WO2025038763A1 - Ceramic hydroxyapatite chromatography flow through method - Google Patents

Abstract

The present disclosure is directed methods of isolating and/or purifying a protein in a ceramic hydroxyapatite chromatography in a flow-through mode, comprising loading a sample comprising the protein and one or more impurities on a CHT columns in a flow-through mode.

Description

CERAMIC HYDROXYAPATITE CHROMATOGRAPHY FLOW THROUGH METHOD

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Application No. 63/519,751, filed August 15, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of electronically submitted sequence listing in ASCII text file (File Name: 3338_331PC01_SequenceListing_ST26; Size: 50,639 bytes; and Date of Creation: July 30, 2024) filed with the application is incorporated herein by reference in its entirety.

FIELD

[0003] The present application relates to the field of protein isolation and purification using a ceramic hydroxyapatite chromatography in a flow through mode.

BACKGROUND

[0004] The rise in popularity of higher-titer processes within the biologies development space can amplify certain challenges associated with designing and optimizing purification processes. Such challenges and obstacles include, but are not limited to, increases in consumption of raw materials such as buffers or chromatography resins, longer operating times, greater number of purification cycles, increased costs, and product stability concerns. A solution to these challenges also needs to consider maintaining the productivity of the biologies.

[0005] As such, there remains a need in the field of protein purification for methods that maximize yield and purity while reducing the cost and time required to generate the purified sample. BRIEF SUMMARY

[0006] Some aspects of the disclosure are directed to a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a flow-through (F/T) mode of a ceramic hydroxyapatite chromatography (CHT) comprising a) loading the sample on a CHT column, and b) obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

[0007] Certain aspects of the disclosure are directed to a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising a) loading the sample on a CHT column in a flow- through (F/T) mode and b) obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

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

[0009] In some aspects, the molecule of interest in the sample is at an amount that the molecule of interest is not bound to the column during and/or after the loading. In some aspects, the molecule of interest in the sample is at an amount that some molecules are bound to the column during and/or after the loading.

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

[0011] In some aspects, the molecule of interest comprises a protein. In some aspects, the molecule of interest comprises a nucleic acid. In some aspects, the molecule of interest comprises a virus.

[0012] In some aspects, the loaded sample comprises a loading amount of 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/Lresin. In some aspects, the loaded sample comprises a loading amount of between about 50 and about 500, about 50 and about 450, about 50 and about 400, about 50 and about 350, between about 50 and about 300, about 70 and about 300, about 90 and about 300, about 100 and about 300, about 120 and about 300, about 140 and about 300, about 150 and about 300, about 50 and about 250, about 70 and about 250, about 90 and about 250, about 100 and about 250, about 120 and about 250, about 140 and about 250, about 150 and about 250, about 50 and about 200, about 70 and about 200, about 90 and about 200, about 100 and about 200, about 120 and about 200, about 140 and about 200, or about 150 and about 200 g/Lresin.

[0013] In some aspects, the loading the sample on the CHT column comprises adding the sample to a loading buffer comprising a phosphate salt. In some aspects, the phosphate salt in the loading buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0014] In some aspects, the phosphate salt in the loading buffer is in an amount of 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 aspects, the phosphate salt in the loading buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM. In some aspects, the F/T composition is pooled as a final product. In some aspects, the final product is formulated.

[0015] In some aspects, the method further comprises (c) adding a chase buffer to the CHT column. In some aspects, the chase buffer comprises a phosphate salt. In some aspects, the phosphate salt in the chase buffer comprises sodium phosphate, potassium phosphate, or any combination thereof. In some aspects, the phosphate salt in the chase buffer is in an amount of 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 aspects, the phosphate salt in the chase buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM.

[0016] In some aspects, the method further comprises collecting a chase composition comprising the molecule after (c). In some aspects, the chase composition and the F/T composition are pooled as a final product. In some aspects, the final product is formulated. [0017] In some aspects, the method further comprises (d) adding an elution buffer, if a certain amount of the molecule of interest is bound to the column. In some aspects, the elution buffer comprises a phosphate salt. In some aspects, the phosphate salt in the elution buffer comprises sodium phosphate, potassium phosphate, or any combination thereof. In some aspects, the phosphate salt in the elution buffer is in an amount of 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 aspects, the phosphate salt in the elution buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM.

[0018] In some aspects, 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 aspects, 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 aspects, the pH of the loading buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH in the loading buffer is about 7.5. In some aspects, 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 aspects, 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 aspects, the pH of the chase buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH of the chase buffer is about 7.5. In some aspects, the pH of 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 aspects, 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 aspects, the pH of the elution buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH in the elution buffer is about 7.5.

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

[0020] In some aspects, the conductivity of the chase buffer is similar to or higher than the conductivity of the loading buffer. In some aspects, the conductivity of the chase buffer is similar to or lower than the conductivity of the loading buffer. In some aspects, the conductivity of the elution buffer is similar to or higher than the loading buffer. In some aspects, the conductivity of the elution buffer is similar to or lower than the loading buffer. [0021] In some aspects, wherein the molecule of interest comprises a protein comprising an antibody or 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 clotting 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 an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CDl la, 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 specifically binding to an antigen presented on a tumor. In some aspects, the bispecific molecule comprises a molecule specifically binding to BCMA and CD3, a molecule specifically binding to CD47 and CD20, a molecule specifically binding to NKG2D and FLT3, or a combination thereof.

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

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

[0028] In some aspects, the impurity comprises a virus, a high molecular weight aggregate (HMW), a low molecular weight aggregate (LMW), a host cell protein (HCP), residual deoxyribose nucleic acid (rDNA), residual protein A (rProA), or any combination thereof.

[0029] In some aspects, the methods reduce a risk of polysorbate 80 degradation in the final product. [0030] Some aspects of the disclosure are directed to a molecule of interest isolated by the methods.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0031] FIG. 1 A shows schematic diagrams of the molecules used in the present disclosure. FIG. IB shows molecular structures and information of bispecific antibodies (bsAbs) and Fc-fusion protein used in the disclosure. The figure includes molecular weight (MW) in kilodaltons (kDa), process-related impurity challenges such as host cell proteins (HCP) and residual deoxyribose nucleic acid (rDNA), and product-related impurity challenges such as low molecular weight (LMW) aggregates, high molecular weight (HMW) aggregates, and residual protein A (rProA).

[0032] FIG. 2 shows the comparison of observed load breakthrough for each bsAb and sodium phosphate condition. Columns in the figure represent CHT F/T column loading at which the load breakthrough was observed. Plotted line represents percent difference of the load breakthrough between 5 mM and 20 mM sodium phosphate conditions for each bsAb. [0033] FIGs. 3A and 3B show HMW impurity data for bsAb D. FIG. 3A shows the chromatogram of the 20 mM sodium phosphate condition for bsAb D that has the absorbance of ultraviolet light at 280 nanometers (UV 280) in milli-Absorbance Units (mAU) on the right-hand side axis, representing the concentration of protein eluting from the CHT column. This curve is sectioned by the fractions that were individually collected and tested for impurities. Overlaid on top of the curve and within each section is the HMW percentage of the load material and each respective, individual pool fraction based on size exclusion chromatography using ultra performance liquid chromatography (SEC-UPLC) results; this corresponds to the left-hand axis. The dashed slope represents the percentage of the linear sodium phosphate elution gradient. FIG. 3B shows the same HMW percentage information as the chromatogram in FIG. 3A, but for both 5 mM and 20 mM sodium phosphate conditions for bsAb D. Each column represents one individual sample, with the first being the load followed by each fraction, just like the columns overlaid on the above chromatogram. The chases of each phosphate concentration condition are sectioned to indicate when they occurred since they do not align with the same fractions collected across both runs. The chase during the 5 mM sodium phosphate run was collected with fractions 7 and 8, while the chase during the 20 mM sodium phosphate run was collected with fractions 9 and 10.

[0034] FIG. 4A shows HMW impurity data for bsAb B at both 5 mM and 20 mM sodium phosphate conditions arranged by collected fractions. FIG. 4B shows HMW impurity data for bsAb C at both 5 mM and 20 mM sodium phosphate conditions arranged by collected fractions. For both FIG. 4 A and FIG. 4B, each point corresponds to the HMW percentage measured via SEC-UPLC of each individual sample for the load and pool fractions.

[0035] FIGs. 5A, 5B, 5C, and 5D represent LMW impurity data for bsAbs A (FIG. 5A), B (FIG. 5B), C (FIG. 5C), and D (FIG. 5D), respectively, at both 5 mM and 20 mM sodium phosphate conditions arranged by collected fractions. Each point corresponds to the LMW percentage measured via non-reduced capillary electrophoreses sodium dodecyl sulfate (CAL-NR) of each individual sample for the load and pool fractions. The chases of each phosphate concentration condition for bsAb D are sectioned to indicate when they occurred since they do not align with the same fractions collected across both runs. The chase during the 5 mM sodium phosphate run of bsAb D was collected with fractions 7 and 8, while the chase during the 20 mM sodium phosphate run of bsAb D was collected with fractions 9 and 10.

[0036] FIGs. 6A and 6B show rDNA impurity data for bsAb B for both 5 mM (FIG. 6A) and 20 mM (FIG. 6B) sodium phosphate conditions arranged by collected fractions. Each point corresponds to the rDNA concentration measured via quantitative polymerase chain reaction (qPCR) of each individual sample for the load and pool fractions. Both column charts contain the same data, however the bottom chart has the y-axis adjusted to a logarithmic scale to show the lesser values more clearly.

[0037] FIGs. 7A and 7B show CHT F/T chromatograms for Fc-fusion A at both bench-scale (FIG. 7A) and 500 liter-scale (FIG. 7B). Both show consistently shaped UV280 curves regardless of scale. The bench-scale column used was 0.66 cm inner diameter (ID) x 20 cm bed height (BH). The 500 liter-scale column used was 30 cm ID x 19.1 cm BH.

DETAILED DESCRIPTION

[0038] The present disclosure provides methods of and insight on the benefits of the flow- through (F/T) mode of operation approach for a mixed-mode ceramic hydroxyapatite (CHT) chromatography with complex biologies in the downstream purification process. Flow-through CHT chromatography allows for the purification of a range of complex biologies including bispecific antibodies (bsAbs) and Fc-fusion proteins with significantly higher-productivity than in a traditional bind and elute (BZE) mode of operation. Additionally, these results demonstrate increased stability of product pools via risk mitigation in polysorbate 80 (PS80) degradation, and robust viral clearance capabilities by showcasing significant surrogate viral clearance across multiple surrogate models by up to 3 log reduction values (LRV).

[0039] In some aspects, a greater column loading approach was applied to CHT resin by forgoing the traditional mode of operation for this resin, BZE, and applying the F/T mode of operation, which is atypical in the context of CHT. The results of the present disclosure indicate efficient separation and purification of therapeutic proteins from process- and product-related impurities on par with BZE, while still adopting significantly increased productivity afforded by the F/T mode of operation.

[0040] Some aspects of the present disclosure are directed to methods of isolating a molecule of interest in a sample comprising the molecule and an impurity in a flow-through mode of a ceramic hydroxyapatite chromatography (CHT) comprising: a) loading the sample with a CHT column; and b) obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

[0041] Some aspects of the present disclosure are directed to methods of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising: a) loading the sample with a CHT column in a flow- through mode; and b) obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

I. Terms

[0042] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0043] The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple." [0044] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: 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).

[0045] The terms "about" or "comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, z.e., the limitations of the measurement system. For example, "about" or "comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of' can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of' should be assumed to be within an acceptable error range for that particular value or composition.

[0046] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.

[0047] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately" refers to 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) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0048] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. 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 one of skill with a general dictionary of many of the terms used in this disclosure.

[0050] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined are more fully defined by reference to the specification in its entirety.

[0051] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0052] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the degree of purity of a protein of interest from a composition or sample comprising the protein of interest and one or more impurities. Typically, the degree of purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition. In some aspects, the protein of interest is a first charge variant of a protein, e.g., a charge variant of an antibody, and the one or more impurities comprises a second charge variant of the same protein.

[0053] The term "chromatography," as used herein, refers to a dynamic separation technique, which separates a target molecule such as a target protein (e.g. , a charge variant of a protein, e.g., an antibody) from other molecules in the mixture (e.g., other charge variants) and allows it to be isolated. Typically, in a chromatography method, a liquid mobile phase transports a sample containing the target molecule of interest across or through a stationary phase (normally solid) medium. Differences in partition or affinity to the stationary phase causes the temporary binding of selected molecules to the stationary phase while the mobile phase carries different molecules out at different times.

[0054] The term "flow-through" or "F/T" refers to chromatography process wherein a protein of interest in sample comprising the protein is passed through chromatography columns instead of binding to the column and then being eluted from the columns. To the contrary, the term “bind-elute” or “B/E” means that the protein in the sample is loaded on chromatography columns and allowed to bind to the columns. The protein of interest that are bound to the columns can be unbound by applying certain conditions and can be eluted for collection.

[0055] The term “ceramic hydroxyapatite chromatography” or “CHT” refers to a mixedmode chromatography that utilizes ceramic hydroxyapatite as the chromatography columns. The ions present on the surface of hydroxyapatite make it an ideal candidate with unique selectivity, separation and purification of biomolecule mixtures. The combined presence of calcium ions (C- sites) and phosphate sites (P-sites) provide metal affinity and ion exchange properties respectively. The C-sites on the surface of the resin undergo metal affinity interactions with phosphate or carboxyl groups present on the proteins. Concurrently, these positively charged C-sites tend to repel positively charged functional groups (e.g., amino groups) on proteins. P-sites undergo cationic exchange with positively charged functional groups on proteins. They exhibit electrostatic repulsion with negatively charged functional groups on proteins. For the elution of molecules buffer with high concentration of phosphate and sodium chloride is used. The nature of different charged ions on the surface of hydroxyapatite provides the framework for unique selectivity and binding of proteins, facilitating robust separation of proteins.

[0056] The term "loading" as used herein, refers to applying a solution, e.g., a mixture comprising a protein product and a contaminant, as described herein, to a chromatography matrix. In some embodiments, the term "loading" is synonymous with "contacting" a solution onto a chromatography column.

[0057] The term "is applied to," when used in the context of a gradient being applied to a chromatography matrix, broadly means that a gradient is formed, directly or indirectly, within and/or around a chromatography matrix. In some aspects, the chromatography matrix is present in a column, and the gradient is formed within the column. In some aspects, a gradient that is applied to a chromatography matrix is formed internally within a column, as opposed to a gradient which is formed externally and then added to a column. In certain aspects, a gradient that is applied to the chromatography matrix forms within a column as a result of more than one buffer being added to the chromatography matrix. In other aspects, a gradient that is applied to the chromatography matrix is formed externally and then added to the column.

[0058] The terms "culture", "cell culture" and "eukaryotic cell culture" as used herein refer to a cell population, either surface-attached or in suspension that is maintained or grown in a medium (see definition of "medium" below) under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein can refer to the combination comprising the cell population and the medium in which the population is suspended.

[0059] As used herein, the terms "expression" or "expresses" are used to refer to transcription and translation occurring within a cell. The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell, or both.

[0060] The term "antibody" refers, in some aspects, to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, e.g., naturally- occurring IgG antibodies, the heavy chain constant region is comprised of a hinge and three domains, CHI, CH2 and CH3. In some antibodies, e.g., naturally-occurring IgG antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "antibody" can include a bispecific antibody or a multispecific antibody.

[0061] An "IgG antibody", e.g., a human IgGl, IgG2, IgG3 and IgG4 antibody, as used herein has, in some aspects, the structure of a naturally-occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally-occurring IgG antibody of the same subclass. For example, an IgGl, IgG2, IgG3 or IgG4 antibody may consist of two heavy chains (HCs) and two light chains (LCs), wherein the two HCs and LCs are linked by the same number and location of disulfide bridges that occur in naturally-occurring IgGl, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bridges).

[0062] An immunoglobulin can be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. Immunoglobulins, e.g., IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" includes, by way of example, both naturally-occurring and non-naturally-occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies and wholly synthetic antibodies.

[0063] The term "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0064] The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as

(a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom,

(b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.

[0065] As used herein, "isotype" refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.

[0066] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0067] As used herein, the term “protein”, “protein of interest”, or "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The terms "polypeptide" or "protein" or “protein of interest” or "product" or "product protein" or "amino acid residue sequence" are used interchangeably. The term "polypeptide" refers to any chain or 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 a molecule comprised of one or more polypeptides, which can in some instances be associated by bonds other than amide bonds. On the other hand, a protein can also be a single polypeptide chain. In this latter instance the single polypeptide chain can in some instances comprise two or more polypeptide subunits fused together to form a protein. The terms "polypeptide" and "protein" also refer to the products of post-expression modifications, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide or protein can be derived from a natural biological source or produced by recombinant technology. [0068] The terms "polynucleotide" or "nucleotide" as used herein are intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA, cDNA, or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA or RNA, which has been removed from its native environment, for example, a recombinant polynucleotide encoding an antigen binding protein contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

[0069] The term "impurity" or "impurities," as used herein, refers to one or more molecule, e.g., polypeptide, nucleic acid molecule, small molecule, or any combination thereof, present in a mixture with a target molecule, e.g., a target species of a polypeptide, e.g., a target charge variant of a polypeptide. In some aspects, the impurity is a different polypeptide, e.g., a polypeptide that has a different structure, sequence, or function than a target polypeptide. In some aspects, the impurity is a different species of a target polypeptide, e.g., a LMW species, or a HMW species. In some aspects, the impurity is a host cell protein, fragments of a protein of interest, a virus, or any other substances in a sample to be purified that are not desirable.

[0070] The term "purity," as used herein, refers to the degree to which a composition, e.g., a sample or solution comprising a target polypeptide, comprises one or more impurities. For example, a solution comprising a target polypeptide wherein 98% of the substance in the solution is a protein of interest, and 2% of the substance is impurities has a purity of 98%.

II. Methods of the Disclosure

[0071] Some aspects of the present disclosure are directed to methods of isolating a molecule of interest in a sample comprising the molecule and an impurity in a flow-through (F/T) mode of a ceramic hydroxyapatite chromatography (CHT) comprising: a) loading the sample on a CHT column; and b) obtaining a F/T composition comprising the molecule. In some aspects, the F/T composition after the F/T comprises the molecule of interest and a lower amount of the impurity compared to the sample prior to the loading.

[0072] Some aspects of the present disclosure are directed to methods of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising: a) loading the sample with a CHT column in a flow- through (F/T) mode; and b) obtaining a F/T composition comprising the molecule. In some aspects, the F/T composition comprises the molecule and a lower amount of the impurity compared to the sample prior to the loading.

[0073] CHT resin has two different types of sites distributed throughout the matrix’s crystal structure: positively charged calcium and negatively charged phosphoryl groups. The calcium sites interact with negatively charged carboxyl groups by forming a covalent bond stronger than regular ion interactions by upwards of 15 to 60 times; this is known as a metal affinity interaction, which primarily affects acidic proteins and can be weakened by adding buffering salts such as phosphate in the mobile phase. Alternatively, phosphoryl groups interact with positively charged amine groups via the normal ionic attraction of cation exchange; this primarily affects basic proteins and can be weakened by increasing pH, adding neutral salts such as chloride or buffering salts like phosphate into the mobile phase. The metal affinity and cation exchange interactions respective of the calcium and phosphoryl sites directly compete with each other, as the positive charge of the calcium sites repel the amine groups sharing the same charge similarly to and simultaneously as the negative charge of the phosphoryl sites repelling the negative carboxyl groups. Additionally, while increasing the concentration of chloride (or other neutral salts) in the mobile phase only weakens the cation exchange interactions and does not weaken metal affinity interactions, it can also increase the strength of the metal affinity interactions by ionically shielding the repulsive, negatively charged phosphoryl sites of the resin.

[0074] In some aspects, the methods disclosed herein result in increased purity of the molecule of interest, as compared to conventional methods. In some aspects, the methods disclosed herein result in increased purity of the molecule of interest, as compared to bind and elute (BZE) methods. In some aspects, the purity of the molecule of interest is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, as compared to BZE methods.

[0075] In some aspects, the methods disclosed herein result in increased stability of the molecule of interest, as compared to conventional methods. In some aspects, the methods disclosed herein result in increased stability of the molecule of interest, as compared to BZE methods. In some aspects, the stability of the molecule of interest is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, as compared to BZE methods. In some aspects, the methods reduce the risk of polysorbate 80 degradation in the sample.

[0076] In some aspects, the methods disclosed herein have increased productivity relative to conventional methods, e.g, BZE methods. In some aspects the productivity is increased by at least about at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 30-fold, relative to conventional methods (e.g, BZE methods).

[0077] In some aspects, the methods disclosed herein maintain the productivity of the molecule of interest relative to conventional methods, e.g., BZE methods, while reducing the amount of impurities better than the conventional methods, e.g., BZE methods.

[0078] In some aspects, the CHT is a polishing step. In some aspects, the polishing step for isolating the molecule of interest comprises two polishing steps, one being the CHT and another being a different chromatography, i.e., a chromatography other than a CHT. In some aspects, the polishing step for isolating the molecule of interest comprises two polishing steps, one being the CHT and another being a second CHT, wherein the two CHTs are identical. In some aspects, the two CHTs are different. In some aspects, the final product after the polishing step is formulated as a drug product.

[0079] In some aspects, the method further comprises, before or after the CHT of the present disclosure, an affinity chromatography, an ion exchange chromatography, a cation exchange chromatography, an anion exchange chromatography, a hydrophobic interaction chromatography, a filtration, or any combination thereof.

[0080] In some aspects, the method further comprises subjecting the isolated molecule of interest to one or more analytical characterization. As such, some aspects of the present disclosure are directed to methods for enriching a molecule of interest for analytical characterization, comprising: (a) separating the molecule of interest from a mixture comprising the molecule of interest and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode in a chromatographic separation system; and (b) subjecting the molecule of interest from (a) to analytical characterization. Some aspects of the present disclosure are directed to methods for conducting analytical characterization of a molecule of interest, comprising: (a) separating the molecule of interest from a mixture comprising the molecule of interest and one or more impurities, comprising contacting the mixture with two or more chromatography columns in a continuous operation mode in a chromatographic separation system; and (b) conducting analytical characterization of the molecule of interest from (a).

[0081] In some aspects, the analytical characterization comprises a HPLC system, capillary isoelectric focusing (cIEF) gel electrophoresis, imaged capillary isoelectric focusing (iCIEF), cation exchange chromatography (CEX), anion exchange chromatography (AEX), MFI, SEC-MALS, SEC, mass spectrometry, or any combination thereof.

[0082] In some aspects, the amount of molecule of interest loaded on the column is an amount that does not allow any of the molecule of interest to bind to the CHT column: e.g., the amount of molecule of interest loaded on the CHT column is low that no significant amount of the molecule of interest binds to the CHT column. In some aspects, the amount of the molecule of interest loaded on the CHT column is an amount that does not exceed the dynamic binding capacity (DBC) of the column for the molecule of interest. As used herein, the term “dynamic binding capacity” or “DBC” of a chromatography column describes the maximum amount of target molecule that you can load onto your column without causing unnecessary loss, measured under realistic experimental conditions (default flow-rate, real protein sample).

[0083] In some aspects, the disclosure includes a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising: [0084] (a) loading the sample on a CHT column in a flow-through (F/T) mode;

[0085] (b) obtaining a F/T composition comprising the molecule, wherein only the F/T composition is pooled as a final product. In some aspects, the F/T composition comprises the molecule and a lower amount of the impurity compared to the sample prior to the loading.

[0086] In some aspects, the methods of the present disclosure includes a chase step. In some aspects, the chase step is optional. In some aspects, the composition collected in the chase step can be pooled as a final product. In some aspects, the disclosure includes a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising:

[0087] (a) loading the sample on a CHT column in a flow-through (F/T) mode;

[0088] (b) obtaining a F/T composition comprising the molecule;

[0089] (c) adding a chase buffer in the CHT column; and

[0090] (d) obtaining a chase composition comprising the molecule, wherein the F/T composition and the chase composition are pooled as a final product. In some aspects, the F/T composition and/or the chase composition comprises the molecule and a lower amount of the impurity compared to the sample prior to the loading and/or chasing, respectively.

[0091] In some aspects, the amount of molecule of interest loaded on the column is an amount that allows some of the molecule of interest to bind to the CHT column: e.g., the amount of molecule of interest loaded on the CHT column is high that a significant amount of the molecule of interest binds to the CHT column. In some aspects, the amount of molecule of interest loaded on the column is an amount that exceeds the dynamic binding capacity (DBC) of the column for the molecule of interest.

[0092] Therefore, in some aspects of the disclosure, especially when the amount of the molecule of interest added on the CHT column is high enough to allow any significant amount of the molecule of interest to be bound to the column, the methods of the present disclosure comprise an elution step to obtain the molecule of interest that is bound to the CHT column to be collected in a final product. In some aspects, the disclosure includes a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising:

[0093] (a) loading the sample on a CHT column in a flow-through (F/T) mode;

[0094] (b) obtaining a F/T composition comprising the molecule; [0095] (c) adding an elution buffer in the CHT column; and

[0096] (d) obtaining an elution composition comprising the molecule, wherein the F/T composition and the elution composition are pooled as a final product. In some aspects, the method further comprises optionally adding a chase buffer between (b) and (c) on the CHT column and collecting a chase composition comprising the molecule.

[0097] In some aspects, the disclosure includes a method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising:

[0098] (a) loading the sample on a CHT column in a flow-through (F/T) mode;

[0099] (b) obtaining a F/T composition comprising the molecule;

[0100] (c) adding a chase buffer in the CHT column;

[0101] (d) obtaining a chase composition comprising the molecule;

[0102] (e) adding an elution buffer in the CHT column; and

[0103] (f) obtaining an elution composition comprising the molecule, wherein the F/T composition, the chase composition, and the elution composition are pooled as a final product.

[0104] In some aspects, the loaded sample comprises a loading amount of 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/Lresin.

[0105] In some aspects, the loaded sample in the present methods comprises a loading amount of between about 50 and about 500, about 50 and about 450, about 50 and about 400, about 50 and about 350, between about 50 and about 300, about 70 and about 300, about 90 and about 300, about 100 and about 300, about 120 and about 300, about 140 and about 300, about 150 and about 300, about 50 and about 250, about 70 and about 250, about 90 and about 250, about 100 and about 250, about 120 and about 250, about 140 and about 250, about 150 and about 250, about 50 and about 200, about 70 and about 200, about 90 and about 200, about 100 and about 200, about 120 and about 200, about 140 and about 200, or about 150 and about 200 g/Lresin.

[0106] In some aspects, the loaded sample in the present methods comprises a loading amount 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/Lresin.

[0107] In some aspects, the loaded sample in the present methods comprises a loading amount of about 200 g/Lresin. In some aspects, the loaded sample in the present methods comprises a loading amount of about 150 g/Lresin. In some aspects, the loaded sample in the present methods comprises a loading amount of about 250 g/Lresin. In some aspects, the loaded sample in the present methods comprises a loading amount of about 300 g/Lresin.

[0108] In some aspects, the methods of the present disclosure can further comprise a washing step. In some aspects, the methods of the disclosure can include any other steps that is necessary.

A. Buffers

[0109] The methods disclosed herein utilizes various buffers: loading buffer, chase buffer, and/or elution buffer as necessary. In some aspects, the buffers in various steps (e.g., loading, chasing, or eluting) can be the same. In some aspects, the buffers in different steps can be different. In some aspects, the pH of various buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) can be the same. In some aspects, the pH of various buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) can be different. In some aspects, the conductivity of various buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) can be the same. In some aspects, the conductivity of various buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) can be different.

[0110] In some aspects, the buffers comprise a phosphate salt. In some aspects, the amount of the phosphate salt in the buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) is the same. In some aspects, the buffers comprise a phosphate salt. In some aspects, the amount of the phosphate salt in the buffers (e.g., loading buffer, chase buffer, elution buffer, and/or wash buffer) is different.

Loading Buffer:

[OHl] In some aspects, loading the sample on the CHT column comprises adding the sample to a loading buffer comprising a phosphate salt. In some aspects, the phosphate salt in the loading buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0112] In some aspects, the phosphate salt in the loading buffer is in an amount of 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.

[0113] In some aspects, the phosphate salt in the loading buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM. In some aspects, the phosphate salt in the loading buffer is in an amount of 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 aspects, the phosphate salt in the loading buffer is in an amount between about 20 mM and about 50 mM.

[0114] In some aspects, 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 aspects, 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 aspects, the pH of the loading buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH in the loading buffer is about 7.5.

[0115] In some aspects, the loading buffer comprises a chloride at a concentration less than about 500, less than about 450, less than about 400, less than about 350, less than about 300, less than about 250, less than about 200, less than about 190, less than about 180, less than about 170, less than about 160, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, or less than about 100 mM. In some aspects, the loading buffer comprises a chloride at a concentration less than about 100 mM, less than about 90 mM, less than about 80 mM, less than about 70 mM, less than about 60 mM, or less than about 50 mM. In some aspects, the loading buffer comprises no chloride.

Chase Buffer:

[0116] In some aspects, the method of the disclosure comprises adding a chase buffer on the CHT column after the loading, wherein the chase buffer comprises a phosphate salt. In some aspects, the phosphate salt in the chase buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0117] In some aspects, the phosphate salt in the chase buffer is in an amount of 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.

[0118] In some aspects, the phosphate salt in the chase buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM. In some aspects, the phosphate salt in the loading buffer is in an amount of 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 aspects, the phosphate salt in the chase buffer is in an amount between about 20 mM and about 50 mM.

[0119] In some aspects, 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 aspects, the pH in 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 aspects, the pH of the chase buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH in the chase buffer is about 7.5.

[0120] In some aspects, the chase buffer comprises a chloride at a concentration less than about 500, less than about 450, less than about 400, less than about 350, less than about 300, less than about 250, less than about 200, less than about 190, less than about 180, less than about 170, less than about 160, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, or less than about 100 mM. In some aspects, the chase buffer comprises a chloride at a concentration less than about 100 mM, less than about 90 mM, less than about 80 mM, less than about 70 mM, less than about 60 mM, or less than about 50 mM. In some aspects, the chase buffer comprises no chloride.

Elution Buffer:

[0121] In some aspects, the method of the disclosure comprises adding an elution buffer on the CHT column after the loading or chasing, wherein the elution buffer comprises a phosphate salt. In some aspects, the phosphate salt in the elution buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0122] In some aspects, the phosphate salt in the elution buffer is in an amount of 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.

[0123] In some aspects, the phosphate salt in the elution buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM. In some aspects, the phosphate salt in the elution buffer is in an amount of 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 aspects, the phosphate salt in the elution buffer is in an amount between about 20 mM and about 50 mM.

[0124] In some aspects, 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 aspects, 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 aspects, the pH of the elution buffer is between about pH 6.3 and about pH 7.7. In some aspects, the pH in the elution buffer is about 7.5. [0125] In some aspects, the elution buffer comprises a chloride at a concentration less than about 500, less than about 450, less than about 400, less than about 350, less than about 300, less than about 250, less than about 200, less than about 190, less than about 180, less than about 170, less than about 160, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, or less than about 100 mM. In some aspects, the elution buffer comprises a chloride at a concentration less than about 100 mM, less than about 90 mM, less than about 80 mM, less than about 70 mM, less than about 60 mM, or less than about 50 mM. In some aspects, the elution buffer comprises no chloride.

B. Sample

[0126] In some aspects, the sample that is subject to the methods of the present disclosure comprises a molecule of interest to be isolated or purified and one or more impurities. In some aspects, the impurities in the sample is in a traceable amount that requires a polishing step.

Molecule of Interest

[0127] In some aspects, a molecule of interest that is to be isolated or purified can be any molecule with a biological activity. In some aspects, the molecule of interest is a nucleic acid, a protein, a virus, or any combination thereof. In some aspects, the molecule of interest is a nucleic acid. In some aspects, the molecule of interest comprises which comprises DNA, RNA (e.g., mRNA), plasmid, vector, siRNA, shRNA, antisense oligonucleotide, or any combination thereof. In some aspects, the molecule of interest comprises a vector.

[0128] In some aspects, a molecule of interest that is to be isolated or purified can be a virus or a viral vector. In some aspects, the molecule of interest comprises an adeno-associated virus, a lentivirus, an adenovirus, or any combination thereof. In some aspects, the molecule of interest comprises a retrovirus. In some aspects, the molecule of interest for the present methods comprises an adeno-associated virus (AAV). In some aspects, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrhlO, AAV10, AAVRH10, AAV11, and AAV12.

[0129] In some aspects, the molecule of interest for the present methods comprises a lentivirus. [0130] In some aspects, the molecule of the interest has been subjected to a prior purification process before being subjected to the methods disclosed herein. In some aspects the molecule of the interest has been subjected to, e.g., partially purified by, a prior affinity chromatography. In some aspects, the prior affinity chromatography comprises a protein A affinity chromatography.

[0131] In some aspects, the methods disclosed herein can be used to isolate and/or purify any polypeptide. In some aspects, the polypeptide is a protein.

[0132] In some aspects, the molecule of interest comprises a protein comprising an antibody or 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 clotting factor, a chemokine, an enzyme, a hormone, or any combination thereof. In some aspects, the molecule of interest can be an antibody or antigen-binding portion thereof prior to a conjugation to a drug. In some aspects, the molecule of interest can be an antibody drug conjugate after the conjugation.

[0133] In some aspects, the molecule of interest comprises a fusion protein. In some aspects, the fusion protein comprises an Fc-fusion protein, an albumin fusion protein, or any combination thereof. In some aspects, the molecule of interest comprises an immunoglobulin component fused to a biologically active polypeptide. In some aspects, the immunoglobulin component comprises a fragment of an antibody. In some aspects, the immunoglobulin component comprises a fragment of the constant region of an antibody. In some aspects, the immunoglobulin component comprises an Fc.

[0134] In some aspects, the molecule of interest comprises an immunoglobulin fused to a growth factor, a clotting factor, a cytokine, a chemokine, an enzyme, a hormone, or any combination thereof. In some aspects, the molecule of interest comprises an Fc fused to a growth factor. In some aspects, the molecule of interest comprises an Fc fused to an interleukin.

[0135] In some aspects, the molecule of interest 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, Granulocytemacrophage colony-stimulating factor, IL5, or any combination thereof. In some aspects, the molecule of interest comprises an Fc fusion protein comprising TGF-beta. [0136] In some aspects, the molecule of interest is an immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor is PD-1, PD-L1, CTLA-4, LAG-3, or any combination thereof.

[0137] In some aspects, the molecule of interest comprises an antibody or an antigen-binding portion thereof, including but not limited to, a bispecific molecule or a multispecific molecule comprising the antibody or antigen-binding portion thereof. In some aspects, the antibody or antigen-binding portion thereof binds a tumor antigen. In some aspects, the antibody or antigen-binding portion thereof binds a checkpoint inhibitor. In some aspects, the antibody or antigen-binding portion thereof binds an antigen selected from PD-1, PD- Ll, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD1 la, tissue factor (TF), MICA/B PSCA, IL-8, EGFR, HER3, HER4, BCMA, CD3, CD47, NKG2D, FLT3, and any combination thereof.

[0138] In some aspects, the antibody or antigen-binding portion thereof specifically binds PD-1. Various human monoclonal antibodies that bind specifically to PD-1 with high affinity have been disclosed in U.S. Patent Nos. 8,008,449, 6,808,710, 7,488,802, 8,168,757 and 8,354,509, US Publication No. 2016/0272708, and PCT Publication Nos. WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2017/025016, WO 2017/106061, WO 2017/19846, WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540, each of which is incorporated by reference in its entirety.

[0139] In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as toripalimab; see Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), BGB-A317 (Beigene; also known as Tislelizumab; see WO 2015/35606 and US 2015/0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et al., J. Hematol. Oncol. 10X36 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics, see WO 2017/19846), BCD-100 (Biocad; Kaplon et al., mAbs 70(2): 183-203 (2018), and IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540).

[0140] In one aspect, the anti-PD-1 antibody is nivolumab. In another aspect, the anti-PD-1 antibody is pembrolizumab.

[0141] In some aspects, the antibody or antigen-binding portion thereof specifically binds PD-L1. Examples of anti-PD-Ll antibodies include, but are not limited to, the antibodies disclosed in US Patent No. 9,580,507. In certain aspects, the anti-PD-Ll antibody is selected from the group consisting of BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013/173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see US 8,217,149; see, also, Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDI-4736; see WO 2011/066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO 2013/079174), STI-1014 (Sorrento; see WO2013/181634), CX-072 (Cytomx; see WO2016/149201 ), KN035 (3D Med/Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017/034916), BGB-A333 (BeiGene; see Desai et al., JCO 36 (15suppl):TPS3113 (2018)), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0142] In certain aspects, the PD-L1 antibody is atezolizumab (TECENTRIQ®). In certain aspects, the PD-L1 antibody is durvalumab (IMFINZI™). In certain aspects, the PD-L1 antibody is avelumab (BAVENCIO®).

[0143] In some aspects, the antibody or antigen-binding portion thereof specifically binds CTLA-4. Human monoclonal antibodies that bind specifically to CTLA-4 with high affinity have been disclosed in U.S. Patent Nos. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described in, for example, U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121 and International Publication Nos. WO 2012/122444, WO 2007/113648, WO 2016/196237, and WO 2000/037504, each of which is incorporated by reference herein in its entirety. In certain aspects, the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Patent No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016/196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In particular aspects, the anti-CTLA-4 antibody is ipilimumab. In particular aspects, the CTLA-4 antibody is tremelimumab. In particular aspects, the CTLA-4 antibody is MK-1308. In particular aspects, the CTLA-4 antibody is AGEN-1884.

[0144] In some aspects, the antibody or antigen-binding portion thereof specifically binds LAG-3. Antibodies that bind to LAG-3 have been disclosed in Int'l Publ. No.

WO/2015/042246 and U.S. Publ. Nos. 2014/0093511 and 2011/0150892, each of which is incorporated by reference herein in its entirety. Non-limiting examples of anti-LAG-3 antibodies include but are not limited to 25F7 (described in U.S. Publ. No. 2011/0150892), BMS-986016, IMP731 (H5L7BW), MK-4280 (28G-10), REGN3767, humanized BAP050,

IMP-701 (LAG-5250), TSR-033, BI754111, MGD013, or FS-118. These and other anti- LAG-3 antibodies useful in the claimed invention can be found in, for example

WO20 16/028672, W02017/106129, WO2017/062888, W02009/044273

WO20 18/069500, WO2016/126858, WO2014/179664, WO2016/200782 WO20 15/200119, WO2017/019846, WO2017/198741, WO2017/220555 WO20 17/220569, WO2018/071500, W02017/015560, WO2017/025498 WO2017/087589, WO20 17/087901, W02018/083087, WO2017/149143

WO20 17/219995, US2017/0260271, WO2017/086367, WO2017/086419

WO2018/034227, and W02014/140180, each of which is incorporated by reference herein in its entirety.

[0145] In some aspects, the antibody or antigen-binding portion thereof specifically binds CD137. Antibodies that bind to CD137 have been disclosed in U.S. Publ. No.

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

[0146] In some aspects, the antibody or antigen-binding portion thereof specifically binds KIR. Examples of anti-KIR antibodies have been disclosed in Int'l Publ. Nos. WO/2014/055648, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO 2012/071411 and WO/2012/160448, each of which is incorporated by reference herein in its entirety. One anti-KIR antibody useful in the present disclosure is lirilumab (also referred to as BMS-986015, IPH2102, or the S241P variant of 1-7F9), first described in Int'l Publ. No. WO 2008/084106. An additional anti-KIR antibody useful in the present disclosure is 1- 7F9 (also referred to as IPH2101), described in Int'l Publ. No. WO 2006/003179.

[0147] In some aspects, the antibody or antigen-binding portion thereof specifically binds GITR. Examples of anti-GITR antibodies have been disclosed in Int'l Publ. Nos. WO/2015/031667, WO2015/184,099, WO2015/026,684, WO 11/028683 and

WO/2006/105021, U.S. Pat. Nos. 7,812,135 and 8,388,967 and U.S. Publ. Nos. 2009/0136494, 2014/0220002, 2013/0183321 and 2014/0348841, each of which is incorporated by reference herein in its entirety. In one aspect, an anti-GITR antibody useful in the present disclosure is TRX518 (described in, for example, Schaer et al. Curr Opin Immunol. (2012) Apr; 24(2): 217-224, and WO/2006/105021). In another aspect, the anti- GITR antibody is selected from MK4166, MK1248, and antibodies described in WO1 1/028683 and U.S. 8,709,424. In certain aspects, an anti-GITR antibody is an anti- GITR antibody that is disclosed in WO2015/031667. In certain aspects, an anti-GITR antibody is an anti-GITR antibody that is disclosed in WO2015/184099, e.g., antibody Hum231#l or Hum231#2, or the CDRs thereof, or a derivative thereof (e.g., pabl967, pab!975 or pab!979). In certain aspects, an anti-GITR antibody is an anti-GITR antibody that is disclosed in JP2008278814, W009/009116, WO2013/039954, US20140072566, US20140072565, US20140065152, or WO2015/026684, or is INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (Medlmmune). In certain aspects, an anti-GITR antibody is an anti-GITR antibody that is described in PCT/US2015/033991 (e.g., an antibody comprising the variable regions of 28F3, 18E10 or 19D3).

[0148] In some aspects, the antibody or antigen-binding portion thereof specifically binds TIM3. In some aspects, the anti-TIM3 antibody is selected from the anti-TIM3 antibodies disclosed in Int'l Publ. Nos.WO2018013818, WO/2015/117002 (e.g., MGB453, Novartis), WO/2016/161270 (e.g., TSR-022, Tesaro/AnaptysBio), WO2011155607,

WO2016/144803 (e.g., STI-600, Sorrento Therapeutics), WO2016/071448, WO17055399; W017055404, WO17178493, WO18036561, W018039020 (e.g., Ly-3221367, Eli Lilly), WO2017205721, WO17079112; WO17079115; WO17079116, WO11159877,

W013006490, W02016068802 W02016068803, WO20 16/111947, and

WO/2017/031242, each of which is incorporated by reference herein in its entirety.

[0149] In some aspects, the antibody or antigen-binding portion thereof specifically binds 0X40 (also known as CD 134, TNFRSF4, ACT35 and/or TXGP1L). In some aspects, the anti-OX40 antibody is BMS-986178 (Bristol-Myers Squibb Company), described in Int'l Publ. No. WO20160196228. In some aspects, the anti-OX40 antibody is selected from the anti-OX40 antibodies described in Int'l Publ. Nos. WO95012673, WO199942585, WO14148895, WO15153513, WO15153514, WO13038191, WO16057667,

WO03 106498, WO12027328, WO13028231, W016200836, WO 17063162, WO17134292, WO 17096179, WO 17096281, and WO 17096182, each of which is incorporated by reference herein in its entirety.

[0150] In some aspects, the antibody or antigen-binding portion thereof specifically binds NKG2A. In some aspects, the anti-NKG2A antibody is BMS-986315. In some aspects, the anti-NKG2A antibody is selected from the anti-NKG2A antibodies described in, for example, WO 2006/070286 (Innate Pharma S.A.; University of Genova); U.S. Patent No. 8,993,319 (Innate Pharma S.A.; University of Genova); WO 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); WO 2008/009545 (Novo Nordisk A/S); US. Patent Nos. 8,206,709; 8,901,283; 9,683,041 (Novo Nordisk A/S); WO 2009/092805 (Novo Nordisk A/S); U.S. Patent Nos. 8,796,427 and 9,422,368 (Novo Nordisk A/S); WO 2016/134371 (Ohio State Innovation Foundation); WO 2016/032334 (Janssen); WO 2016/041947 (Innate); WO 2016/041945 (Academisch Ziekenhuis Leiden H.O.D.N. LUMC); WO 2016/041947 (Innate Pharma); and WO 2016/041945 (Innate Pharma), each of which is incorporated by reference herein in its entirety.

[0151] In some aspects, the antibody or antigen-binding portion thereof specifically binds ICOS In some aspects, the anti-ICOS antibody is BMS-986226. In some aspects, the anti- ICOS antibody is selected from anti-ICOS antibodies described in, for example, WO 2016/154177 (Jounce Therapeutics, Inc.), WO 2008/137915 (Medlmmune), WO 2012/131004 (INSERM, French National Institute of Health and Medical Research), EP3147297 (INSERM, French National Institute of Health and Medical Research), WO 2011/041613 (Memorial Sloan Kettering Cancer Center), EP 2482849 (Memorial Sloan Kettering Cancer Center), WO 1999/15553 (Robert Koch Institute), U.S. Patent Nos. 7,259,247 and 7,722,872 (Robert Kotch Institute); WO 1998/038216 (Japan Tobacco Inc.), US. Patents. 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 WO 2017/220988 (Kymab Limited), each of which is incorporated by reference herein in its entirety.

[0152] In some aspects, the antibody or antigen-binding portion thereof specifically binds TIGIT. In some aspects, the anti-TIGIT antibody is BMS-986207. In some aspects, the anti-TIGIT antibody is clone 22G2, as described in WO 2016/106302. In some aspects, the anti-TIGIT antibody is MTIG7192A/RG6058/RO7092284, or clone 4.1D3, as described in WO 2017/053748. In some aspects, the anti-TIGIT antibody is selected from the anti- TIGIT antibodies described in, for example, WO 2016/106302 (Bristol-Myers Squibb Company) and WO 2017/053748 (Genentech).

[0153] In some aspects, the antibody or antigen-binding portion thereof specifically binds CSF1R. In some aspects, the anti-CSFIR antibody is an antibody species disclosed in any of international publications WO2013/132044, W02009/026303, WO2011/140249, or W02009/112245, such as cabiralizumab, RG7155 (emactuzumab), AMG820, SNDX 6352 (UCB 6352), CXIIG6, IMC-CS4, JNJ-40346527, MCS110, or the anti-CSFIR antibody in the methods is replaced with an anti-CSFIR inhibitor or anti-CSFl inhibitor such as BLZ- 945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486, ARRY-382, or PLX-73086. [0154] In some aspects, the molecule of interest comprises a bispecific molecule. In some aspects, the bispecific or multispecific molecule comprises a first binding moiety and a second binding moiety, wherein the first binding moiety comprises a molecule specifically binding to an antigen presented on a tumor. In some aspects, a second binding moiety in the bispecific or multispecific molecule comprises a molecule specifically binding to a protein expressed on an immune cell, e.g., T cell. In some aspects, the first binding moiety in the bispecific or multispecific molecule comprises a molecule specifically binding to an antigen on a tumor and the second binding moiety in the bispecific or multispecific molecule comprises a molecule specifically binding to a protein expressed on an immune cell, e.g., T cell. In some aspects the protein expressed on an immune cell, e.g., T cell, comprises CD3. In some aspects, the molecule of interest to be isolated or purified comprises any one or more structures in FIGs. 1 A and IB.

[0155] Bispecific antibodies present several challenges in downstream processing. The impurities associated with bispecific antibody products, such as mispaired species, half antibodies, antibody fragments, and aggregates, are difficult to eliminate due to their similar size and physicochemical properties compared to the target product. Ingavat et al., Bioresour Bioprocess. 2023 Dec 13;10(l):93. Additionally, engineered bispecific antibodies are known to be more prone to aggregation, resulting in less stability compared to their parental mAbs. Id. Given the differences between mAbs and bsAbs, traditional purification methods used for mAbs may not be as effective for bsAbs. Id. Furthermore, "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(l):98; Chen et al., Bioresour Bioprocess. 2022;9(l):72; Kimerer et al., J Chromatogr A. 2019; 1601 : 121— 132. This issue is exacerbated when bispecific antibodies are subjected to high loading conditions, which can significantly impact productivity by necessitating lower loading levels to reduce aggregation.

[0156] Therefore, in some aspects, a present method provides a method of purifying a bispecific antibody in a sample comprising the bispecific antibody and an impurity comprising (1) loading the sample on a CHT column, and (2) obtaining a flow-through composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading. [0157] In some aspects, a present method provides a method of purifying a bispecific antibody in a sample comprising the bispecific antibody and an impurity comprising running the sample through a ceramic hydroxyapatite chromatography in a flow through mode.

[0158] In some aspects, the loading the sample on the CHT column or running a CHT chromatography comprises adding the sample to a loading buffer comprising a phosphate salt. In some aspects, the phosphate salt in the loading buffer comprises sodium phosphate, potassium phosphate, or any combination thereof.

[0159] In some aspects, the bispecific molecule comprises a molecule targeting BCMA and CD3, a molecule targeting CD47 and CD20, a molecule targeting NKG2D and FLT3, or a combination thereof.

[0160] In some aspects, the bispecific molecule comprises a molecule targeting BCMA and CD3. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule is Alnuctamab, CC-93269, or BMS-986349. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a polypeptide comprising 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 sequences of SEQ ID NOs: 1-4.

[0161] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide comprising 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. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0162] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a second polypeptide comprising 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. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a second polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0163] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a third polypeptide comprising 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: 3. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a third polypeptide having the amino acid sequence of SEQ ID NO: 3. [0164] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a fourth polypeptide comprising 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: 4. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a fourth polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0165] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a fifth polypeptide comprising 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: 4. In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a fifth polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0166] In some aspects, the anti-BCMA and anti-CD3 bispecific molecule comprises a first polypeptide comprising 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 comprising 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, a third polypeptide comprising 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: 3, a fourth polypeptide comprising 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: 4, and a fifth polypeptide comprising 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: 4.

[0167] In some aspects, 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. Table 1. Anti-BCMA and Anti-CD3 Bispecific Molecule Amino Acid Sequences

[0168] In some aspects, the bispecific molecule comprises a molecule targeting CD47 and CD20. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule is CC-96673 and BMS-986358.

[0169] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises a polypeptide comprising 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 sequences of one or more of SEQ ID NOs: 5-24. In some aspects, the anti-CD47 and anti- CD20 bispecific molecule comprises a polypeptide having the sequence of one or more of SEQ ID NOs: 5-24.

[0170] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises light chain CDRs comprising 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 NOs: 5-7 and 15-17. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises light chain CDRs having the amino acid sequence of SEQ ID NO: 5-7 and 15-17.

[0171] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises a light chain CDR1 comprising 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 comprising 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, a light chain CDR3 comprising 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, a light chain CDR1 comprising 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: 15, a light chain CDR2 comprising 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: 16, and a light chain CDR3 comprising 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: 17. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises 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.

[0172] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises heavy chain CDRs comprising 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 NOs: 8-10 and 18-20. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises heavy chain CDRs having the amino acid sequence of SEQ ID NO: 8-10 and 18-20.

[0173] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises a heavy chain CDR1 comprising 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 comprising 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, a heavy chain CDR3 comprising 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, a heavy chain CDR1 comprising 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: 18, a heavy chain CDR2 comprising 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: 19, and a heavy chain CDR3 comprising 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: 20. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises 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.

[0174] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises variable light chains comprising 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 NOs: 11 and 21. In some aspects, the anti-CD47 and anti- CD20 bispecific molecule comprises variable light chains having the sequence of SEQ ID NOs: 11 and 21. [0175] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises variable heavy chains comprising 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 NOs: 12 and 22. In some aspects, the anti-CD47 and anti- CD20 bispecific molecule comprises variable heavy chains having the sequence of SEQ ID NOs: 12 and 22.

[0176] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises light chains comprising 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 NOs: 13 and 23. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises light chains having the sequence of SEQ ID NOs: 13 and

23.

[0177] In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises heavy chains comprising 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 NOs: 14 and 24. In some aspects, the anti-CD47 and anti-CD20 bispecific molecule comprises heavy chains having the sequence of SEQ ID NOs: 14 and

24.

Table 2. Anti-CD47 and Anti-CD20 Bispecific Molecule Amino Acid Sequences

[0178] In some aspects, the bispecific molecule comprises a molecule targeting NKG2D and FLT3. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule is DF4001 and BMS-986450.

[0179] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a polypeptide comprising 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 sequences of one or more of SEQ ID NOs: 25-43. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a polypeptide having the sequence of one or more of SEQ ID NOs: 25-43.

[0180] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises light chain CDRs comprising 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 NOs: 28-30 and 36-38. In some aspects, the anti-NKG2D and anti- FLT3 bispecific molecule comprises light chain CDRs having the amino acid sequence of SEQ ID NO: 28-30 and 36-38. [0181] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a light chain CDR1 comprising 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 comprising 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, a light chain CDR3 comprising 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, a light chain CDR1 comprising 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: 36, a light chain CDR2 comprising 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: 37, and a light chain CDR3 comprising 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: 38. In some aspects, the anti- NKG2D and anti-FLT3 bispecific molecule comprises 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.

[0182] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises heavy chain CDRs comprising 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 NOs: 25-27 and 33-35. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises heavy chain CDRs having the amino acid sequence of SEQ ID NO: 25-27 and 33-35.

[0183] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a heavy chain CDR1 comprising 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 comprising 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, a heavy chain CDR3 comprising 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, a heavy chain CDR1 comprising 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: 33, a heavy chain CDR2 comprising 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: 34, and a heavy chain CDR3 comprising 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: 35.

[0184] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises 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.

[0185] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises variable light chains comprising 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 NOs: 32 and 40. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises variable light chains having the sequence of SEQ ID NOs: 32 and 40.

[0186] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises variable heavy chains comprising 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 NOs: 31 and 39. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises variable heavy chains having the sequence of SEQ ID NOs: 31 and 39.

[0187] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a polypeptide comprising 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 sequences of SEQ ID NOs: 41-43. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises the amino acid sequences of SEQ ID NOs: 41-43.

[0188] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide comprising 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: 41. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 41.

[0189] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a second polypeptide comprising 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: 42. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a second polypeptide having the amino acid sequence of SEQ ID NO: 42.

[0190] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a third polypeptide comprising 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: 43. In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a third polypeptide having the amino acid sequence of SEQ ID NO: 43.

[0191] In some aspects, the anti-NKG2D and anti-FLT3 bispecific molecule comprises a first polypeptide comprising 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: 41, a second polypeptide comprising 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: 42, and a third polypeptide comprising 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: 43. In some aspects, 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. Table 3. Anti-NKG2D and Anti-FLT3 Bispecific Molecule Amino Acid Sequences

[0192] The present disclosure also includes a molecule of interest isolated or purified by the present disclosure. In some aspects, the disclosure includes the final product comprising the F/T composition, and optionally the chase composition and/or the elution composition. In some aspects, the disclosure includes the F/T composition only as a final product. In some aspects, the product obtained from the present methods is subject to a formulation as a drug product.

[0193] In some aspects, the disclosure also includes a methods of treating a disease or condition in a subject in need thereof.

Impurities

[0194] The impurities to be isolated out includes any process related impurities or product related impurities. In some aspects, the impurity in a sample subject to the present disclosure comprises a virus, a high molecular weight aggregate (HMW), a low molecular weight aggregate (LMW), a host cell protein (HCP), residual deoxyribose nucleic acid (rDNA), residual protein A (rProA), or any combination thereof.

[0195] In some aspects, the present F/T CHT method reduces the amount of HMW that was in the sample by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 1% to about 50%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 5% to about 40%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 10% to about 35%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 10% to about 30%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 10% to about 20%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 15% to about 25%. In some aspects, the present F/T CHT method reduces the amount of HMW in the sample by about 20% to about 30%.

[0196] In some aspects, the present F/T CHT method reduces the amount of LMW that was in the sample by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by up to about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1%. In some aspects the present F/T CHT method reduces the amount of LMW in the sample by about 1% to about 20%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 1% to about 30%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 1% to about 10%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 5% to about 10%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 5% to about 20%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 10% to about 40%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 20% to about 30%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 10% to about 25%. In some aspects, the present F/T CHT method reduces the amount of LMW in the sample by about 15% to about 25%.

[0197] In some aspects, the present F/T CHT method reduces the amount of HCP that was in the sample by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by about 10% to about 50%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by about 50% to about 90%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 20% to 80%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 10% to 50%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 1% to 30%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 5% to 50%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 10% to 20%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 1% to 20%. In some aspects, the present F/T CHT method reduces the amount of HCP in the sample by 10% to 40%.

[0198] In some aspects, the present F/T CHT method has a viral clearance by 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 Log Reduction Value (LRV). The LRV indicates a viral clearance defined as the difference between the total virus amount in the input sample and the product-containing faction after purification. In some aspects, the present F/T CHT method has a viral clearance of about 2.8 LRV. In some aspects the present F/T CHT method has a viral clearance between about 1 LRV and about 4 LRV, about 2 LRV and about 3 LRV, about 2 LRV and about 4 LRV, or about 1 LRV and about 3 LRV.

[0199] In some aspects, the virus to be cleared comprises an adeno-associated virus, a lentivirus, an adenovirus, or any combination thereof. In some aspects, the virus to be cleared comprises a retrovirus. In some aspects, the virus to be cleared comprises an adeno- associated virus (AAV). In some aspects, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAVrh9, AAV9, AAVrhlO, AAV10, AAVRH10, AAV11, and AAV12. In some aspects, the virus to be cleared comprises a lentivirus.

[0200] In some aspects, the present F/T CHT method reduces the amount of rProA that was in the sample by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by about 10% to about 50%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by about 50% to about 90%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 20% to 80%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 30% to 80%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 40% to 80%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 50% to 80%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 20% to 70%. In some aspects, the present F/T CHT method reduces the amount of rProA in the sample by 20% to 60%.

[0201] In some aspects, the present methods are capable of reducing the risk of degradation of one or more ingredients in the sample, e.g., inactive ingredients, e.g., polysorbate 80. In some aspects, one or more ingredients in the sample, e.g., polysorbate 80, can turn into one or more molecules that are not desirable as a part of the final product, e.g., oleic acid for polysorbate 80 degradation. In some aspects, one or more ingredients in the sample, e.g., polysorbate 80, can turn into one or more contaminations that are not desirable as a part of the final product, e.g., oleic acid for polysorbate 80 degradation, within 24 hours or 48 hours. In some aspects, the amount of the one or more contaminations, e.g., oleic acid due to polysorbate 80 degradation, from the final product or the F/T composition, especially for the fusion protein, after the present F/T CHT is lower than that of the BZE final product or BZE composition. In some aspects, the concentration of oleic acid in the final product as polysorbate 80 degradation or in the F/T composition after the present method is a low risk amount, e.g., less than about 1.75 pg/mL, over a 48 hours period.

[0202] Various aspects of the disclosure are described in further detail in the following subsections. The present disclosure is further illustrated by the following examples which should not be construed as further limiting.

Examples

EXAMPLE 1 : ADOPTING FLOW-THROUGH METHODS FOR BIOLOGICS

[0203] In this study, five different complex biologies were chosen to analyze the capabilities of CHT resin when operated using flow-through mode. Among these, four were bispecific molecules (referred to as bsAb A, B, C, and D) comprising three different overall structures, with the fifth molecule being an Fc-fusion protein (referred to as Fc-fusion A). The bsAb A is CD3xBCMA bispecific mAb, in particular, alnuctamab, also known as CC-93269 or BMS-986349. The bsAb B is CD47xCD20 bispecific Ab, which is also known as CC- 96673 or BMS-986358. The bsAb C is NKG2DxFLT3 bispecific Ab, which is also known as DF4001 or BMS-986450. The bsAb D is FcRH5xCD3 bispecific Ab, which is also known as FcRH5 T cell engager or BMS-986448. Like most antibodies, bsAbs are y- shaped molecules, but instead of having two, identical “arms” of the y-shape like monoclonal antibodies (mAbs), they have two distinct (or bispecific) arms. See FIG. 1 A. For example, a bsAb with one arm of IgG subclass 2 (IgG 2) and one arm of IgG 1 would be referred to as an IgG(2+l). Of the four bsAbs used in this study, there are three different overall structures: IgG(2+l), IgG(l+l), and IgG(Fab-scFv). See FIG. 1A and FIG. IB. These bsAbs have a range in overall isoelectric point (pl) of 7.7 to 8.1, with Fc-fusion A having a pl of 5.5. The bsAbs already have the bind and elute (BZE) processes established, with the column loading ranging from 14 to 20 grams per liter of CHT resin (g/Lresin). Based on the prior experiments, Fc-fusion A had a binding capacity as high as only 5 g/Lresin or less. A summary including the molecular structures and relevant, aforementioned information can be found in FIGs. 1A and IB. As the column loading amount of these established BZE processes was low (and likely to cause bottleneck problems in the overall down-stream purification process), this study was conducted to test a flow through (F/T) CHT process instead of the BZE CHT process. The study was to increase the column loading amount that can increase the productivity after the process while maintaining the similar impurity clearance seen as a result of the BZE bsAb processes. To increase loading and to ensure sufficient load breakthrough, the loading amount of the CHT F/T column was targeted to be 200 g/Lresin. An operating pH of 7.5 was selected as it is relatively close to the bsAbs' pl, and it is expected to ensure stability of the sodium phosphate buffer matrix commonly used with CHT. A linear gradient elution using sodium chloride was performed after the load flow-through and chase to gain a higher resolution of impurity separation and better understanding of potential separation trends and behavior. The load flow-through, chase, and gradient chloride elution were all collected using multiple fractions. Lastly, sodium phosphate load and overall operating concentrations of 5 millimolar (mM) and 20 mM were chosen to observe any potential effect that phosphate concentration may have.

EXAMPLE 2. EFFECT OF PHOSPHATE ON CHT BINDING

[0204] This example was performed to observe the effect of phosphate concentration in the load and mobile phase on CHT binding. FIG. 2 shows that an increase in phosphate concentration in the load and mobile phase reduced its binding capacity as phosphate weakens both metal affinity and cation exchange interactions. Binding capacity was calculated based on the mass of protein loaded onto the CHT column when breakthrough of the load was observed according to UV of the respective chromatogram. While load concentration must be considered as it varied between the 5 mM and 20 mM sodium phosphate conditions across most of the bsAbs tested, this is not true for all. BsAb C had negligible difference in load concentrations, at 8.61 milligrams of protein per milliliter (mg/mL) and 8.62 mg/mL for 5 mM and 20 mM sodium phosphate conditions, respectively, however, a percent difference in binding capacity of approximately 10% was observed. A juxtaposition of load protein concentration and percent difference in binding capacity for all bsAbs used in this study can be seen in Table 4. The data may suggest little to no impact caused by load concentration, but more of an impact caused by sodium phosphate concentration and molecular-dependence. Table 4.

i • i | | | |

EXAMPLE 3. BISPECIFIC ANTIBODY SELECTIVITY STUDY RESULTS

[0205] CHT F/T impurity clearance also has an observable impact caused by phosphate concentration and, even more so, molecular-dependence. Across multiple impurity types and molecules, CHT F/T shows strong separation with promising opportunity for optimization. This molecular-dependent separation can be observed with bsAb D in FIG. 3A and FIG. 3B, which detail the HMW aggregates removal even at such a high load percentage of 30% with many product pool fractions around 15% or less. Higher overall purity could be achieved in a more typical, non-fractionated pool by simply beginning collection slightly later and ending slightly sooner. Further separation can be observed in FIG. 4, where similar trends to bsAb D’s HMW separation are found in bsAbs B and C, with the large exception being the very beginning of the flow-through HMW percentage being much greater than the load for bsAb D only. This abnormally high value suggests further molecular-dependence, with a hypothesis being a specific type of fragment unique to bsAb D of higher negative charge being repelled from the CHT’s phosphoryl sites more strongly. It is also worth mentioning the much lower HMW percentage that bsAbs B and C began with in their load material.

[0206] Quality data for other impurity types are seen in FIGs. 5A to 5D and FIGs. 6A and 6V for LMW aggregates and rDNA respectively. Strong LMW selectivity was observed across bsAbs A, B, and C, with many samples measuring around half of the LMW percentage as that of the load material. BsAb D showed minor LMW selectivity, with most samples’ measured LMW percentages not deviating from that of the load material significantly. Differences in LMW separation trends further indicate the molecular- dependent selectivity of CHT F/T. The trends in rDNA separation of bsAb B in FIGs. 6A and 6B began similarly for both phosphate concentration conditions, however they diverged later on due to most of the rDNA eluting from the column during the linear sodium chloride gradient for 5mM sodium phosphate while most rDNA bound more strongly for 20mM sodium phosphate and came out in the collected column strip.

[0207] Additionally, bsAb F/T data were compared to BZE and can be seen in Table 5.

Table 5.

[0208] Table 5 shows the summary of bsAb CHT F/T data with BZE data available for comparison. Some results were not available (N/A) due to lack of testing and/or no impurity challenge, and others were below the limit of quantitation (LOQ). HCP defined lower limit of quantitation is 60 ng/mL. The collected fractions of the F/T chromatography runs were collected and cumulated based on their protein mass and impurity data. Furthermore, collection criteria adjustments were simulated by omitting certain fractions towards the beginning or end of the chromatography runs that were relatively high in impurities; the cumulative yields reflected these omissions as well. This was done to mimic the performance of the optimized BZE processes. The phosphate concentration condition chosen to include in the table for each bsAb in F/T varied across bsAbs to prioritize the condition that resulted in more desirable balance of impurity clearance and yield. All F/T impurity data are from the same 5mM and 20mM sodium phosphate chromatography runs that this document has detailed up to this point, with the exception of the F/T HCP data for bsAbs C and D. These values come from confirmation runs performed after the aforementioned chromatography runs to verify separation behavior. These confirmation runs also operated under 200 g/Lresin loading and a 20mM sodium phosphate concentration at pH 7.5, but with a set sodium chloride concentration throughout rather than a gradient. BZE bsAb chromatography runs used optimized conditions from their already established BZE processes. The load material used in the BZE chromatography runs had higher purity than that of the F/T runs, as they used representative material respective of their established processes.

[0209] In most cases, CHT F/T proved to be useful when targeting the same impurities as one would with a BZE process for each respective molecule. For example, bsAb A’ s primary BZE impurity challenge was in removing LMW aggregates, which CHT F/T did sufficiently. Some exceptions where CHT performed better in BZE compared to F/T were with HCP for bsAb C and LMW aggregates for bsAb D. However, it is important to note that the purity of the load material for the BZE chromatography runs was more representative and purer, compared to the more challenging material used in these F/T runs. Furthermore, the BZE chromatography runs used optimized and established conditions, while the F/T used non-established development conditions. Potential optimization could include a chase buffer condition at a higher conductivity than the load and equilibration (EQ) buffer. One could also adjust the collection criteria to avoid collecting more impurityrich fractions of the F/T pool. Another optimization could be to reduce the column loading, however, productivity would still be significantly greater than BZE operation even when halving the column loading to 100 g/Lresin. Therefore, CHT F/T shows desirable separation and purification, even at higher column loading upwards of 200 g/Lresin. More detailed information on methods can be found in figure captions.

EXAMPLE 4. FC-FUSION CASE STUDY RESULTS

[0210] While the bsAb selectivity study serves as a proof of concept for adapting complex biologies with BZE processes to higher-productivity CHT F/T, the Fc-fusion case study shows more than just clearance of common process- and product- related impurities. For example, Table 6 shows the improved stability offered by CHT F/T by reducing the risk of degradation of polysorbate 80 (PS80), a common surfactant added to improve stability in drug substance (DS) within biologies manufacturing. CHT F/T surpassed POROS 50 HQ anion exchange (AEX) F/T chromatography in this aspect, at similar DS concentrations and column loading.

[0211] Table 6 shows evaluation of PS80 degradation risk for CHT F/T and AEX F/T with Fc-fusion A. Risk level is defined by ranges of measured oleic acid (OA) concentration increasing over a period of 48 hours. Below 1.75 pg/mL OA is defined as low risk, 1.75 - 7.0 pg/mL as medium risk, and greater than 7.0 pg/mL as high risk.

[0212] PS80 degradation risk through enzymatic hydrolysis is estimated based on the lipase activity by measuring the concentration of free oleic acid released from the PS80 degradation over 48-hour incubation. The in-process material or final drug substance is spiked with PS80 at 0.5 mg/mL target level. The spiked material is then incubated at 25°C for up to 48 hours. Samples at initial, 24-hour and 48-hour time intervals are taken for oleic acid measurement based on the following protocol. Briefly, 150 pL of 2.67 pg/mL¹³Cis- OA internal standard working solution in isopropanol is added to 50 pL of sample, mixed well and centrifuge at RT with 10,000 rpm for 10 min. Take the supernatant for LC/MS single ion monitoring analysis. The risk category assignment is based on the observed free oleic acid growth over 48 hours combined with known PS80 degradation data from multiple existing long term stability studies. Based on an empirical evaluation, if 0.5 mg/ml PS80 gets completely hydrolyzed, it will generate -175 pg /mL oleic acid. Below 1.75 pg/mL oleic acid growth over 48 hour is defined as low risk (corresponding to 1% PS80 loss in 48 hours and stable PS80 profile over long-term storage from existing programs), 1.75 - 7.0 pg/mL as medium risk (some noticeable PS80 loss over long term storage from existing programs), and greater than 7.0 pg/mL as high risk (significant PS80 loss over long term storage from existing programs).

Table 6

[0213] 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.

Table 7.

[0214] Table 7 shows surrogate viral clearance data for CHT F/T with Fc-fusion A. LRVs are averages of two data sets (n=2) for each surrogate model and resin. Load material for each run was spiked with a known quantity of concentrated stock of surrogate virus. The load and CHT F/T product pools were tested using qPCR to calculate LRVs.

[0215] Table 7 shows that CHT F/T achieved an average of 3.7 LRV clearance when using a minute mouse virus (MVM) surrogate, also known as a mock virus particle (MVP). The other surrogate model is for xenotropic murine leukemia virus (X-MuLV) and is referred to as a retrovirus-like particle (RVLP); CHT F/T averaged 2.8 LRV with this model. CHT F/T performed as well or better when compared to Capto Phenyl High Sub hydrophobic interaction chromatography (HIC) F/T. Some of the CHT F/T chromatography column strips were collected and tested, which showed significantly greater amounts of MVP and RVLP than in the product pools. This suggests even further that CHT F/T shows promising potential for viral clearance capabilities because of the stronger binding of the surrogate viruses to the column. More background information on surrogate virus models and their methodology can be found in Table 8 below. Table 8.

[0216] Table 8 shows summary of simplified background information on viruses and their respective surrogate models. Equipment, additional information, and user manuals including method protocols were supplied by Cygnus.

[0217] Fc-fusion A has CHT F/T performance akin to or better than that of the bsAbs used in this study. A chromatography run using a pre-optimization development condition with more challenging load material showcases this performance in Table 9. Strong separation of HCP, rDNA, and HMW aggregates were observed. Minor separation of rProA was observed, while LMW aggregates were not separated. These observations further suggest molecular-dependence.

Table 9.

[0218] Table 9 shows summary of impurity results of a Fc-fusion A CHT F/T preoptimization development chromatography run. The load material used was also higher in impurities than in most representative, optimized chromatography runs. The defined lower limits of quantitation (LOQ) for HCP and rDNA are 60 ng/mL and 1 pg/mL, respectively. [0219] CHT F/T has not only been tested at bench-scale, but it also has a successfully completed run at 500 liter- (500 L) scale. FIG. 7A and 7B show two CHT F/T chromatograms processing Fc-fusion A: one of a representative, optimized bench-scale run, and the other at 500 L-scale using the same operating conditions. The consistency in shape of the UV280 absorbance peak across scales suggest little to no changes in performance or behavior, especially in combination with the data in Table 10 below.

Table 10.

[0220] Table 10 shows overall CHT F/T yield and product quality data for representative average (n=8) at bench-scale and a successfully completed 500 liter-scale run. Values show consistency across both scales. Values below the limit of quantitation (LOQ) as defined by each assay are marked as such. The defined lower limits of quantitation for HCP and rDNA are 60 ng/mL and 1 pg/mL, respectively.

[0221] Yield and product quality averaged across 8 representative CHT F/T bench-scale chromatography runs are compared to that of the completed 500 L-scale run, and they have no significant differences. The consistency in absorbance peaks, yield, quality, and robustness present promising capabilities for scaling CHT F/T moving forward.

[0222] To summarize, this study proves that CHT does not need to be strictly limited to the traditional BZE mode of operation that was conventionally used. While BZE provides various benefits, operating CHT in F/T can be similarly effective performance-wise, but at a much higher-productivity afforded by its significantly greater column loading (upwards of ten times or greater). The bsAb screening study resulted in the observation of the strong, molecular-dependent separation that CHT F/T is capable of across various molecules, phosphate concentrations, impurity types, and impurity levels. Data suggest that the CHT F/T process can significantly improve the down-stream purification process: potentially without having to sacrifice much productivity (if any) while reducing impurity levels by roughly 50% (or more in some cases).

[0223] Additionally, in the context of Fc-fusion A, CHT F/T shows even more benefits such as increased product stability via risk reduction in PS80 degradation, and significant surrogate viral clearance suggesting promising potential results for actual viruses. Not only this, but the consistency in absorbance curves, yields as high as 94%, product quality below the limit of quantitation, and process robustness at both bench- and 500 L-scales demonstrate the promising scalability of CHT F/T processes and the confidence one can have when considering this mode of operation in another context.

[0224] Overall, CHT F/T has strong performance comparable to the conventional BZE mode of operation, but has greatly increased productivity. This could serve as a reasonable solution to limit bottlenecks within biologies manufacturing processes. With the aforementioned promising qualities, hopefully CHT F/T can successfully be applied to not only more complex biologies such as bsAbs and Fc-fusion proteins at larger scales, but also other biologies like mAbs.

***

[0225] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0226] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0227] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined herein. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

[0228] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Database entries and electronic publications disclosed in the present disclosure are incorporated by reference in their entireties. The version of the database entry or electronic publication incorporated by reference in the present application is the most recent version of the database entry or electronic publication that was publicly available at the time the present application was filed. The database entries corresponding to gene or protein identifiers (e.g., genes or proteins identified by an accession number or database identifier of a public database such as Genbank, Refseq, or Uniprot) disclosed in the present application are incorporated by reference in their entireties. The gene or protein-related incorporated information is not limited to the sequence data contained in the database entry. The information incorporated by reference includes the entire contents of the database entry in the most recent version of the database that was publicly available at the time the present application was filed. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a flow-through (F/T) mode of a ceramic hydroxyapatite chromatography (CHT) comprising a. loading the sample on a CHT column, and b. obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

2. A method of isolating a molecule of interest in a sample comprising the molecule and an impurity in a ceramic hydroxyapatite chromatography (CHT) comprising a. loading the sample on a CHT column in a flow-through (F/T) mode and b. obtaining a F/T composition comprising the molecule and a lower amount of the impurity compared to the sample prior to the loading.

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

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

5. The method of any one of claims 1 to 4, wherein the molecule of interest in the sample is at an amount that the molecule of interest is not bound to the column during and/or after the loading.

6. The method of any one of claims 1 to 4, wherein the molecule of interest in the sample is at an amount that some molecules are bound to the column during and/or after the loading.

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

8. The method of any one of claims 1 to 7, wherein the molecule of interest comprises a protein.

9. The method of any one of claims 1 to 7, wherein the molecule of interest comprises a nucleic acid.

10. The method of any one of claims 1 to 7, wherein the molecule of interest comprises a virus.

11. The method of any one of claims 1 to 10, wherein the loaded sample comprises a loading amount of 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/Lresin.

12. The method of any one of claims 1 to 11, wherein the loaded sample comprises a loading amount of between about 50 and about 500, about 50 and about 450, about 50 and about 400, about 50 and about 350, between about 50 and about 300, about 70 and about 300, about 90 and about 300, about 100 and about 300, about 120 and about 300, about 140 and about 300, about 150 and about 300, about 50 and about 250, about 70 and about 250, about 90 and about 250, about 100 and about 250, about 120 and about 250, about 140 and about 250, about 150 and about 250, about 50 and about 200, about 70 and about 200, about 90 and about 200, about 100 and about 200, about 120 and about 200, about 140 and about 200, or about 150 and about 200 g/Lresin.

13. The method of any one of claims 1 to 12, wherein the loading the sample on the CHT column comprises adding the sample to a loading buffer comprising a phosphate salt.

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

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

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

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

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

20. The method of claim 19, wherein the chase buffer comprises a phosphate salt.

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

22. The method of claim 20 or 21 , wherein the phosphate salt in the chase buffer is in an amount of 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 of any one of claims 20 to 22, wherein the phosphate salt in the chase buffer is in an amount of between about 5 and about 50, about 5 and about 45, about 5 and about 40, about 5 and about 35, about 5 and about 30, about 10 and about 30, about 15 and about 30, about 20 and about 30, about 25 and about 30, about 5 and about 20, about 10 and about 20, about 15 and about 20, about 5 and about 15, or about 10 and about 15 mM.

24. The method of any one of claims 19 to 23, further collecting a chase composition comprising the molecule after (c).

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

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

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

28. The method of claim 27, wherein the elution buffer comprises a phosphate salt.

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

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

32. The method of 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 of any one of claims 13 to 31, wherein 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.

34. The method of any one of claims 13 to 31, wherein the pH of the loading buffer is between about pH 6.3 and about pH 7.7.

35. The method of any one of claims 13 to 31, wherein the pH in the loading buffer is about 7.5.

36. The method of any one of claims 19 to 35, wherein 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.

37. The method of 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 of any one of claims 19 to 35, wherein the pH of the chase buffer is between about pH 6.3 and about pH 7.7.

39. The method of any one of claims 19 to 35, wherein the pH of the chase buffer is about 7.5.

40. The method of any one of claims 19 to 35, wherein the pH of 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.

41. The method of any one of claims 27 to 40, wherein 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.

42. The method of any one of claims 27 to 40, wherein the pH of the elution buffer is between about pH 6.3 and about pH 7.7.

43. The method of any one of claims 27 to 40, wherein the pH in the elution buffer is about 7.5.

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

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

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

47. The method of any one of claims 19 to 46, wherein the conductivity of the chase buffer is similar to or higher than the conductivity of the loading buffer.

48. The method of any one of claims 19 to 46, wherein the conductivity of the chase buffer is similar to or lower than the conductivity of the loading buffer.

49. The method of any one of claims 27 to 48, wherein the conductivity of the elution buffer is similar to or higher than the loading buffer.

50. The method of any one of claims 27 to 48, wherein the conductivity of the elution buffer is similar to or lower than the loading buffer.

51. The method of any one of claims 1 to 8 and 11 to 50, wherein the molecule of interest comprises a protein comprising an antibody or 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 clotting factor, a chemokine, an enzyme, a hormone, or any combination thereof.

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

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

54. The method of claim 51, wherein 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.

55. The method of claim 51, wherein the antibody or antigen binding portion thereof binds an antigen selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CDl la, tissue factor (TF), MICA/B PSCA, IL-8, EGFR, HER3, HER4, and any combination thereof.

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

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

58. The method of claim 51, wherein the bispecific molecule comprises a molecule specifically binding to BCMA and CD3, a molecule specifically binding to CD47 and CD20, a molecule specifically binding to NKG2D and FLT3, or a combination thereof.

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

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

61. The method of any one of claims 1 to 60, wherein the impurity comprises a virus, a high molecular weight aggregate (HMW), a low molecular weight aggregate (LMW), a host cell protein (HCP), residual deoxyribose nucleic acid (rDNA), residual protein A (rProA), or any combination thereof.

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

63. A molecule of interest isolated by the method of any one of claims 1 to 62.