108] In some embodiments, the molecule of interest is subjected to a prior purification process before being subjected to the methods disclosed herein. In some embodiments, the molecule of interest is subjected to prior affinity chromatography, for example, partially purified by prior affinity chromatography. In some embodiments, the prior affinity chromatography includes protein A affinity chromatography.

[0109] In some embodiments, any polypeptide can be isolated and / or purified using the methods disclosed herein. In some embodiments, the polypeptide is a protein.

[0110] In some embodiments, the molecule of interest includes proteins comprising an antibody or its antigen-binding moiety, an antibody-drug conjugate (ADC), a bispecific molecule, a multispecific molecule, a fusion protein, a cytokine, an immunomodulator, a growth factor, a coagulation factor, a chemokine, an enzyme, a hormone, or any combination thereof. In some embodiments, the molecule of interest may be an antibody or its antigen-binding moiety before conjugation with a drug. In some embodiments, the molecule of interest may be an antibody-drug conjugate after conjugation.

[0111] In some embodiments, the molecule of interest comprises a fusion protein. In some embodiments, the fusion protein comprises an Fc fusion protein, an albumin fusion protein, or any combination thereof. In some embodiments, the molecule of interest 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 the constant region of an antibody. In some embodiments, the immunoglobulin component comprises Fc.

[0112] In some embodiments, the molecule of interest comprises immunoglobulin fused to a growth factor, coagulation factor, cytokine, chemokine, enzyme, hormone, or any combination thereof. In some embodiments, the molecule of interest comprises Fc fused to a growth factor. In some embodiments, the molecule of interest comprises Fc fused to an interleukin.

[0113] In some embodiments, the molecule of interest comprises cytokines. In some embodiments, the cytokines comprise IL10, IL6, IL18, IL12, IL4, TGF-beta, IL17, IL8, IL1B, IL13, IL15, IL2, IL7, IL11, IL22, IL21, IL9, IL-1 receptor, IL3, TNF, IFN-gamma, granulocyte-macrophage colony-stimulating factor, IL5, or any combination thereof. In some embodiments, the molecule of interest comprises an Fc fusion protein containing TGF-β.

[0114] In some embodiments, the molecule of interest is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is PD-1, PD-L1, CTLA-4, LAG-3, or any combination thereof.

[0115] In some embodiments, the molecule of interest comprises an antibody or its antigen-binding moiety (including, but not limited to, a bispecific molecule or a multispecific molecule containing an antibody or its antigen-binding moiety). In some embodiments, the antibody or its antigen-binding moiety binds to a tumor antigen. In some embodiments, the antibody or its antigen-binding moiety binds to a checkpoint inhibitor. In some embodiments, the antibody or its antigen-binding moiety 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, BCMA, CD3, CD47, NKG2D, FLT3, and any combination thereof.

[0116] In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-1. Various human monoclonal antibodies that bind specifically 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 and 8,354,509, U.S. Patent Publication No. 2016 / 0272708 and International Publication Nos. 2012 / 145493, 2008 / 156712 and 2 Pamphlets No. 015 / 112900, No. 2012 / 145493, No. 2015 / 112800, No. 2014 / 206107, No. 2015 / 35606, No. 2015 / 085847, No. 2014 / 179664, No. 2017 / 020291, No. 2017 / 020858, No. 2016 / 19 Pamphlet No. 7367, Pamphlet No. 2017 / 024515, Pamphlet No. 2017 / 025051, Pamphlet No. 2017 / 123557, Pamphlet No. 2016 / 106159, Pamphlet No. 2014 / 194302, Pamphlet No. 2017 / 040790, Pamphlet No. 2017 / 133540, Pamphlet No. 2017 / 132827, Pamphlet No. 2017 / 024465 This information is disclosed in the pamphlets, pamphlets No. 2017 / 106061, 2017 / 19846, 2017 / 024465, international release pamphlets No. 2017 / 025016, international release pamphlets No. 2017 / 025016, international release pamphlets No. 2017 / 132825, and international release pamphlets No. 2017 / 133540, each of which is incorporated in its entirety by reference.

[0117] In some embodiments, anti-PD-1 antibodies include nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck, also known as KEYTRUDA®, lambrolizumab, and MK-3475; see International Publication No. 2008 / 156712), PDR001 (Novartis; see International Publication No. 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see International Publication No. 2012 / 145493), semiprimab (Regeneron; also known as REGN-2810; see International Publication No. 2015 / 112800), and JS001 (TAIZHOU JUNSHI PHARMA (also known as tripalimab, see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene, also known as tislelizumab, see International Publication No. 2015 / 35606 and U.S. Patent Application Publication No. 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine, also known as SHR-1210, see International Publication No. 2015 / 085847, see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical (also known as ANB011, see International Publication No. 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals, also known as WBP3055, Si-Yang Liu et al., J. Hematol. Oncol.)See 10:136 (2017), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics, see International Publication No. 2014 / 194302), AGEN2034 (Agenus, see International Publication No. 2017 / 040790), MGA012 (Macrogenics, see International Publication No. 2017 / 19846), BCD-100 (Biocad, Kaplon et al., mAbs) The selection is made from the group consisting of 10(2):183-203(2018) and IBI308 (see Innovent, International Publication Nos. 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540).

[0118] In one embodiment, the anti-PD-1 antibody is nivolumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab.

[0119] In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-L1. Examples of anti-PD-L1 antibodies include, but are not limited to, those disclosed in U.S. Patent No. 9,580,507. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent No. 7,943,743 and International Publication No. 2013 / 173223), atezolizumab (also known as Roche, TECENTRIQ®; see MPDL3280A, RG7446; see U.S. Patent No. 8,217,149; Herbst et al. (2013) J Clin Oncol) See also 31(suppl):3000), durvalumab (also known as AstraZeneca, IMFINZI, MEDI-4736; see International Publication No. 2011 / 066389), avelumab (also known as Pfizer, BAVENCIO, MSB-0010718C; see International Publication No. 2013 / 079174), STI-1014 (Sorrento; see International Publication No. 2013 / 181634), CX-072 (Cytomx; see International Publication No. 2016 / 149201), KN035 (3DMed / Alphamab; Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly The selection is made from the group consisting of Co. (see, for example, International Publication No. 2017 / 034916), BGB-A333 (BeiGene, Desai et al., JCO36(15 above):TPS3113(2018)), and CK-301 (Checkpoint Therapeutics), see Gorelik et al., AACR:Abstract 4606(Apr 2016).

[0120] In certain embodiments, the PD-L1 antibody is atezolizumab (TECENTRIQ®). In certain embodiments, the PD-L1 antibody is durvalumab (IMFINZI®). In certain embodiments, the PD-L1 antibody is avelumab (BAVENCIO®).

[0121] In some embodiments, the antibody or its antigen-binding moiety 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, for example, in U.S. Patents No. 5,977,318, No. 6,051,227, No. 6,682,736, and No. 7,034,121, and in International Publications 2012 / 122444, 2007 / 113648, 2016 / 196237, and 2000 / 037504, respectively, and are incorporated herein by reference as a whole. In certain embodiments, the CTLA-4 antibody is selected from ipilimumab (YERVOY®, also known as MDX-010, 10D1, see U.S. Patent No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc., see International Publication No. 2016 / 196237), and tremelimumab (AstraZeneca, also known as tisilimmab, see CP-675,206, International Publication No. 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 certain embodiments, the CTLA-4 antibody is AGEN-1884.

[0122] In some embodiments, the antibody or its antigen-binding moiety specifically binds to LAG-3. Antibodies that bind to LAG-3 are described in International Publication No. 2015 / 042246, U.S. Patent Application Publication No. 2014 / 0093511, and U.S. Patent Application Publication No. 2011 / 0150892, respectively, and are incorporated herein by reference in their entirety. Non-limiting examples of anti-LAG-3 antibodies include, but are not limited to, 25F7 (U.S. 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.For these and other anti-LAG-3 antibodies useful in the claimed invention, see, for example, International Publication Nos. 2016 / 028672, 2017 / 106129, 2017 / 062888, 2009 / 044273, 2018 / 069500, 2016 / 126858, 2014 / 179664, 2016 / 200782, 2015 / 200119, 2017 / 019846, 2017 / 198741, 2017 / 220555, and 2017 / 220569. These can be found in International Publication No. 2018 / 071500, International Publication No. 2017 / 015560, International Publication No. 2017 / 025498, International Publication No. 2017 / 087589, International Publication No. 2017 / 087901, International Publication No. 2018 / 083087, International Publication No. 2017 / 149143, International Publication No. 2017 / 219995, U.S. Patent Application Publication No. 2017 / 0260271, International Publication No. 2017 / 086367, International Publication No. 2017 / 086419, International Publication No. 2018 / 034227, and International Publication No. 2014 / 140180, the entirety of which is incorporated herein by reference.

[0123] In some embodiments, the antibody or its antigen-binding moiety specifically binds to CD137. Antibodies that bind to CD137 are described in U.S. Patent Publication No. 2005 / 0095244 and U.S. Patents No. 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 incorporated herein by reference in its entirety. In some embodiments, the anti-CD137 antibody is urelumab (BMS-663513), disclosed 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), which is 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 described in U.S. Patent Nos. 7,214,493, 6,303,121, 6,569,997, 6,905,685, or 6,355,476. In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492, 3H3, or BMS-469497, or 3E1, as described in U.S. Patent No. 6,362,325. In some embodiments, the anti-CD137 antibody is described in U.S. Patent No. 6,974,863 (e.g., 53A2). In some embodiments, the anti-CD137 antibody is described in U.S. Patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E1). In some embodiments, the antibody is Pfizer's PF-05082566 (PF-2566).

[0124] In some embodiments, the antibody or its antigen-binding moiety specifically binds to KIR. Examples of anti-KIR antibodies are disclosed in International Publication Nos. 2014 / 055648, 2005 / 003168, 2005 / 009465, 2006 / 072625, 2006 / 072626, 2007 / 042573, 2008 / 084106, 2010 / 065939, 2012 / 071411, and 2012 / 160448, which are incorporated herein by reference in their entirety. One anti-KIR antibody useful in this disclosure is ririlumab (also known as BMS-986015, IPH2102, or the S241P variant of 1-7F9), which is first described in International Publication No. 2008 / 084106. Another anti-KIR antibody useful in this disclosure is 1-7F9 (also known as IPH2101), which is described in International Publication No. 2006 / 003179.

[0125] In some embodiments, the antibody or its antigen-binding moiety specifically binds to GITR. Examples of anti-GITR antibodies are described in International Publication No. 2015 / 031667, International Publication No. 2015 / 184,099, International Publication No. 2015 / 026684, International Publication No. 11 / 028683, and International Publication No. 2006 / 105021, U.S. Patent Nos. 7,812,135 and 8,388,967, and U.S. Patent Application Publication Nos. 2009 / 0136494, 2014 / 0220002, 2013 / 0183321, and 2014 / 0348841, which are incorporated herein by reference in their entirety. In one embodiment, the anti-GITR antibody useful in this disclosure is TRX518 (e.g., described in Schaer et al. Curr Opin Immunol. (2012) Apr;24(2):217-224 and International Publication No. 2006 / 105021). In another embodiment, the anti-GITR antibody is selected from MK4166, MK1248, and the antibodies described in International Publication No. 11 / 028683 and U.S. Patent No. 8,709,424. In a particular embodiment, the anti-GITR antibody is the anti-GITR antibody disclosed in International Publication No. 2015 / 031667. In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in International Publication No. 2015 / 184099, for example, antibody Hum231#1 or Hum231#2 or its CDR or derivative thereof (e.g., pab1967, pab1975 or pab1979). In certain embodiments, the anti-GITR antibody includes an anti-GITR antibody disclosed in Japanese Patent Publication No. 2008278814, International Publication No. 09 / 009116, International Publication No. 2013 / 039954, U.S. Patent No. 20140072566, U.S. Patent No. 20140072565, U.S. Patent No. 20140065152, or International Publication No. 2015 / 026684, or includes INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (MedImmune).In certain embodiments, the anti-GITR antibody is an anti-GITR antibody described in PCT / US2015 / 033991 (for example, an antibody containing the variable regions 28F3, 18E10, or 19D3).

[0126] In some embodiments, the antibody or its antigen-binding moiety specifically binds to TIM3. In some embodiments, the anti-TIM3 antibody is described in International Publication No. 2018013818, International Publication No. 2015 / 117002 (e.g., MGB453, Novartis), International Publication No. 2016 / 161270 (e.g., TSR-022, Tesaro / AnaptysBio), International Publication No. 2011155607, International Publication No. 2016 / 144803 Fret (e.g., STI-600, SorrentoTherapeutics), International Publication No. 2016 / 071448, International Publication No. 17055399, International Publication No. 17055404, International Publication No. 17178493, International Publication No. 18036561, International Publication No. 18039020 (e.g., Ly-3221367, Eli Selected from the anti-TIM3 antibodies disclosed in Lilly's International Publication No. 2017205721, International Publication No. 17079112, International Publication No. 17079115, International Publication No. 17079116, International Publication No. 11159877, International Publication No. 13006490, International Publication No. 2016068802, International Publication No. 2016068803, International Publication No. 2016 / 111947 and International Publication No. 2017 / 031242, each in its entirety is incorporated herein by reference.

[0127] In some embodiments, the antibody or its antigen-binding moiety 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 No. 20160196228. In some embodiments, the anti-OX40 antibody is described in International Publication Nos. 95012673, 199942585, 14148895, 15153513, 15153514, 13038191, 16057667, 03106498, and 12027328. Anti-OX40 antibodies are selected from those described in the brochures, International Publication No. 13028231, International Publication No. 16200836, International Publication No. 17063162, International Publication No. 17134292, International Publication No. 17096179, International Publication No. 17096281, and International Publication No. 17096182, and the entirety of each is incorporated herein by reference.

[0128] In some embodiments, the antibody or its antigen-binding moiety specifically binds to NKG2A. In some embodiments, the anti-NKG2A antibody is BMS-986315. In some embodiments, anti-NKG2A antibodies are, for example, described in International Publication No. 2006 / 070286 (Innate Pharma SA; University of Genova), U.S. Patent No. 8,993,319 (Innate Pharma SA; University of Genova), International Publication No. 2007 / 042573 (Innate Pharma S / A; 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), International Publication No. 2008 / 009545 (Novo Nordisk A / S), U.S. Patent No. 8,206,709, U.S. Patent No. 8,901,283, and U.S. Patent No. 9,683,041 (Novo Nordisk Anti-NKG2A antibodies are selected from those described in International Publication No. 2009 / 092805 (Novo Nordisk A / S), U.S. Patent No. 8,796,427 and No. 9,422,368 (Novo Nordisk A / S), International Publication No. 2016 / 134371 (Ohio State Innovation Foundation), International Publication No. 2016 / 032334 (Janssen), International Publication No. 2016 / 041947 (Innate), International Publication No. 2016 / 041945 (Academisch Ziekenhuis Leiden H. ODNLUMC), International Publication No. 2016 / 041947 (Innate Pharma), and International Publication No. 2016 / 041945 (Innate Pharma), and the entirety of each is incorporated herein by reference.

[0129] In some embodiments, the antibody or its antigen-binding moiety specifically binds to ICOS. In some embodiments, the anti-ICOS antibody is BMS-986226. In some aspects, anti-ICOS antibodies are described, for example, in International Publication No. 2016 / 154177 (JounceTherapeutics, Inc.), International Publication No. 2008 / 137915 (MedImmune), International Publication No. 2012 / 131004 (INSERM, French National Institute of Health and Medical Research), European Patent No. 3147297 (INSERM, French National Institute of Health and Medical Research), International Publication No. 2011 / 041613 (Memorial Sloan Kettering Cancer Center), European Patent No. 2482849 (Memorial Sloan Kettering Cancer Center), and International Publication No. 1999 / 15553 (Robert Koch Selected from anti-ICOS antibodies described in U.S. Patent Nos. 7,259,247 and 7,722,872 (Robert Kotch Institute), International Publication No. 1998 / 038216 (Japan Tobacco Inc.), U.S. Patent Nos. 7,045,615, 7,112,655 and 8,389,690 (Japan Tobacco Inc.), U.S. Patent Nos. 9,738,718 and 9,771,424 (GlaxoSmithKline), and International Publication No. 2017 / 220988 (Kymab Limited), the entirety of each is incorporated herein by reference.

[0130] In some embodiments, the antibody or its antigen-binding moiety 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 International Publication No. 2016 / 106302. In some embodiments, the anti-TIGIT antibody is MTIG7192A / RG6058 / RO7092284 or clone 4.1D3, as described in International Publication No. 2017 / 053748. In some embodiments, the anti-TIGIT antibody is selected from anti-TIGIT antibodies described, for example, in International Publication No. 2016 / 106302 (Bristol-Myers Squibb Company) and International Publication No. 2017 / 053748 (Genentech).

[0131] In some embodiments, the antibody or its antigen-binding moiety specifically binds to CSF1R. In some embodiments, the anti-CSF1R antibody is an antibody species disclosed in any of the following brochures: International Publication No. 2013 / 132044, International Publication No. 2009 / 026303, International Publication No. 2011 / 140249, or International Publication No. 2009 / 112245, e.g., kabilizumab, RG7155 (emactozumab), AMG820, SNDX 6352 (UCB The anti-CSF1R antibody in the method is either 6352), CXIIG6, IMC-CS4, JNJ-40346527, MCS110, or can be replaced with an anti-CSF1R inhibitor or anti-CSF1 inhibitor such as BLZ-945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486, ARRY-382, or PLX-73086.

[0132] In some embodiments, the target molecule comprises a bispecific molecule. In some embodiments, the bispecific or multispecific molecule comprises a first binding site and a second binding site, the first binding site comprising a molecule that specifically binds to an antigen presented on a tumor. In some embodiments, the second binding site in the bispecific or multispecific molecule comprises a molecule that specifically binds to a protein expressed on immune cells, such as T cells. In some embodiments, the first binding site in the bispecific or multispecific molecule comprises a molecule that specifically binds to an antigen on a tumor, and the second binding site in the bispecific or multispecific molecule comprises a molecule that specifically binds to a protein expressed on immune cells, such as T cells. In some embodiments, the protein expressed on immune cells, such as T cells, comprises CD3. In some embodiments, the target molecule to be isolated or purified comprises one or more structures from Figures 1A and 1B.

[0133] Bispecific antibodies present several challenges in downstream processing. Impurities associated with bispecific antibody products, such as mispair species, hemiantibodies, antibody fragments, and aggregates, are difficult to eliminate because their size and physicochemical properties are similar to those of the target product. (Ingavat et al., Bioresour Bioprocess. 2023 Dec 13;10(1):93). Furthermore, manipulated bispecific antibodies are known to be prone to aggregation and have lower stability compared to their parent mAb. (Ibid.). Considering the differences between mAbs and bsAbs, conventional purification methods used for mAbs may not be effective for bsAbs. (Ibid.). In addition, "chromatography-induced aggregation" has been observed during both protein A and cation exchange chromatography (CEX) for bsAb purification, as reported by Serene Chen et al. and Lucas K. Kimerer et al. Chen et al., Bioresour Bioprocess. 2022;9(1):98; Chen et al., Bioresour Bioprocess. 2022;9(1):72; Kimerer et al., J Chromatogr A. 2019;1601:121-132. This problem is exacerbated when bispecific antibodies are subjected to high loading conditions, which can significantly impact productivity by requiring lower loading levels to reduce aggregation.

[0134] Therefore, in some embodiments, the present method provides a method for purifying a bispecific antibody in a sample containing a bispecific antibody and impurities, comprising the steps of (1) loading the sample onto a CHT column and (2) obtaining a flow-through composition containing the molecule and a smaller amount of impurities compared to the sample before loading.

[0135] In some embodiments, the present invention provides a method for purifying a bispecific antibody in a sample containing a bispecific antibody and impurities, the method comprising passing the sample through ceramic hydroxyapatite chromatography in flow-through mode.

[0136] In some embodiments, loading a sample onto a CHT column or performing CHT chromatography involves adding the sample to a loading buffer containing a phosphate. In some embodiments, the phosphate in the loading buffer includes sodium phosphate, potassium phosphate, or any combination thereof.

[0137] In some embodiments, the bispecific molecules include molecules targeting BCMA and CD3, molecules targeting CD47 and CD20, molecules targeting NKG2D and FLT3, or combinations thereof.

[0138] In some embodiments, the bispecific molecule includes a molecule that targets BCMA and CD3. In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule is alnuctamab, CC-93269, or BMS-986349. In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule includes a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 1-4.

[0139] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 1.

[0140] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a second polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 2.

[0141] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a third polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 3.

[0142] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a fourth polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 4.

[0143] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a fifth polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 4.

[0144] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1; a second polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 2; and at least 60%, at least 65%, at least 70%, at least Each comprises a third polypeptide having 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 4; a fourth polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 4; and a fifth polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 4.

[0145] In some embodiments, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 1, a second polypeptide having the amino acid sequence of SEQ ID NO: 2, a third polypeptide having the amino acid sequence of SEQ ID NO: 3, a fourth polypeptide having the amino acid sequence of SEQ ID NO: 4, and a fifth polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0146] [Table 1]

[0147] In some embodiments, the bispecific molecules include molecules that target CD47 and CD20. In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules are CC-96673 and BMS-986358.

[0148] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules comprise a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with one or more amino acid sequences of SEQ ID NOs. 5 to 24.

[0149] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a light chain CDR having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 5-7 and 15-17.

[0150] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include: a light chain CDR1 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 5; a light chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 6; and a light chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 7. The light chain CDR3 contains oneness, the light chain CDR1 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 15, the light chain CDR2 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 16, and the light chain CDR3 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD47 and anti-CD20 bispecific molecule includes a light chain CDR1 having the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 6, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 7, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0151] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a heavy chain CDR having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 8-10 and 18-20.

[0152] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include: a heavy chain CDR1 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9; and a heavy chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10. It includes a heavy chain CDR3 containing a sex and a heavy chain CDR1 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 18; a heavy chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 19; and a heavy chain CDR3 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20.

[0153] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a variable light chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 11 and 21.

[0154] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a variable heavy chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 12 and 22.

[0155] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a light chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 13 and 23.

[0156] In some embodiments, the anti-CD47 and anti-CD20 bispecific molecules include a heavy chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 14 and 24.

[0157] [Table 2]

[0158] [Table 3]

[0159] [Table 4]

[0160] In some embodiments, the bispecific molecules include molecules that target NKG2D and FLT3. In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules are DF4001 and BMS-986450.

[0161] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules comprise a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with one or more amino acid sequences of SEQ ID NOs. 25-43.

[0162] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a light chain CDR having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequences of SEQ ID NOs. 28-30 and 36-38.

[0163] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a light chain CDR1 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 29, and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 30. The light chain CDR1 contains a light chain CDR3 with identity, and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 36, a light chain CDR2 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 37, and a light chain CDR3 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule includes a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 29, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 30, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 37, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0164] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a heavy chain CDR having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequences of SEQ ID NOs. 25-27 and 33-35.

[0165] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a heavy chain CDR1 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 26, and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 27. The heavy chain CDR1 contains a heavy chain CDR3 with identity, and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR2 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 34, and a heavy chain CDR3 contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 35.

[0166] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 27, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 34, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 35.

[0167] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a variable light chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 32 and 40.

[0168] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules include a variable heavy chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 31 and 39.

[0169] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecules contain polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequences of SEQ ID NOs. 41-43.

[0170] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 41.

[0171] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a second polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 42.

[0172] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a third polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 43.

[0173] In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 41; a second polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 42; and a third polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 41, a second polypeptide having the amino acid sequence of SEQ ID NO: 42, and a third polypeptide having the amino acid sequence of SEQ ID NO: 43.

[0174] [Table 5]

[0175] [Table 6]

[0176] This disclosure also includes the target molecule isolated or purified by this disclosure. In some embodiments, this disclosure includes a final product comprising an F / T composition and optionally a chase composition and / or elution composition. In some embodiments, this disclosure includes only an F / T composition as the final product. In some embodiments, the product obtained from this method is used for formulation as a pharmaceutical product.

[0177] In some embodiments, the disclosure also includes methods for treating a disease or condition in which such treatment is required.

[0178] impurities The impurities isolated may include any process-related or product-related impurities. In some embodiments, the impurities in the sample covered by this disclosure may include viruses, high molecular weight aggregates (HMWs), low molecular weight aggregates (LMWs), host cell proteins (HCPs), residual deoxyribose nucleic acids (rDNA), residual protein A (rProA), or any combination thereof.

[0179] In some embodiments, the F / T CHT method reduces the amount of HMW present in the sample by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 1% to approximately 50%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 5% to approximately 40%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 10% to approximately 35%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 10% to approximately 30%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 10% to approximately 20%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 15% to 25%. In some embodiments, the F / T CHT method reduces the amount of HMW in the sample by approximately 20% to 30%.

[0180] In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by up to approximately 30%, approximately 25%, approximately 20%, approximately 15%, approximately 10%, approximately 5%, or approximately 1%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 1% to approximately 20%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 1% to approximately 30%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 1% to approximately 10%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 5% to 10%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 5% to 20%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 10% to 40%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 20% to 30%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 10% to 25%. In some embodiments, the F / T CHT method reduces the amount of LMW in the sample by approximately 15% to 25%.

[0181] In some embodiments, the F / T CHT method reduces the amount of HCP present in the sample by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by approximately 10% to approximately 50%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by approximately 50% to approximately 90%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 20% to 80%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 10% to 50%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 1% to 30%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 5% to 50%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 10% to 20%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 1% to 20%. In some embodiments, the F / T CHT method reduces the amount of HCP in the sample by 10% to 40%.

[0182] In some embodiments, the F / T CHT method has a viral clearance of logarithmically reduced values ​​(LRV) of at least about 0.5, at least about 1, at least about 1.5, 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, or at least about 5.0. LRV represents viral clearance, which is defined as the difference between the total viral load in the input sample and the fraction containing the purified product. In some embodiments, the F / T CHT method has a viral clearance of about 2.8 LRV. In some embodiments, the F / T CHT method has a viral clearance of about 1 LRV to about 4 LRV, about 2 LRV to about 3 LRV, about 2 LRV to about 4 LRV, or about 1 LRV to about 3 LRV.

[0183] In some embodiments, the viruses to be cleared include adeno-associated viruses, lentiviruses, adenoviruses, or any combination thereof. In some embodiments, the viruses to be cleared include retroviruses. In some embodiments, the viruses to be cleared include adeno-associated viruses (AAVs). In some embodiments, the AAV serotypes are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrh10, AAV10, AAVRH10, AAV11, and AAV12. In some embodiments, the viruses to be cleared include lentiviruses.

[0184] In some embodiments, the F / T CHT method reduces the amount of rProA present in the sample by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by approximately 10% to approximately 50%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by approximately 50% to approximately 90%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 20% to 80%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 30% to 80%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 40% to 80%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 50% to 80%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 20% to 70%. In some embodiments, the F / T CHT method reduces the amount of rProA in the sample by 20% to 60%.

[0185] In some embodiments, the method can reduce the risk of degradation of one or more components in the sample, e.g., inactive components, e.g., polysorbate 80. In some embodiments, one or more components in the sample, e.g., polysorbate 80, may become one or more undesirable molecules as part of the final product, e.g., oleic acid due to polysorbate 80 degradation. In some embodiments, one or more components in the sample, e.g., polysorbate 80, may become one or more undesirable contaminants as part of the final product, e.g., oleic acid due to polysorbate 80 degradation, within 24 or 48 hours. In some embodiments, the amount of one or more contaminants due to polysorbate 80 degradation, e.g., oleic acid, from the final product or F / T composition after F / T CHT, particularly from the fusion protein, is lower than the amount of the B / E final product or B / E composition. In some embodiments, the concentration of oleic acid in the final product or F / T composition as polysorbate 80 degradation after the method is a low-risk amount over 48 hours, e.g., less than about 1.75 μg / mL.

[0186] Various aspects of this disclosure are described in further detail in the following subsections. The disclosure is further illustrated by the following embodiments, which should not be construed as further limitations. [Examples]

[0187] Example 1: Adoption of a flow-through method for biopharmaceuticals In this study, five different complex biopharmaceuticals were selected to analyze the capabilities of CHT resin when manipulated using flow-through mode. Of these, four were bispecific molecules containing three different overall structures (referred to as bsAb A, B, C, and D), and the fifth molecule was an Fc fusion protein (referred to as Fc fusion A). bsAb A is a CD3xBCMA bispecific mAb, specifically alnuctamab, also known as CC-93269 or BMS-986349. bsAb B is a CD47×CD20 bispecific Ab, also known as CC-96673 or BMS-986358. bsAb C is an NKG2DxFLT3 bispecific Ab, also known as DF4001 or BMS-986450. bsAb D is an FcRH5×CD3 bispecific Ab, also known as FcRH5 T cell engager or BMS-986448. Like most antibodies, bsAbs are y-type molecules, but instead of having two identical "arms" like y-type monoclonal antibodies (mAbs), they have two distinct (or bispecific) arms. See Figure 1A. For example, a bsAb with one arm of IgG subclass 2 (IgG2) and one arm of IgG1 is called IgG(2+1). Of the four bsAbs used in this study, three have different overall structures: IgG(2+1), IgG(1+1), and IgG(Fab-scFv). See Figures 1A and 1B. These bsAbs have a total isoelectric point (pI) range of 7.7 to 8.1, with Fc-fusion A having a pI of 5.5. The bsAbs already have established binding and elution (B / E) processes, and the column loading amount is CHT resin (g / L). 樹脂 ) It ranges from 14 to 20 grams per liter. Based on previous experiments, Fc fusion A is only 5 g / L 樹脂The following binding capacities were observed. A summary including the molecular structure and related information can be found in Figures 1A and 1B. Because the column loading amounts of these established B / E processes were low (and potentially caused bottleneck problems throughout the downstream purification process), this study was conducted to test a flow-through (F / T) CHT process instead of the B / E CHT process. We sought to increase the column loading amount so that post-process productivity could be increased while maintaining impurity clearances similar to those seen as a result of the B / E bsAb process. To increase the loading and ensure sufficient loading breakthrough, the loading amount for the CHT F / T column was set to 200 g / L. 樹脂 The goal was to achieve the desired outcome. A working pH of 7.5 was selected, as it is relatively close to the pI of bsAbs, and is expected to ensure the stability of the sodium phosphate buffer matrix, which is commonly used with CHT. After loading flow-through and chase, linear gradient elution with sodium chloride was performed to obtain higher resolution for impurity separation and a better understanding of potential separation trends and behaviors. Loading flow-through, chase, and gradient chloride elution were all collected using multiple fractions. Finally, sodium phosphate loading and overall working concentrations of 5 mmol (mM) and 20 mM were selected to observe any potential effects that phosphate concentration may have.

[0188] Example 2. Effect of phosphate on CHT binding This example was performed to observe the effect of phosphate concentrations in the loading and mobile phases on CHT binding. Figure 2 shows that increasing phosphate concentrations in the loading and mobile phases reduced the binding capacity because phosphates weaken both metal affinity and cation exchange interactions. Binding capacity was calculated based on the mass of protein loaded onto the CHT column when loading breakthrough was observed according to the UV of each chromatogram. Loading concentrations should be assumed to have varied between 5 mM and 20 mM sodium phosphate conditions for most of the bsAbs tested, although this was not the case for all. BsAb C showed negligible differences in loading concentrations of 8.61 mg protein / milliliter (mg / mL) and 8.62 mg / mL for 5 mM and 20 mM sodium phosphate conditions, respectively, but a percentage difference of approximately 10% in binding capacity was observed. Table 4 shows a juxtaposition of loading protein concentrations and percentage differences in binding capacity for all bsAbs used in this study. The data suggest little to no effect caused by loading concentration, but may suggest an effect caused by sodium phosphate concentration and molecular dependence.

[0189] [Table 7]

[0190] Example 3. Results of bispecific antibody selectivity test CHT F / T impurity clearance is also affected by observable influences caused by phosphate concentration and further by molecular dependence. Across multiple impurity types and molecules, CHT F / T exhibits strong separation, with promising opportunities for optimization. This molecular-dependent separation can be observed in bsAb D in Figures 3A and 3B, detailing the removal of HMW aggregates even at such a high loading rate of 30% with many product pool fractions of approximately 15% or less. In more typical unfractionated pools, higher overall purity could be achieved by starting collection slightly later and ending slightly earlier. Further separation can be observed in Figure 4, where similar trends to the HMW separation of bsAb D are seen in bsAb B and C, with a major exception being that the percentage of flow-through HMW is much greater than that of bsAb D alone. This unusually high value suggests further molecular dependence and supports the hypothesis that certain types of fragments specific to bsAb D with higher negative charge are more strongly repelled from the phosphoryl moiety of CHT. It is also worth mentioning the much lower HMW percentages that bsAb B and C started with their loading substances.

[0191] Quality data for other impurity types are shown in Figures 5A–5D for LMW aggregates and in Figures 6A and 6V for rDNA, respectively. Strong LMW selectivity was observed across bsAb A, B, and C, with many samples measuring approximately half of the LMW percentage of the loading material. BsAb D showed slight LMW selectivity, and the measured LMW percentage for most samples did not deviate significantly from the LMW percentage of the loading material. The differences in the trends of LMW separation further indicate molecular-dependent selectivity of CHT F / T. Trends in rDNA separation for bsAb B in the figure. Figures 6A and 6B started similarly for both phosphate concentration conditions but later branched due to the majority of rDNA eluting from the column during a linear sodium chloride gradient of 5 mM sodium phosphate, while most rDNA bound more strongly to 20 mM sodium phosphate and eluted into the collected column strip.

[0192] Furthermore, the bsAb F / T data can be compared with B / E data, as shown in Table 5.

[0193] [Table 8]

[0194] Table 5 presents a summary of bsAb CHT F / T data with B / E data available for comparison. Some results were unavailable (N / A) due to lack of testing and / or impurity exposure, while others were below the limit of quantification (LOQ). The limit of quantification, as defined by HCP, is 60 ng / mL. Fractions from collected F / T chromatography runs were gathered and accumulated based on their protein mass and impurity data. Furthermore, adjustments to the recovery criteria were simulated by omitting certain fractions towards the beginning or end of chromatography runs with relatively high impurity levels. Cumulative yields also reflect these omissions. This was done to mimic the performance of an optimized B / E process. The phosphate concentration conditions selected for inclusion in the table for each bsAb during F / T varied among bsAbs to prioritize conditions that yielded a more desirable balance of impurity clearance and yield. All F / T impurity data, with the exception of the F / T HCP data for bsAbs C and D, are from the same 5 mM and 20 mM sodium phosphate chromatography runs detailed throughout this document. These values ​​are obtained from confirmation runs performed after the aforementioned chromatography to verify the separation behavior. These confirmation runs use 200 g / L 樹脂 The process was also performed under loading conditions and under a 20 mM sodium phosphate concentration at pH 7.5, but the sodium chloride concentration was set throughout the entire process, rather than as a gradient. B / E bsAb chromatography uses optimized conditions derived from the already established B / E process. The loading material used in B / E chromatography was of higher purity than that used in F / T chromatography because it consisted of representative materials from each established process.

[0195] In most cases, CHT F / T proved useful when targeting the same impurities as the B / E process for each molecule. For example, the main B / E impurity challenge for bsAb A was the removal of LMW aggregates, which CHT F / T performed well. Some exceptions where CHT performed better in B / E compared to F / T were HCP for bsAb C and LMW aggregates for bsAb D. However, it is important to note that the purity of the loading material in the B / E chromatography runs was more representative and purer compared to the more challenging material used in these F / T runs. Furthermore, the B / E chromatography runs used optimized and established conditions, while the F / T runs used unestablished development conditions. Potential optimizations include including a chase buffer with higher conductivity than the loading and equilibrium (EQ) buffers. The collection criteria can also be adjusted to avoid collecting more impurity-rich fractions in the F / T pool. Another optimization could be reducing the column loading volume, although the column loading volume is 100 g / L. 樹脂 Even when halved, productivity remains significantly higher than B / E operation. Therefore, CHT F / T is 200 g / L 樹脂 Even with column loading amounts exceeding [a certain threshold], the desired separation and purification are observed. More detailed information on the method can be found in the figure caption.

[0196] Example 4. Case study results of FC fusion materials While bsAb selectivity studies serve as proof-of-concept for adapting complex biopharmaceuticals with B / E processes to higher productivity CHT F / T, case studies of Fc fusion demonstrate more than just clearance of common process-related and product-related impurities. For example, Table 6 shows the improved stability provided by CHT F / T by reducing the risk of degradation of polysorbate 80 (PS80), a common surfactant added to improve the stability of active pharmaceutical ingredients (DS) in biopharmaceutical manufacturing. CHT F / T outperformed POROS 50 HQ anion exchange (AEX) F / T chromatography in this embodiment at similar DS concentrations and column loadings.

[0197] Table 6 shows the assessment of PS80 degradation risk for CHT F / T and AEX F / T with Fc fusion A. Risk levels are defined by the range of measured oleic acid (OA) concentrations increasing over 48 hours. OA below 1.75 μg / mL is defined as low risk, 1.75–7.0 μg / mL as medium risk, and OA above 7.0 μg / mL as high risk.

[0198] The risk of PS80 degradation by enzymatic hydrolysis is estimated based on lipase activity by measuring the concentration of free oleic acid released from PS80 degradation over a 48-hour incubation period. PS80 is added to the process material or final drug substance at a target level of 0.5 mg / mL. The added material is then incubated at 25°C for up to 48 hours. Samples are taken at initial, 24-hour, and 48-hour time intervals for oleic acid measurement according to the following protocol: Briefly, 2.67 μg / mL in 150 μL of isopropanol. 13 C 18- Add 50 μL of OA internal standard working solution to the sample, mix thoroughly, and centrifuge at 10,000 rpm for 10 minutes at room temperature. Take the supernatant for LC / MS single-ion monitoring analysis. Risk category assignment is based on the observed increase in free oleic acid over 48 hours, combined with known PS80 degradation data from multiple existing long-term stability studies. Based on empirical assessment, complete hydrolysis of 0.5 mg / ml of PS80 produces approximately 175 μg / mL of oleic acid. An increase of less than 1.75 μg / mL of oleic acid over 48 hours is defined as low risk (corresponding to a 1% decrease in PS80 over 48 hours and a stable PS80 profile over long-term storage from existing programs), 1.75–7.0 μg / mL as medium risk (some significant decrease in PS80 over long-term storage from existing programs), and greater than 7.0 μg / mL as high risk (significant decrease in PS80 over long-term storage from existing programs).

[0199] [Table 9]

[0200] Additional impurity clearance can be observed in Table 7, which shows the ability of CHT F / T to remove surrogate viruses across multiple surrogate models.

[0201] [Table 10]

[0202] Table 7 shows surrogate virus clearance data for CHT F / T with Fc fusion A. LRV is the average of two datasets (n=2) for each surrogate model and resin. A known amount of enriched surrogate virus stock was added to the loading material for each run. LRV was calculated by testing the loading and CHT F / T product pools using qPCR.

[0203] Table 7 shows that CHT F / T achieved an average LRV clearance of 3.7 LRV when using a micro-mouse virus (MVM) surrogate, also known as a simulated viral particle (MVP). Another surrogate model was used against heterotropic mouse leukemia virus (X-MuLV), called retrovirus-like particles (RVLP). CHT F / T achieved an average of 2.8 LRV with this model. CHT F / T performed similarly or better than Capto Phenyl High Sub hydrophobic interaction chromatography (HIC) F / T. When several CHT F / T chromatography column strips were collected and tested, they showed significantly higher levels of MVP and RVLP than the product pool. This further suggests that CHT F / T shows promising potential for viral clearance due to stronger surrogate virus binding to the column. Further background information on surrogate virus models and their methodologies can be found in Table 8 below.

[0204] [Table 11]

[0205] Table 8 provides a simplified summary of background information regarding the viruses and their respective surrogate models. User manuals, including instrumentation, additional information, and method protocols, were provided by Cygnus.

[0206] Fc fusion A exhibits CHT F / T performance equivalent to or better than bsAb used in this test. Chromatographic studies using pre-optimization development conditions with more challenging loading materials demonstrate this performance, as shown in Table 9. Strong separation of HCP, rDNA, and HMW aggregates was observed. Slight separation of rProA was observed, but LMW aggregates were not. These observations further suggest molecular dependence.

[0207] [Table 12]

[0208] Table 9 summarizes the impurity results from the Fc-fusion A CHT F / T pre-optimization development chromatography. The loading material used also showed higher impurity levels than the most representative optimized chromatography. The limits of quantification (LOQ) for HCP and rDNA were 60 ng / mL and 1 pg / mL, respectively.

[0209] CHT F / T has not only been tested on a bench scale, but also has a run completed without issue on a 500-liter (500L) scale. Figures 7A and 7B show two CHT F / T chromatograms processing Fc fusion A. One is a representative optimized bench-scale run, and the other is on a 500L scale using the same operating conditions. The consistency of the UV280 absorbance peak shape between scales, especially in conjunction with the data in Table 10 below, suggests little to no change in performance or behavior.

[0210] [Table 13]

[0211] Table 10 shows the overall CHT F / T yield and product quality data for representative means in bench-scale and 500-liter scale runs that were completed without issues (n=8). The values ​​are consistent across both scales. Values ​​below the limit of quantification (LOQ) defined by each assay are marked as such. The limits of quantification for HCP and rDNA are 60 ng / mL and 1 pg / mL, respectively.

[0212] Average yields and product quality across eight representative CHT F / T bench-scale chromatography runs were compared to those of a completed 500L scale run and showed no significant difference. The consistency of absorbance peaks, yield, quality, and robustness demonstrate promising ability for scaling forward CHT F / T.

[0213] In summary, this study demonstrates that CHT does not need to be strictly limited to the conventional B / E operating mode that has been used. While B / E offers various advantages, operating CHT with F / T can be equally effective in terms of performance, but offers significantly higher productivity provided by much greater column loading (more than 10 times). Results from bsAb screening studies showed that CHT F / T enables strong molecule-dependent separation across a wide range of molecules, phosphate concentrations, impurity types, and impurity levels. The data suggest that the CHT F / T process can significantly improve downstream purification processes, potentially reducing impurity levels by approximately 50% (and sometimes more) without sacrificing considerable productivity (if any).

[0214] Furthermore, in the context of Fc fusion A, CHT F / T exhibits even more benefits, including increased product stability due to reduced risk of PS80 degradation and significant surrogate virus elimination, suggesting promising potential outcomes for actual viruses. Beyond this, the consistency of the absorbance curve, high yield of up to 94%, sub-quantification product quality, and process robustness at both bench and 500L scales demonstrate the promising scalability of the CHT F / T process and the reliability it can have when considering this mode of operation in other contexts.

[0215] Overall, CHT F / T offers robust performance comparable to conventional B / E operation modes, but with significantly improved productivity. This could serve as a reasonable solution for limiting bottlenecks in biopharmaceutical manufacturing processes. Given these promising qualities, it is desirable that CHT F / T be applied without issue to other biopharmaceuticals such as mAbs, as well as more complex biopharmaceuticals such as bsAb and Fc fusion proteins on a larger scale.

[0216] It should be understood that the detailed description section, rather than the summary and abstract section, is intended to be used to interpret the claims. The summary and abstract section may describe one or more, but not all, exemplary embodiments of the present disclosure as contemplated by the inventors, and is therefore not intended in any way to limit the scope of the present disclosure and the accompanying claims.

[0217] The scope and width of this disclosure should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims and equivalents.

[0218] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art, unless otherwise defined herein. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described herein.

[0219] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety. Database entries and electronic publications disclosed herein are incorporated in their entirety. The versions of database entries or electronic publications incorporated by reference herein are the most recent versions available at the time of filing of this application. Database entries corresponding to gene or protein identifiers disclosed herein (e.g., genes or proteins identified by accession numbers or database identifiers in public databases such as Genbank, Refseq, or Uniprot) are incorporated in their entirety. The incorporated gene or protein-related information is not limited to the sequence data contained in the database entries. The incorporated information includes the entire contents of the database entries in the most recent versions available in the databases at the time of filing of this application. In case of any inconsistency, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope of this application.

Claims

1. A method for isolating a target molecule and an impurity from a sample in the flow-through (F / T) mode of ceramic hydroxyapatite chromatography (CHT), a. Loading the sample into the CHT column, b. To obtain an F / T composition comprising the molecule and a smaller amount of the impurity compared to the sample before loading. A method that includes this.

2. A method for isolating a target molecule and an impurity from a sample using ceramic hydroxyapatite chromatography (CHT), a. Load the sample into the CHT column in flow-through (F / T) mode. b. To obtain an F / T composition comprising the molecule and a smaller amount of the impurity compared to the sample before loading. A method that includes this.

3. The method according to claim 1 or 2, wherein the CHT is a polishing step.

4. The method according to claim 3, further comprising an additional polishing step.

5. The method according to any one of claims 1 to 4, wherein the amount of the target molecule in the sample is such that the target molecule does not bind to the column during and / or after loading.

6. The method according to any one of claims 1 to 4, wherein the target molecule in the sample is in an amount such that several molecules bind to the column during and / or after loading.

7. The method according to any one of claims 1 to 6, further comprising affinity chromatography, ion exchange chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, filtration, or any combination thereof, before and after the CHT.

8. The method according to any one of claims 1 to 7, wherein the target molecule includes a protein.

9. The method according to any one of claims 1 to 7, wherein the target molecule includes a nucleic acid.

10. The method according to any one of claims 1 to 7, wherein the target molecule includes a virus.

11. The loaded sample contains at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, or at least about 500 g / L 樹脂 The method according to any one of claims 1 to 10, including the loading amount.

12. The loaded samples are approximately 50-500, 50-450, 50-400, 50-350, 50-300, 70-300, 90-300, 100-300, 120-300, 140-300, 150-300, 50-250, 70-250, 90-250, 100-250, 100-250, 120-250, 140-250, 150-250, 50-200, 70-200, 90-200, 100-200, 120-200, 120-200, 140-200, or 150-200 g / L. 樹脂 The method according to any one of claims 1 to 11, including the loading amount.

13. The method according to any one of claims 1 to 12, wherein loading the sample onto the CHT column comprises adding the sample to a loading buffer containing a phosphate.

14. The method according to claim 13, wherein the phosphate in the loading buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

15. The method according to claim 14, wherein the amount of the phosphate in the loading buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

16. The method according to claim 14, wherein the amount of phosphate in the loading buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM.

17. The method according to any one of claims 1 to 16, wherein the F / T composition is pooled as the final product.

18. The method according to claim 17, wherein the final product is formulated.

19. (c) The method according to any one of claims 1 to 18, further comprising adding a chase buffer to the CHT column.

20. The method according to claim 19, wherein the chase buffer comprises a phosphate.

21. The method according to claim 20, wherein the phosphate in the chase buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

22. The method according to claim 20 or 21, wherein the amount of phosphate in the chase buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

23. The method according to any one of claims 20 to 22, wherein the amount of phosphate in the chase buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM.

24. (c) The method according to any one of claims 19 to 23, wherein the chase composition containing the molecule is further recovered.

25. The method according to claim 24, wherein the chase composition and the F / T composition are pooled as the final product.

26. The method according to claim 25, wherein the final product is formulated.

27. (d) The method according to any one of claims 1 to 26, further comprising adding an elution buffer when a specific amount of the target molecule is bound to the column.

28. The method according to claim 27, wherein the elution buffer contains a phosphate.

29. The method according to claim 28, wherein the phosphate in the elution buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

30. The method according to claim 29, wherein the amount of the phosphate in the elution buffer is at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, or at least 50 mM.

31. The method according to claim 29, wherein the amount of phosphate in the elution buffer is about 5 to about 50, about 5 to about 45, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 5 to about 20, about 10 to about 20, about 15 to about 20, about 5 to about 15, or about 10 to about 15 mM.

32. The method according to any one of claims 13 to 31, wherein the loading buffer has a pH of 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, or at least about 8.

5.

33. The method according to any one of claims 13 to 31, wherein the pH of the loading buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.

5.

34. The method according to any one of claims 13 to 31, wherein the pH of the loading buffer is approximately pH 6.3 to approximately pH 7.

7.

35. The method according to any one of claims 13 to 31, wherein the pH of the loading buffer is approximately 7.

5.

36. The method according to any one of claims 19 to 35, wherein the pH of the chase buffer is 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, or at least about 8.

5.

37. The method according to any one of claims 19 to 35, wherein the pH of the chase buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.

5.

38. The method according to any one of claims 19 to 35, wherein the pH of the chase buffer is approximately pH 6.3 to approximately pH 7.

7.

39. The method according to any one of claims 19 to 35, wherein the pH of the chase buffer is approximately 7.

5.

40. The method according to any one of claims 19 to 35, wherein the pH of the elution buffer is 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, or at least about 8.

5.

41. The method according to any one of claims 27 to 40, wherein the pH of the elution buffer is about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.

5.

42. The method according to any one of claims 27 to 40, wherein the pH of the elution buffer is approximately pH 6.3 to approximately pH 7.

7.

43. The method according to any one of claims 27 to 40, wherein the pH of the elution buffer is approximately 7.

5.

44. The method according to any one of claims 13 to 43, wherein the loading buffer contains chloride at a concentration of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM.

45. The method according to any one of claims 19 to 44, wherein the chase buffer contains chloride at a concentration of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM.

46. The method according to any one of claims 27 to 45, wherein the elution buffer contains chloride at a concentration of less than approximately 500 mM, less than approximately 450 mM, less than approximately 400 mM, less than approximately 350 mM, less than approximately 300 mM, less than approximately 250 mM, less than approximately 200 mM, less than approximately 190 mM, less than approximately 180 mM, less than approximately 170 mM, less than approximately 160 mM, less than approximately 150 mM, less than approximately 140 mM, less than approximately 130 mM, less than approximately 120 mM, less than approximately 110 mM, or less than approximately 100 mM.

47. The method according to any one of claims 19 to 46, wherein the conductivity of the chase buffer is equal to or greater than that of the loading buffer.

48. The method according to any one of claims 19 to 46, wherein the conductivity of the chase buffer is the same as or less than the conductivity of the loading buffer.

49. The method according to any one of claims 27 to 48, wherein the conductivity of the elution buffer is the same as or greater than that of the loading buffer.

50. The method according to any one of claims 27 to 48, wherein the conductivity of the elution buffer is the same as or less than that of the loading buffer.

51. The method according to any one of claims 1 to 8 or 11 to 50, wherein the target molecule comprises a protein containing an antibody or its antigen-binding portion, an antibody-drug conjugate (ADC), a bispecific molecule, a multispecific molecule, a fusion protein, a cytokine, an immunomodulator, a growth factor, a coagulation factor, a chemokine, an enzyme, a hormone, or any combination thereof.

52. The method according to claim 51, wherein the fusion protein comprises an Fc fusion protein, an albumin fusion protein, or any combination thereof.

53. The method according to claim 51, wherein the fusion protein comprises a cytokine.

54. The method according to claim 51, wherein the cytokines include IL10, IL6, IL18, IL12, IL4, TGF beta, IL17, IL8, IL1B, IL13, IL15, IL2, IL7, IL11, IL22, IL21, IL9, IL-1 receptor, IL3, TNF, IFN gamma, granulocyte-macrophage colony-stimulating factor, IL5, or any combination thereof.

55. The method according to claim 51, 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.

56. The method according to claim 51, wherein the protein is an immune checkpoint inhibitor.

57. The method according to claim 51, wherein the bispecific molecule comprises a first binding portion and a second binding portion, and the first binding portion comprises a molecule that specifically binds to an antigen presented on a tumor.

58. The method according to claim 51, wherein the bispecific molecule includes a molecule that specifically binds to BCMA and CD3, a molecule that specifically binds to CD47 and CD20, a molecule that specifically binds to NKG2D and FLT3, or a combination thereof.

59. The method according to any one of claims 1 to 7, 9, or 11 to 50, wherein the target molecule is a nucleic acid comprising DNA, RNA (e.g., mRNA), plasmid, vector, siRNA, shRNA, antisense oligonucleotide, or any combination thereof.

60. The method according to any one of claims 1 to 7, 9, or 11 to 50, wherein the target molecule is a virus comprising an adeno-associated virus, a lentivirus, an adenovirus, or any combination thereof.

61. The method according to any one of claims 1 to 60, wherein the impurities include viruses, high molecular weight aggregates (HMW), low molecular weight aggregates (LMW), host cell proteins (HCP), residual deoxyribose nucleic acids (rDNA), residual protein A (rProA), or any combination thereof.

62. The method according to any one of claims 1 to 61, which reduces the risk of polysorbate 80 degradation in the final product.

63. A target molecule isolated by the method described in any one of claims 1 to 62.

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