Methods for protein purification

Abstract

The present disclosure relates generally to methods for purifying protein preparations (e.g., antibody preparations) that contain 0-glucuronidase. In some embodiments, the purified protein (e.g., antibody) is capable of being conjugated to and / or for use in producing a molecule comprising a glucuronide, such as conjugation to an agent via a glucuronide-containing β-glucuronidase cleavable linker. The methods may involve the use of specific chromatography resins, or size exclusion chromatography (SEC), optionally in addition to a protein A purification step.

Description

METHODS FOR PROTEIN PURIFICATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 555,865, filed February 20, 2024, the disclosure of which is incorporated by reference herein in its entirety.1. FIELD

[0002] The present disclosure relates generally to methods for purifying protein preparations (e.g., antibody preparations) that contain P-glucuronidase. In some embodiments, the purified protein (e.g., antibody) is capable of being conjugated to and / or for use in producing a molecule comprising a glucuronide, such as conjugation to an agent via a glucuronide-containing P- glucuronidase cleavable linker. The methods may involve the use of specific chromatography resins, or size exclusion chromatography (SEC), optionally in addition to a protein A purification step.2. BACKGROUND

[0003] Conjugated proteins such as antibody-drug conjugates (ADCs) have been widely developed for therapeutic purposes. Yet, manufacturing conjugated proteins such as ADCs still faces several challenges, thus potentially slowing down their maturation into approved drugs. One of the biggest technical difficulties is to provide consistently efficacious drug products.

[0004] Accordingly, there remains a need to develop improved methods for producing conjugation products such as ADCs effectively and efficiently, for example, by providing suitable protein (e.g., antibody) intermediates for use in a conjugation.3. SUMMARY

[0005] The present disclosure is based, at least in part, on the unexpected finding that the use of certain specific chromatography resins during protein purification effectively removed P- glucuronidase from protein preparations. The present disclosure is also based, at least in part, on the unexpected finding that the use of size exclusion chromatography (SEC) during protein purification also effectively removed -glucuronidase from protein preparations.

[0006] Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus and has been known for its ability to bind many proteins especially antibodies. Accordingly, protein A chromatography is now the most commonlyused purification method in industrial-scale downstream processing of antibodies. Yet, as described in this disclosure, surprisingly, protein A chromatography combined with the commonly used ion exchange chromatography did not sufficiently remove P-glucuronidase from antibodies. The presence of P-glucuronidase can release glucuronic acid from substrates, such as a glucuronide-containing P-glucuronidase cleavable linker, thereby removing the protection to the linker (in particular, a linker containing a cleavable peptide motif) provided by the glucuronic acid, for example, against proteolysis.

[0007] The present disclosure provides improved methods of protein (e.g., antibody) purification for proteins (e.g., antibodies) to be used in producing a molecule comprising a glucuronide, such as proteins (e.g., antibodies) to be conjugated to an agent (e.g., a cytotoxic drug, immunomodulating agent, kinase inhibitor, protein degrader, peptide, oligonucleotide, or another biologically-active moiety) via a glucuronide-containing P-glucuronidase cleavable linker, by removing P-glucuronidase from a protein (e.g., an antibody) preparation. In some embodiments, the protein (e.g., antibody) preparation has been purified by contacting with a protein A affinity chromatography resin. Additionally or alternatively, the method comprises subjecting the protein (e.g., antibody) preparation to an anion exchange (AEX) chromatography as described herein. Additionally or alternatively, the method comprises subjecting the protein (e.g., antibody) preparation to a multimodal chromatography as described herein. Additionally or alternatively, the method comprises subjecting the protein (e.g., antibody) preparation to a hydrophobic interaction chromatography (HIC) as described herein. Additionally or alternatively, the method comprises subjecting the protein (e.g., antibody) preparation to a cation exchange (CEX) chromatography as described herein. Additionally or alternatively, the method comprises subjecting the protein (e.g., antibody) preparation to a size exclusion chromatography (SEC) as described herein.

[0008] The present disclosure provides a method of purifying a protein, wherein the method comprises subjecting a P-glucuronidase-containing protein preparation to anion exchange (AEX) chromatography or multimodal chromatography using a resin. In some embodiments, a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group. Additionally or alternatively, the purified protein is capable of beingconjugated to and / or for use in conjugation to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

[0009] The present disclosure also provides a method of producing a conjugated protein, wherein the method comprises: (1) purifying a protein by subjecting a P-glucuronidase- containing protein preparation to AEX chromatography or multimodal chromatography using a resin; and (2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker. In some embodiments, a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group.

[0010] In some embodiments, the ligand comprises a phenyl functional group and a hydroxy functional group. In some embodiments, the ligand comprises a phenyl functional group and a quaternary ammonium functional group. In some embodiments, the ligand comprises a hydroxy functional group and a quaternary ammonium cation functional group. In some embodiments, the ligand comprises a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group. In certain embodiments, the ligand comprises a N-benzyl-2- hydroxy-N-methylethan-1 -ammoniumyl moiety. In certain embodiments, the ligand is

[0011] In certain embodiments, the resin described herein is Capto™ Adhere.

[0012] The present disclosure further provides a method of purifying a protein, wherein the method comprises subjecting a P-glucuronidase-containing protein preparation to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin. In some embodiments, a ligand of the resin comprises a phenyl functional group. Additionally or alternatively, the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0013] The present disclosure also provides a method of producing a conjugated protein, wherein the method comprises: (1) purifying a protein by subjecting a P-glucuronidase- containing protein preparation to AEX chromatography or HIC using a resin; and (2) conjugatingthe purified protein to an agent via a glucuronide-containing P-glucuronidase cleavable linker. In some embodiments, a ligand of the resin comprises a phenyl functional group.

[0014] In some embodiments, the ligand comprises a phenoxy moiety. In certain embodiments, the ligand is phenoxy.

[0015] In some embodiments, the resin described herein is Capto™ Phenyl. In certain embodiments, the resin is Capto™ Phenyl (high sub).

[0016] The present disclosure further provides a method of purifying a protein, wherein the method comprises subjecting a P-glucuronidase-containing protein preparation to a multimodal chromatography using a resin. Additionally or alternatively, the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P- glucuronidase cleavable linker. In some embodiments, a ligand of the resin comprises a phosphate group, calcium (e.g., Ca2+), a hydroxyl group, or any combination thereof. In further embodiments, a ligand of the resin comprises a phosphate. In further embodiments, a ligand of the resin comprises a phosphate group and calcium. In further embodiments, a ligand of the resin comprises a phosphate group, calcium, and a hydroxyl group. In some embodiments, the multimodal chromatography is a cation exchange (CEX) chromatography. In further embodiments, a ligand of the resin comprises calcium. In further embodiments, a ligand of the resin comprises calcium and a hydroxyl group. In some embodiments, the multimodal chromatography is an anion exchange (AEX) chromatography. In some embodiments, the resin is EconoFit CHT XT.

[0017] The present disclosure also provides a method of producing a conjugated protein, wherein the method comprises: (1) purifying a protein by subjecting a P-glucuronidase- containing protein preparation to a multimodal chromatography using a resin; and (2) conjugating the purified protein to an agent via a glucuronide-containing P-glucuronidase cleavable linker. In some embodiments, a ligand of the resin comprises a phosphate group, calcium (e.g., Ca2), a hydroxyl group, or any combination thereof. In further embodiments, a ligand of the resin comprises a phosphate group. In further embodiments, a ligand of the resin comprises a phosphate group and calcium. In further embodiments, a ligand of the resin comprises a phosphate group, calcium, and a hydroxyl group. In some embodiments, the multimodal chromatography is a cation exchange (CEX) chromatography. In some embodiments, the resin is EconoFit CHT XT.

[0018] In various embodiments, the chromatography is performed at a pH of between 5 and 8. In certain embodiments, the chromatography is performed at a pH of about 5.5. In certain embodiments, the chromatography is performed at a pH of about 6.5. In certain embodiments, the chromatography is performed at a pH of about 7.5.

[0019] In various embodiments, the method described herein further comprises a protein A purification step before the AEX, multimodal or HIC chromatography step, wherein optionally the protein A purification step produces the protein preparation.

[0020] In various embodiments, the protein preparation has been subject to a protein A purification step.

[0021] In various embodiments, the method described herein further comprises, after the AEX, multimodal or HIC chromatography step, a cation exchange (CEX) chromatography step, wherein optionally the purified protein preparation obtained from the AEX, multimodal or HIC chromatography step is subjected to the CEX chromatography step. In some embodiments, the CEX chromatography uses a resin, and a ligand of the resin comprises a sulfonate functional group. In certain embodiments, the ligand comprises a sulfopropyl moiety. In certain embodiments, the ligand is. In some embodiments, the resin isPraesto® SP65. In some embodiments, the resin is SP Sepharose™ Fast Flow ion exchange chromatography media.

[0022] In various embodiments, the method described herein results in no or undetectable [3- glucuronidase activity in the purified protein preparation. In some embodiments, the method described herein results in no loss of glucuronide on the conjugation product of the protein antibody.

[0023] The present disclosure further provides a method of purifying a protein, wherein the method comprises subjecting a P-glucuronidase-containing protein preparation to size exclusion chromatography (SEC). In some embodiments, the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing [3- glucuronidase cleavable linker, and wherein fractions eluting molecules larger than the protein are purified away.

[0024] The present disclosure also provides a method of producing a conjugated protein, wherein the method comprises: (1) purifying a protein by subjecting a [3-glucuronidase-containing protein preparation to SEC, wherein fractions eluting molecules larger than the protein are purified away; and (2) conjugating the purified protein to an agent via a glucuronide- containing P-glucuronidase cleavable linker.

[0025] In some embodiments, the method described herein results in less than 1% loss of glucuronide on the conjugation product of the purified protein.

[0026] In various embodiments, the agent is a cytotoxic drug.

[0027] In various embodiments, the protein is a therapeutic protein. In various embodiments, the protein is an antibody, an Fc-fusion protein, a bispecific binder, an scFv, a VHH, a VHH-Fc fusion protein (also referred to herein as VHH-Fc), or a protein ligand (for example, for targeting a cell-associated receptor(s) such as cell surface receptor(s)). In various embodiments, the protein is an antibody.

[0028] In various embodiments, the conjugation product of the purified protein is an antibody-drug conjugate (ADC).

[0029] In various embodiments, the glucuronide-containing P-glucuronidase cleavable linker comprises a cleavable peptide motif In certain embodiments, the cleavable peptide motif is protected from proteolysis by a glucuronic acid. In certain embodiments, the glucuronide- containing P-glucuronidase cleavable linker comprises a dipeptide unit shielded by a neighboring glucuronic acid.

[0030] In various embodiments, the method further comprises controlling P-glucuronidase concentration and / or P-glucuronidase activity level in the purified protein preparation.

[0031] In some embodiments, the controlling step comprises controlling residence time in (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC.

[0032] In some embodiments, the controlling step comprises controlling residence time in the CEX chromatography.

[0033] In some embodiments, the controlling step comprises controlling residence time in the protein A purification step.

[0034] In some embodiments, the controlling step comprises modulating pH at which (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC, is performed.

[0035] In some embodiments, the controlling step comprises modulating pH at which the CEX chromatography is performed.

[0036] In some embodiments, the controlling step comprises modulating pH at which the protein A purification step is performed.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 : An exemplary calculation of glucuronide loss using the hydrophobic interaction chromatography (HIC) method.

[0038] FIG. 2 : ADCs containing a tandem-cleavage linker-payload were observed to have multiple peaks corresponding to a drug-to-antibody ratio (DAR) of 2. Mass spectrometry analysis of the various DAR2 peaks indicated a loss of 176 Da (DAR2 #2) and ~2x 176 Da (DAR2 #3), corresponding to the molecular weight of one or two glucuronic acid(s).

[0039] FIG. 3 : HIC chromatogram of monoclonal antibody (mAb) conjugated to a tandemcleavage linker-payload or a tandem-cleavage linker-payload lacking the glucuronic acid (*).

[0040] FIG. 4A: Structure of Ac-glucuronide-PABA. FIG. 4B: HIC chromatogram of ADCs containing tandem-cleavage linker-payload conjugated in the presence of Ac-glucuronide- PABA.

[0041] FIG. 5 : A process diagram for the four-arm study.

[0042] FIG. 6: Antibody SEC chromatogram. Highlighted fractions were pooled and analyzed.5. DETAILED DESCRIPTION

[0043] The present disclosure provides improved methods of protein (e.g., antibody) purification for proteins (e.g., antibodies) to be used in producing a molecule comprising a glucuronide, such as proteins e.g., antibodies) to be conjugated to an agent (e.g., a cytotoxic drug, immunomodulating agent, kinase inhibitor, protein degrader, peptide, oligonucleotide, or another biologically-active moiety) via a glucuronide-containing P-glucuronidase cleavable linker. Such methods may involve the use of specific chromatography resins, or size exclusion chromatography (SEC), optionally in addition to a protein A purification step.

[0044] As it would be understood, the section or subsection headings as used herein are for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.5.1. Definitions

[0045] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dtibel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following descriptions of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0046] The term “resin” as used herein refers to the solid phase of chromatography, such as column chromatography. A resin may be packed in a column or a non-column container.

[0047] The term “column” refers to any enclosure or housing adapted for use in a chromatography process that is suitable for retaining all or part of a chromatography resin and enabling sample and / or buffer to contact the chromatography resin. A wide range of materials are suitable for use in a column and are known in the art.

[0048] The term “chromatography” refers to any kind of technique e.g., a preparative technique) which separates a product of interest (e.g., macromolecules, including proteins, in particular antibodies) from other molecules present in a mixture by differential partitioning between a mobile phase and a stationary phase.

[0049] “Multimodal chromatography,” also known as mixed-mode chromatography (MMC), refers to chromatographic method for separating one analyte from another by utilizing more than one form of interaction between the stationary phase and analytes, such as cationic bond donating (cation-exchange multimodal chromatography) and / or metal affinity.

[0050] The term “depth filtration” refers to removing particles from a solution using a series of sequentially arranged filters with reduced pore sizes. In some embodiments, depth filter three- dimensional matrix creates a maze-like path through which the sample passes. The principleretention mechanisms of depth filters rely on random adsorption and mechanical entrapment throughout the depth of the matrix. In various embodiments, the filter membranes or sheets may be wound cotton, polypropylene, rayon cellulose, fiberglass, sintered metal, porcelain, diatomaceous earth, or other known components.

[0051] The term “protein preparation” refers to a composition comprising a protein of interest ready for downstream purification. In some embodiments, a protein preparation is a composition obtained via filtering cultures of cells secreting the protein (also referred to herein as a clarified harvest), for example, by removing cells and cell debris and / or by depth filtration. In some embodiments, without wishing to be bound by the theory, a protein preparation that is harvested via filtering cultures of cells secreting the protein (for example, by depth filtration) comprises contaminants released by cells in the same cultures, and such contaminants include - glucuronidase. In further embodiments, a protein preparation is or has been purified by contacting with a protein A affinity chromatography resin. Additionally or alternatively, a protein preparation is an antibody preparation.

[0052] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein, and are used in the broadest sense and specifically cover, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. “VHH,” as used herein, refers to a domain antibody derived from a variable region of a heavy chain only antibody.

[0053] The terms “multispecific binder” and “bispecific binders” refer to multispecific and bispecific binding agents, respectively, and include, but are not limited to multispecific and bispecific antibodies.

[0054] The term “ADC” refers to an antibody-drug conjugate, in which the antibody is coupled to another moiety which includes a drug (also referred to as a payload), such as a cytotoxic agent or another biologically-active compound.

[0055] As used herein, “drug” refers to a compound that has biological activity, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, and thelike), immunomodulating agent, kinase inhibitor, protein degrader, peptide, oligonucleotide, or another biologically-active moiety.

[0056] As used herein, a ligand of a resin refers to a functional group e.g., a phenyl functional group, a hydroxy functional group, a quaternary ammonium functional group, a phosphate group, a hydroxyl group, calcium, or a sulfonate functional group) of a resin specifically and reversibly bound to a protein (e.g., an antibody) and the protein is to be purified from a protein preparation by subjecting the preparation to the resin.

[0057] As used herein, a protein ligand refers to a ligand that is specifically bound to a protein, such as a ligand that is specifically bound to a cell-associated receptor(s) such as cell surface receptor(s).

[0058] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less variation of a given value or range.

[0059] As used herein, comparative terms as used herein, such as reduce, decrease, increase, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 fold, or about 3 fold, or about 4 fold, or about 5 fold, or about 10 fold, or about 20 fold, or about 30 fold, or about 40 fold, or about 100 fold or higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0060] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0061] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0062] The term “between” as used in a phrase as such “between A and B” or “between A- B” refers to a range including both A and B or any subranges thereof.

[0063] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both 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 embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0064] The term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances wherein the circumstance occurs, and the instances wherein the circumstance does not occur.5.2. Methods of Protein Purification

[0065] The present disclosure provides methods for purifying protein (e.g., antibody) preparations that contain P-glucuronidase, which methods may involve the use of specific anion exchange (AEX) chromatography, multimodal chromatography or hydrophobic interaction chromatography (HIC) resins, or the use of size exclusion chromatography (SEC), optionally in addition to a protein A purification step.

[0066] P-glucuronidase, also known as GUSB, GUS, PGlu, or P-D-glucuronoside glucuronosohydrolase (International Union of Biochemistry and Molecular Biology no. EC 3.2.1.31), is a glycoprotein acid hydrolase that belongs to glycosidase Family 2. P-glucuronidase exists as a tetrameric glycoprotein with four identical subunits. It localizes primarily in lysosomes and is found in virtually all mammalian cells. P-glucuronidase acts as an exoglycosidase to cleave glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Therefore, P-glucuronidase catalyzes deconjugation of P-D-glucuronides and degradation of glucuronic acid-containing glycosaminoglycans, playing an essential role in degradation of macromolecules. Moreover, P-glucuronidase is produced excessively by bacteria, for example, E. coli, Peptostreptococcus, Bacteroides, and Clostridium. By uncoupling glucuronides, P-glucuronidase can deconjugate the glucuronide conjugates of drug molecules.

[0067] In some embodiments, the resin described herein is packed in a column such that the corresponding chromatography is a column chromatography. Suitable columns can be in the form of, for example, conduits, tubes, or other structures having a relatively large ratio of flow length to flow cross section. The column can be, for example, in the size of 1 mL, 5 mL, 10 mL,25 mL, 50 mL, 100 mL, 250 mL, 0.5 L, 1 L, 2.5 L, 5 L, 10 L, 25 L, 50 L, 100 L, 250 L, 500 L, 1000 L, or 2000 L. In some embodiments, the resin described herein comprises a matrix and / or a supporting component, for example, agarose, beads, gel, a monolith, a membrane, and / or a nonwoven support. In specific embodiments, the resin described herein comprises an agarose matrix. In preferred embodiments, the resin described herein comprises a highly cross-linked agarose matrix, in particular a highly cross-linked rigid agarose matrix.

[0068] In one aspect, the present disclosure provides a method of purifying a protein (e.g., an antibody), wherein the method comprises subjecting a P-glucuronidase-containing protein (e.g., antibody) preparation e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to anion exchange (AEX) chromatography or multimodal chromatography using a resin. In some embodiments, a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group. Additionally or alternatively, the purified protein (e.g., antibody) is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0069] In another aspect, the present disclosure also provides a method of producing a conjugated protein (e.g., antibody), wherein the method comprises: (1) purifying a protein (e.g., an antibody) by subjecting a P-glucuronidase-containing protein (e.g., antibody) preparation (e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to AEX chromatography or multimodal chromatography using a resin; and (2) conjugating the purified protein (e.g., antibody) to an agent via a glucuronide-containing P-glucuronidase cleavable linker. In some embodiments, a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group.

[0070] In some embodiments, the ligand comprises a phenyl functional group and a hydroxy functional group. In some embodiments, the ligand comprises a phenyl functional group and a quaternary ammonium functional group. In some embodiments, the ligand comprises a hydroxy functional group and a quaternary ammonium cation functional group. In some embodiments, the ligand comprises a phenyl functional group, a hydroxy functional group, and a quaternaryammonium functional group. In certain embodiments, the ligand comprises an N-benzyl-2- hydroxy-N-methylethan-1 -ammoniumyl moiety. In certain embodiments, the ligand comprises or is

[0071] In certain embodiments, the ligand comprises or is 3-(3-(N-methyl-N-benzyl-2- hydroxylethylammonium)-2-hydroxypropoxy)-2-hydroxypropoxy.

[0072] In certain embodiments, the resin described herein comprises a high-flow agarose matrix. In certain embodiments, the resin described herein comprises a highly cross-linked agarose matrix (in particular, a highly cross-linked agarose rigid matrix). In certain embodiments, the resin described herein has a d50v particle size of between 25 pm and 100 pm (d50v is the average particle size of the cumulative volume distribution). In certain embodiments, the resin described herein has a d50v particle size of about 95 pm. In certain embodiments, the resin described herein has a d50v particle size of about 90 pm. In certain embodiments, the resin described herein has a d50v particle size of about 85 pm. In certain embodiments, the resin described herein has a d50v particle size of about 80 pm. In certain embodiments, the resin described herein has a d50v particle size of about 75 pm. In certain embodiments, the resin described herein has a d50v particle size of about 70 pm. In certain embodiments, the resin described herein has a d50v particle size of about 65 pm. In certain embodiments, the resin described herein has a d50v particle size of about 60 pm. In certain embodiments, the resin described herein has a d50v particle size of about 55 pm. In certain embodiments, the resin described herein has a d50v particle size of about 50 pm. In certain embodiments, the resin described herein has a d50v particle size of about 45 pm. In certain embodiments, the resin described herein has a d50v particle size of about 40 pm. In certain embodiments, the resin described herein has a d50v particle size of about 35 pm. In certain embodiments, the resin described herein has a d50v particle size of about 30 pm. In certain embodiments, the resin described herein has a total ionic capacity of 0.09 to 0.12 mmol Cl- / mL medium. In certain embodiments, the resin described herein has a flow velocity of at least 600 cm / h in a 1 m diameter column with 20 cm bed height at 20 °C using process buffers with thesame viscosity as water at < 3 bar (0.3 Mpa). In certain embodiments, the resin described herein has a short-term clean in place (CIP) stability pH interval of 2 to 14, which is the pH interval where the medium can be subjected to cleaning or sanitization in place. Cleaning in place is a procedure that removes impurities such as lipids, endotoxins, nucleic acids and precipitated or denatured proteins that remain in the packed column after regeneration. In certain embodiments, the resin described herein has an operational stability pH interval of 3 to 12, which is the pH interval where the medium can be operated without significant change in function. In certain embodiments, the resin described herein has a working temperature of 4 °C to 30 °C. In certain embodiments, the resin described herein has a chemical stability for all commonly used aqueous buffers, 1 M acetic acid, and / or 1 M sodium hydroxide. In certain embodiments, there is no significant change in ionic binding capacity and carbon content after storage of the resin described herein for one week in 1 M NaOH at 40 °C. In certain embodiments, oxidizing agents and anionic detergents should be avoided when using the resin described herein. In certain embodiments, the suitable storage condition for the resin described herein is 4 °C to 30 °C in 20% ethanol.

[0073] In certain embodiments, the resin described herein is Capto™ Adhere.

[0074] In another aspect, the present disclosure further provides a method of purifying a protein (e.g., an antibody), wherein the method comprises subjecting a P-glucuronidase- containing protein (e.g., antibody) preparation (e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin. In some embodiments, a ligand of the resin comprises a phenyl functional group. Additionally or alternatively, the purified protein (e.g., antibody) is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0075] In another aspect, the present disclosure also provides a method of producing a conjugated protein (e. ., antibody), wherein the method comprises: (1) purifying a protein (e.g., an antibody) by subjecting a P-glucuronidase-containing protein (e.g., antibody) preparation (e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to AEX chromatography or HIC using a resin, wherein a ligand of theresin comprises a phenyl functional group; and (2) conjugating the purified protein (e.g., antibody) to an agent via a glucuronide-containing [3-glucuronidase cleavable linker.

[0076] In some embodiments, the ligand comprises a phenoxy moiety. In certain embodiments, the ligand is phenoxy.

[0077] In certain embodiments, the resin described herein comprises a high-flow agarose matrix. In certain embodiments, the resin described herein comprises a highly cross-linked agarose matrix (in particular, a highly cross-linked agarose rigid matrix). In certain embodiments, the resin described herein has a d50v particle size of between 25 pm and 100 pm. In certain embodiments, the resin described herein has a d50v particle size of about 95 pm. In certain embodiments, the resin described herein has a d50v particle size of about 90 pm. In certain embodiments, the resin described herein has a d50v particle size of about 85 pm. In certain embodiments, the resin described herein has a d50v particle size of about 80 pm. In certain embodiments, the resin described herein has a d50v particle size of about 75 pm. In certain embodiments, the resin described herein has a d50v particle size of about 70 pm. In certain embodiments, the resin described herein has a d50v particle size of about 65 pm. In certain embodiments, the resin described herein has a d50v particle size of about 60 pm. In certain embodiments, the resin described herein has a d50v particle size of about 55 pm. In certain embodiments, the resin described herein has a d50v particle size of about 50 pm. In certain embodiments, the resin described herein has a d50v particle size of about 45 pm. In certain embodiments, the resin described herein has a d50v particle size of about 40 pm. In certain embodiments, the resin described herein has a d50v particle size of about 35 pm. In certain embodiments, the resin described herein has a d50v particle size of about 30 pm. In certain embodiments, the resin described herein has a ligand density of about 27 pmol / mL medium. In certain embodiments, the resin described herein has a dynamic binding capacity of about 27 mg bovine serum albumin (BSA) / mL medium at QB 10% (10% breakthrough). In certain embodiments, the resin described herein has a dynamic binding capacity of about 19 mg BSA / mL resin at 10% breakthrough measured at a residence time of 4 min (150 cm / h) in Tricorn™ 5 / 100 column with 10 cm bed height, 0.1 M sodium phosphate buffer, and 1.2 M ammonium sulfate, pH 7. In certain embodiments, the resin described herein has a hydrophobicity of 45 to 50 min retention of lyzosyme. In certain embodiments, the resin described herein has a suitable liquid velocity of 150 to 350 cm / h using water at roomtemperature. Tn certain embodiments, the resin described herein has a liquid velocity of at least 600 cm / h in a 1 m column with 20 cm bed height at 20°C using process buffers with the same viscosity as water, which corresponds to a residence time of 2 min. In certain embodiments, the resin described herein has a flow velocity of at least 220 cm / h in a 1 m diameter column with a bed height of 20 cm at 20°C measured using process buffers with the same viscosity as water at 300 kPa. In certain embodiments, the resin described herein has a pH working range of 3 to 13. In certain embodiments, the resin described herein has a short-term stability pH interval of 2 to 14, which is the pH interval where the medium can be subjected to cleaning- or sanitization- in place without significant change in function. In certain embodiments, the resin described herein has a long-term stability pH interval of 3 to 13, which is the pH interval where the medium can be operated without significant change in function. In certain embodiments, the resin described herein has a chemical stability in commonly used aqueous buffers: 1 M sodium hydroxide, 1 M acetic acid, 8 M urea, 6 M guanidine hydrochloride, 70% ethanol, and / or 30% isopropanol. In certain embodiments, there is no significant change in function after one month storage of the resin described herein in 1 M NaOH at ambient temperature. In certain embodiments, the suitable storage condition for the resin described herein is 4 °C to 30 °C in 20% ethanol.

[0078] In some embodiments, the resin described herein is Capto™ Phenyl. In certain embodiments, the resin is Capto™ Phenyl (high sub). In certain embodiments, the resin is Capto™ Phenyl ImpRes.

[0079] In another aspect, the present disclosure further provides a method of purifying a protein (e.g., an antibody), wherein the method comprises subjecting a [3-glucuronidase- containing protein (e.g., an antibody) preparation (e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to a multimodal chromatography using a resin. Additionally or alternatively, the purified protein (e.g., an antibody) is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0080] In another aspect, the present disclosure also provides a method of producing a conjugated protein (e.g., an antibody), wherein the method comprises: (1) purifying a protein (e.g., an antibody) by subjecting a -glucuronidase-containing protein (e.g., an antibody) preparation (e.g., a composition obtained via filtering cultures of cells secreting the protein (forexample, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to a multimodal chromatography using a resin; and (2) conjugating the purified protein (e.g., an antibody) to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0081] In some embodiments, a ligand of the resin comprises a phosphate group, calcium (e.g., Ca2+), a hydroxyl group, or any combination thereof. In some embodiments, a ligand of the resin comprises a phosphate group. In some embodiments, a ligand of the resin comprises calcium (e.g., Ca2+). In some embodiments, a ligand of the resin comprises a phosphate group and calcium (e.g., Ca2+). In some embodiments, a ligand of the resin comprises a phosphate group and a hydroxyl group. In some embodiments, a ligand of the resin comprises calcium (e.g., Ca2+) and a hydroxyl group. In some embodiments, a ligand of the resin comprises a phosphate group, calcium (e.g., Ca2+), and a hydroxyl group.

[0082] In some embodiments, the multimodal chromatography comprises any one or more of the following functional groups: Ca21, a phosphate group (PO4), and / or a hydroxyl group (-OH). In certain embodiments, the multimodal chromatography comprises the following functional groups: Ca2+, a phosphate group (PO4), and a hydroxyl group (-OH).

[0083] In some embodiments, the multimodal chromatography is a cation exchange (CEX) multimodal chromatography. In further embodiments, a ligand of the resin comprises a phosphate group. In some embodiments, the multimodal chromatography is an anion exchange (AEX) multimodal chromatography. In further embodiments, a ligand of the resin comprises calcium (e.g., Ca2+). In yet further embodiments, a ligand of the resin comprises a hydroxyl group (-OH).

[0084] In certain embodiments, the resin described herein is EconoFit CHT XT.

[0085] In certain embodiments, the resin described herein comprises a ceramic hydroxyapatite matrix. In some embodiments, hydroxyapatite is a crystalline mineral of calcium and phosphate. In some embodiments, the ligand and the matrix are the same. Additionally or alternatively, the matrix is represented by the following formula: Caio(P04)e(OH)2. In some embodiments, the multimodal chromatography provides cation exchange and calcium affinity interactions. In certain embodiments, the resin described herein has a particle size of between 10 pm and 100 pm. In certain embodiments, the resin described herein has a particle size of about 100 pm. In certain embodiments, the resin described herein has a particle size of about 90 pm.In certain embodiments, the resin described herein has a particle size of about 80 pm. In certain embodiments, the resin described herein has a particle size of about 75 pm. In certain embodiments, the resin described herein has a particle size of about 70 pm. In certain embodiments, the resin described herein has a particle size of about 65 pm. In certain embodiments, the resin described herein has a particle size of about 60 pm. In certain embodiments, the resin described herein has a particle size of about 55 pm. In certain embodiments, the resin described herein has a particle size of about 50 pm. In certain embodiments, the resin described herein has a particle size of about 45 pm. In certain embodiments, the resin described herein has a particle size of about 40 pm. In certain embodiments, the resin described herein has a particle size of about 35 pm. In certain embodiments, the resin described herein has a particle size of about 30 pm. In certain embodiments, the resin described herein has a particle size of about 25 pm. In certain embodiments, the resin described herein has a particle size of about 20 pm. In certain embodiments, the resin described herein has a particle size of about 15 pm. In certain embodiments, the resin described herein has a particle size of about 10 pm. In certain embodiments, the resin described herein has a nominal mean particle size of 40 ± 4 pm. In certain embodiments, the resin binds to molecules of neutral and basic nature. In certain embodiments, the resin described herein has an observed dynamic binding capacity of about 17.4-24.8 mg lysozyme / g resin. In certain embodiments, the resin described herein has an observed dynamic binding capacity at about >60 mg / mL IgG at 100 cm / hr with 5 mM sodium phosphate, 25 mM sodium chloride, pH 7 in a 0.5 x 9.5 cm column. In certain embodiments, the resin described herein has a tap-settled density of about 0.67 g / mL tap settled bed. In certain embodiments, the resin described herein has a typical linear flow rate range of about 50 to about 400 cm / hr. In certain embodiments, the resin described herein has a pH stability range of 6.5 to 14. In certain embodiments, the resin described herein has a base stability of at least 1 year in 1 N NaOH. In certain embodiments, the suitable regeneration condition for the resin described herein is 0.4 M sodium phosphate, pH 7-7.5 and / or 1 M trisodium phosphate, pH 11-12. In certain embodiments wherein a higher concentration of sodium phosphate is needed for regeneration for the resin described herein, the suitable regeneration condition for the resin described herein is about 0.4 to about 1 M potassium phosphate. In certain embodiments, the sanitization condition for the resin described herein is about 1 to about 2 N NaOH. In certainembodiments, the recommended column storage of the resin described herein is about 0.1 N NaOH.

[0086] In various embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of between 5 and 8. In certain embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of about 5.5. In certain embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of about 6.0. In certain embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of about 6.5. In certain embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of about 7.0. In certain embodiments, the AEX, multimodal or HIC chromatography is performed at a pH of about 7.5.

[0087] In various embodiments, the method described herein further comprises a protein A purification step before the AEX, multimodal or HIC chromatography step, wherein optionally the protein A purification step produces the protein (e.g., antibody) preparation. In some embodiments, the protein A purification step uses a resin. In certain embodiments, a ligand of the resin is Repligen® NGL-ImpactTM A ligand. In certain embodiments, the resin described herein comprises a highly cross-linked agarose matrix. In some embodiments, the resin described herein has a dynamic binding capacity of up to 70 mg mAb / mL resin. In some embodiments, the resin described herein has a dynamic binding capacity of up to 80 mg human IgG (hIgG) / mL resin. In some embodiments, the resin described herein has an average particle size of about 50 pm. In some embodiments, the particle size range of the resin described herein is >95% between 35-90 pm. In some embodiments, the resin described herein has a flow rate of up to 200 cm / h. In some embodiments, the resin described herein has a working stability pH range of 3 to 12. In some embodiments, the resin described herein has a short-term stability pH range of 3 to 14. In certain embodiments, the suitable storage condition for the resin described herein is 2 °C to 8 °C in 20% ethanol. In some embodiments, the resin is Praesto® Jetted A50.

[0088] In various embodiments, the method described herein further comprises subjecting the purified protein (e.g., antibody) preparation obtained from the AEX, multimodal or HIC chromatography step to a cation exchange (CEX) chromatography step. In some embodiments, the CEX chromatography step uses a resin, and a ligand of the resin comprises a sulfonate functional group. In certain embodiments, the ligand comprises a sulfopropyl moiety. In certainembodiments, the ligand comprisesIn certain embodiments, the resin described herein comprises a cross-linked agarose matrix (e.g., a 6% highly crosslinked agarose). In certain embodiments, the resin described herein has an ionic capacity of 0.11-0.16 mmol / mL resin. In certain embodiments, the resin described herein has an ionic capacity of 0.18-0.25 mmol / mL resin. In certain embodiments, the resin described herein has a d50v particle size of between 25 pm and 100 pm. In certain embodiments, the resin described herein has a d50v particle size of about 95 pm. In certain embodiments, the resin described herein has a d50v particle size of about 90 pm. In certain embodiments, the resin described herein has a d50v particle size of about 85 pm. In certain embodiments, the resin described herein has a d50v particle size of about 80 pm. In certain embodiments, the resin described herein has a d50v particle size of about 75 pm. In certain embodiments, the resin described herein has a d50v particle size of about 70 pm. In certain embodiments, the resin described herein has a d50v particle size of about 65 pm. In certain embodiments, the resin described herein has a d50v particle size of about 60 pm. In certain embodiments, the resin described herein has a d50v particle size of about 55 pm. In certain embodiments, the resin described herein has a d50v particle size of about 50 pm. In certain embodiments, the resin described herein has a d50v particle size of about 45 pm. In certain embodiments, the resin described herein has a d50v particle size of about 40 pm. In certain embodiments, the resin described herein has a d50v particle size of about 35 pm. In certain embodiments, the resin described herein has a d50v particle size of about 30 pm. In certain embodiments, the resin described herein has a flow velocity of at least 400 cm / h at 3 bar in a 2.6 x 20 cm column (packed at 4 bar). In certain embodiments, the resin described herein has a flow velocity of 400 to 700 cm / h. In certain embodiments, the resin described herein has a binding capacity of >70 mg IgG per mb resin at 6’ residence time. In some embodiments, the resin described herein has a dynamic binding capacity of about 70 mg ribonuclease A / mL medium. In certain embodiments, the resin described herein has a short-term stability pH interval of 3 to 14. In certain embodiments, the resin described herein has a long-term stability pH interval of 4 to 13. In certain embodiments, the resin described herein has a working temperature of 4-30 °C. In certain embodiments, the resin described herein has a chemical stability in commonly used aqueous buffers, 1 M sodium hydroxide, 8 M urea, 6 M guanidine hydrochloride, 70% ethanol, and / or 30% isopropanol. Incertain embodiments, the suitable storage condition for the resin described herein is 4 °C to 30 °C in 20% ethanol, 0.2M sodium acetate. In certain embodiments, oxidizing agents and cationic detergents should be avoided when using the resin described herein. In some embodiments, the resin is Praesto® SP65. In some embodiments, the resin is SP Sepharose™ Fast Flow ion exchange chromatography media.

[0089] In various embodiments, the method described herein results in no or undetectable - glucuronidase activity in the purified protein (e.g., antibody) preparation. In various embodiments, the method described herein results in an at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% reduction in -glucuronidase activity in the purified protein (e.g., antibody) preparation, for example, compared to the unpurified protein (e.g., antibody) preparation or to a protein (e.g, an antibody) preparation purified using a control method (such as without the respective AEX, multimodal or HIC chromatography step).

[0090] In various embodiments, the method described herein results in no loss of glucuronide on the conjugation product of the purified protein (e.g., antibody). In various embodiments, the method described herein results in a less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% loss of glucuronide on the conjugation product of the purified protein (e.g., antibody), for example, compared to the unpurified protein (e.g., antibody) preparation or to a protein (e.g., an antibody) preparation purified using a control method (such as without the respective AEX, multimodal or HIC chromatography step).

[0091] In another aspect, the present disclosure further provides a method of purifying a protein (e.g., an antibody), wherein the method comprises subjecting a P-glucuronidase- containing protein (e.g., antibody) preparation to size exclusion chromatography (SEC). In some embodiments, the purified protein (e.g., antibody) is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker. Additionally or alternatively, fractions eluting molecules larger than the protein (e.g., antibody) are purified away.

[0092] In another aspect, the present disclosure also provides a method of producing a conjugated protein (e.g., antibody), wherein the method comprises: (1) purifying a protein (e.g., an antibody) by subjecting a P-glucuronidase-containing protein (e.g., antibody) preparation(e.g., a composition obtained via filtering cultures of cells secreting the protein (for example, by depth filtration) and optionally purification by contacting with a protein A affinity chromatography resin) to SEC, wherein fractions eluting molecules larger than the protein (e.g., antibody) are purified away; and (2) conjugating the purified protein (e.g., antibody) to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0093] In some embodiments, the method described herein further comprises a protein A purification step before the SEC step, wherein optionally the protein A purification step produces the protein (e.g., antibody) preparation.

[0094] In some embodiments, the method described herein results in an at least 99% reduction in P-glucuronidase activity in the purified protein (e.g., antibody) preparation, for example, compared to the unpurified protein (e.g, antibody) preparation or to a protein (e.g, an antibody) preparation purified using a control method (such as without the SEC chromatography step). In some embodiments, the method described herein results in an at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% reduction in P-glucuronidase activity in the purified protein (e.g., antibody) preparation, for example, compared to the unpurified protein (e.g., antibody) preparation or to a protein (e.g, an antibody) preparation purified using a control method (such as without the SEC chromatography step). In some embodiments, the method described herein results in no or undetectable P- glucuronidase activity in the purified protein (e.g., antibody) preparation.

[0095] In some embodiments, the method described herein results in less than 1% loss of glucuronide on the conjugation product of the purified protein (e.g., antibody), for example, compared to the unpurified protein (e.g., antibody) preparation or to a protein (e.g., an antibody) preparation purified using a control method (such as without the SEC chromatography step). In some embodiments, the method described herein results in a less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, or less than 2% loss of glucuronide on the conjugation product of the purified protein (e.g., antibody), for example, compared to the unpurified protein (e.g., antibody) preparation or to a protein (e.g., an antibody) preparation purified using a control method (such as without the SEC chromatography step). In some embodiments, the method described herein results in no loss of glucuronide on the conjugation product of the purified protein (e.g., antibody).

[0096] In preferred embodiments, the glucuronide-containing P-glucuronidase cleavable linker is a P-glucuronide-containing P-glucuronidase cleavable linker. In various embodiments, removal of the glucuronide from the P-glucuronidase cleavable linker directly triggers agent (e.g., payload) release. In various embodiments, the glucuronide-containing P-glucuronidase cleavable linker comprises a cleavable peptide motif (z.e., a peptide motif that can be cleaved by a protease). In certain embodiments, the cleavable peptide motif is protected from proteolysis by a glucuronic acid (e.g., a neighboring glucuronic acid). In certain embodiments, the glucuronide- containing P-glucuronidase cleavable linker comprises a dipeptide unit shielded by a neighboring glucuronic acid. Additional suitable linkers and drug linker conjugations can be found at, for example, International Patent Application Publication Nos. WO 2020 / 154437, WO 2022 / 109335, WO 2022 / 155347, WO 2022 / 155362, WO 2022 / 187370, and WO 2023 / 009759, each of which is incorporated herein by reference in its entirety. Also, suitable therapeutic conjugates and linker payloads can be found at, for example, International Patent Application Publication Nos. WO 2020 / 154437, WO 2022 / 109335, WO 2022 / 155347, WO 2022 / 155362, WO 2022 / 187370. and WO 2023 / 009759 (e.g., Compound 8), each of which is incorporated herein by reference in its entirety.

[0097] In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a diagnostic protein.

[0098] In some embodiments, the protein is an antibody, an Fc-fusion protein, a bispecific binder, an scFv, a VHH, a VHH-Fc, or a protein ligand (for example, for targeting a cell- associated receptor(s) such as cell surface receptor(s)). In some embodiments, the protein comprises an Fc. Additionally or alternatively, contacting with a Protein A affinity chromatography resin is used to purify a composition comprising the protein (for example, an antibody preparation) yet fails to remove P-glucuronidase sufficiently.

[0099] In some embodiments, the protein is an antibody.

[0100] In some embodiments, the protein (e.g., antibody) described herein targets a tumor antigen (for example, a cell surface tumor antigen). In some embodiments, the protein (e.g., antibody) described herein targets an antigen of a pathogen (e.g., a virus, bacterium, fungus, helminth or protist) (for example, a pathogen’s antigen that is expressed at the cell surface).

[0101] In various embodiments, the agent (to be conjugated to the protein (e.g., antibody)) is a cytotoxic drug, immunomodulating agent, kinase inhibitor, protein degrader, peptide, oligonucleotide, or other biologically-active moiety. In various embodiments, the agent (to be conjugated to the protein (e.g., antibody)) is a drug, for example a cytotoxic drug (such as auristatin or topoisomerase I inhibitor). In various embodiments, the conjugation product of the purified protein (e.g., antibody) is an antibody-drug conjugate (ADC).

[0102] Examples of drugs that may be used in the instant invention include small molecule drugs, such as a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non- peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.

[0103] Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96 / 33212, WO 96 / 14856, and U.S. 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Set. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrol obenzodi azepine (PBD)).

[0104] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CYNOTAN™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0105] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U®), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine(6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0106] Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel (TAXOTERE®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, e.g. etoposide, teniposide, and the like; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, and the like; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; aziri nopyrrol o indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like; and the like.

[0107] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafme.

[0108] Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (TAXOL®), TAXOL® derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0109] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, and the like; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; and the like; adrenocortical suppressants, e.g. aminoglutethimide; 17a-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chi orotriani sene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide(DROGENIL®), toremifene (FARESTON®), and goserelin (ZOLADEX®), and the like. Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0110] Other suitable chemotherapeutic agents include metal complexes, e.g. cisplatin (cis- DDP), carboplatin, and the like; ureas, e.g. hydroxyurea; hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; and the like. Other anti-proliferative agents of interest include immunosuppressants, e.g. mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); gefitinib (IRESSA®, ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7- methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); and the like.

[0111] Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL®, TAXOTERE® (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) can be readily prepared utilizing techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis). Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TAXOTERE® docetaxel, as noted herein) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0112] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0113] Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-a; (7) IFN-y; (8) colonystimulating factors; and (9) inhibitors of angiogenesis.

[0114] The present invention also contemplates use of a chromatography resin described herein, a chromatography step and / or condition described herein, or the size exclusion chromatography method described herein for controlling or modulating the concentration of 0- glucuronidase or the level of 0-glucuronidase activity in the purified protein preparation (z.e., the purified antibody preparation), for example, to achieve a desirable concentration or level.

[0115] In various embodiments, the method further comprises controlling or modulating 0- glucuronidase concentration and / or 0-glucuronidase activity level in the purified protein preparation.

[0116] In some embodiments, the controlling or modulating step comprises controlling residence time in (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC.

[0117] In some embodiments, the controlling or modulating step comprises controlling residence time in the CEX chromatography.

[0118] In some embodiments, the controlling or modulating step comprises controlling residence time in the protein A purification step.

[0119] In some embodiments, the controlling or modulating step comprises modulating pH at which (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC, is performed.

[0120] In some embodiments, the controlling or modulating step comprises modulating pH at which the CEX chromatography is performed.

[0121] In some embodiments, the controlling or modulating step comprises modulating pH at which the protein A purification step is performed.6. ILLUSTRATIVE EMBODIMENTS

[0122] The present disclosure encompasses the following non-limiting embodiments:

[0123] 1. A method of purifying a protein, comprising subjecting a 0-glucuronidase- containing protein preparation to anion exchange (AEX) chromatography or multimodal chromatography using a resin, wherein a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing 0-glucuronidase cleavable linker.

[0124] 2. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a 0-glucuronidase-containing protein preparation to AEX chromatography or multimodal chromatography using a resin, wherein a ligand of the resin comprises any one or more (such as any two or all three) of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group; and(2) conjugating the purified protein to an agent via a glucuronide-containing 0- glucuronidase cleavable linker.

[0125] 3. The method of embodiment 1 or 2, wherein the ligand comprises a phenyl functional group and a hydroxy functional group.

[0126] 4. The method of embodiment 1 or 2, wherein the ligand comprises a phenyl functional group and a quaternary ammonium functional group.

[0127] 5. The method of embodiment 1 or 2, wherein the ligand comprises a hydroxy functional group and a quaternary ammonium cation functional group.

[0128] 6 The method of embodiment 1 or 2, wherein the ligand comprises a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group.

[0129] 7. The method of embodiment 6, wherein the ligand comprises an N-benzyl-2- hydroxy-N-methylethan- 1 -ammoniumyl moiety.

[0130] 8. The method of embodiment 7, wherein the ligand is

[0131] 9 The method of any one of embodiments 1-8, wherein the resin is Capto™ Adhere.

[0132] 10. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin, wherein a ligand of the resin comprises a phenyl functional group, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0133] 11 A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to AEX chromatography or HIC using a resin, wherein a ligand of the resin comprises a phenyl functional group; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

[0134] 12. The method of embodiment 10 or 11, wherein the ligand comprises a phenoxy moiety.

[0135] 13. The method of embodiment 12, wherein the ligand is phenoxy.

[0136] 14. The method of embodiment 13, wherein the resin is Capto™ Phenyl.

[0137] 15. The method of embodiment 14, wherein the resin is Capto™ Phenyl (high sub).

[0138] 16. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to a multimodal chromatography using a resin, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0139] 17. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to a multimodal chromatography using a resin; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

[0140] 18. The method of embodiment 16 or 17, wherein a ligand of the resin comprises a phosphate group, calcium, a hydroxyl group, or any combination thereof.

[0141] 19. The method of any one of embodiments 16-18, wherein a ligand of the resin comprises a phosphate group.

[0142] 20. The method of any one of embodiments 16-19, wherein a ligand of the resin comprises a phosphate group, and calcium.

[0143] 21. The method of any one of embodiments 16-20, wherein a ligand of the resin comprises a phosphate group, calcium, and a hydroxyl group.

[0144] 22. The method of any one of embodiments 19-21, wherein the chromatography is a cation exchange (CEX) chromatography.

[0145] 23. The method of any one of embodiments 16-18, wherein a ligand of the resin comprises calcium.

[0146] 24. The method of any one of embodiments 16-18, wherein a ligand of the resin comprises calcium and a hydroxyl group.

[0147] 25. The method of any one of embodiments 16-18, 20-21 and 23-24, wherein the chromatography is an anion exchange (AEX) chromatography.

[0148] 26. The method of any one of embodiments 16-25, wherein the resin is EconoFitCHT XT.

[0149] 27. The method of any one of embodiments 1-26, wherein the chromatography is performed at a pH of between 5 and 8.

[0150] 28. The method of embodiment 27, wherein the chromatography is performed at a pH of about 5.5.

[0151] 29. The method of embodiment 27, wherein the chromatography is performed at a pH of about 6.5.

[0152] 30. The method of embodiment 27, wherein the chromatography is performed at a pH of about 7.5.

[0153] 31. The method of any one of embodiments 1-30, which further comprises a proteinA purification step before the chromatography step, wherein optionally the protein A purification step produces the protein preparation.

[0154] 32. The method of any one of embodiments 1-31 , which further comprises subjecting the purified protein preparation obtained from the chromatography step to a cation exchange (CEX) chromatography step.

[0155] 33. The method of embodiment 32, wherein the CEX chromatography step uses a resin, and a ligand of the resin comprises a sulfonate functional group.

[0156] 34. The method of embodiment 33, where the ligand comprises a sulfopropyl moiety.

[0157] 35. The method of embodiment 34, wherein the ligand is

[0158] 36. The method of embodiment 35, wherein the resin is Praesto® SP65.

[0159] 37. The method of embodiment 35, wherein the resin is SP Sepharose™ Fast Flow ion exchange chromatography media.

[0160] 38. The method of any one of embodiments 1-37, which results in no or undetectableP-glucuronidase activity in the purified protein preparation.

[0161] 39. The method of any one of embodiments 1-38, which results in no loss of glucuronide on the conjugation product of the purified protein.

[0162] 40. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to size exclusion chromatography (SEC), wherein fractions eluting molecules larger than the protein are purified away, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

[0163] 41. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to SEC, wherein fractions eluting molecules larger than the protein are purified away; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

[0164] 42. The method of embodiment 40 or 41, which results in less than 1% loss of glucuronide on the conjugation product of the purified protein.

[0165] 43. The method of any one of embodiments 1-42, wherein the agent is a cytotoxic drug.

[0166] 44. The method of any one of embodiments 1-43, wherein the protein is a therapeutic protein.

[0167] 45. The method of any one of embodiments 1-44, wherein the protein is an antibody, an Fc-fusion protein, a bispecific binder, an scFv, a VHH, a VHH-Fc, or a protein ligand.

[0168] 46. The method of any one of embodiments 1-45, wherein the protein is an antibody.

[0169] 47. The method of embodiment 46, wherein the conjugation product of the purified protein is an antibody-drug conjugate (ADC).

[0170] 48. The method of any one of embodiments 1-47, wherein the glucuronide-containingP -glucuronidase cleavable linker comprises a cleavable peptide motif.

[0171] 49. The method of embodiment 48, wherein the cleavable peptide motif is protected from proteolysis by a glucuronic acid.

[0172] 50. The method of any one of embodiments 1-49, wherein the glucuronide-containingP-glucuronidase cleavable linker comprises a dipeptide unit shielded by a neighboring glucuronic acid.

[0173] 51. The method of any one of embodiments 1-50, which further comprises controllingP-glucuronidase concentration and / or P-glucuronidase activity level in the purified protein preparation.

[0174] 52. The method of embodiment 51, wherein the controlling step comprises controlling residence time in (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC.

[0175] 53 The method of embodiment 51 or 52, wherein the controlling step comprises controlling residence time in the CEX chromatography.

[0176] 54. The method of any one of embodiments 51-53, wherein the controlling step comprises controlling residence time in the protein A purification step.

[0177] 55. The method of any one of embodiments 51-54, wherein the controlling step comprises modulating pH at which (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC, is performed.

[0178] 56. The method of any one of embodiments 51-55, wherein the controlling step comprises modulating pH at which the CEX chromatography is performed.

[0179] 57. The method of any one of embodiments 51-56, wherein the controlling step comprises modulating pH at which the protein A purification step is performed.

[0180] It is understood that modifications which do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Accordingly, the following Example is intended to illustrate but not limit the present disclosure.7. EXAMPLE

[0181] Antibody preparations were (1) harvested via filtering cultures of cells secreting the antibody (for example, by depth filtration), and (2) purified, such as using Protein A chromatography, as described in the art. After conjugating the antibodies with a payload via a linker, the produced antibody-drug conjugates (ADCs) were then subjected to hydrophobic interaction chromatography (HIC) for purity analysis. As expected, observed was a major HIC peak representing the ADC having the desirable DAR (such as one antibody conjugated to a certain number of payloads as designed in a site-specific conjugation) (see, for example, the DAR2 peaks in FIGs. 2-3), as well as minor peaks representing other possible DAR species (such as antibodies not saturated with payload conjugations) (see, for example, the DAR0 and DARI peaks in FIGs. 2-3). Surprisingly, additional peaks were consistently observed following the major peak (see, for example, the peaks labeled as DAR2 #2 and DAR2 #3 in FIGs. 2-3), indicating certain systematic contamination, which would, in turn, significantly decrease the stability of the conjugation product (e.g., ADC). Accordingly, several experiments were designed and performed to explore this issue (data not shown). As a result, P-glucuronidase contamination was identified as the main cause of this impurity issue of the ADC product. See, for example, Section 7.2.1. Further, solutions to this contamination issue were investigated and provided herein. See, for example, Sections 7.2.2-7.2.8.

[0182] The presence of a process-related contaminant (P-glucuronidase) was detected in certain antibody preparations. When present, this contaminant could degrade a chemical linker used to make a therapeutic conjugate. For example, a linker containing a tandem-cleavage element paired with a cytotoxic payload, such as an auristatin or topoisomerase I inhibitor, couldbe conjugated to an antibody to make an antibody-drug conjugate (ADC). The tandem-cleavage element consists of a dipeptide unit shielded by a neighboring glucuronic acid. The dipeptide is resistant to proteolytic cleavage while the monosaccharide is intact. Once the glucuronic acid has been removed by glucuronidase (a process that in the body would happen inside a lysosome after ADC internalization and trafficking), then the dipeptide would be unshielded and available for cleavage by proteases, such as Cathepsin B, followed by release of the payload.

[0183] When the process-related contaminant (P-glucuronidase) was present in the purified antibody (drug substance intermediate) to be used for conjugation and ADC production, then the enzymatic activity of the contaminant could affect removal of the glucuronic acid from the chemical linker during the conjugation process and / or after the ADC was produced and was in formulation buffer. This degradation process was facilitated by the fact that the glucuronidase enzyme is active in acidic conditions, which mimic the acidic conjugation conditions required for some conjugation approaches, and the acidic formulation (storage) conditions preferred by most proteins, antibodies, and ADCs.7.1. Methods7.1.1. Glucuronidase activity assay to quantify active glucuronidase in monoclonal antibody (mAb) preparations

[0184] Residual P-glucuronidase was monitored throughout the downstream process of antibody preparation by two orthogonal techniques. For the determination of GUSB (P- glucuronidase) concentration by activity, samples were incubated in a sodium acetate buffer at 37 °C with 4-methylumbelliferyl P-D-glucuronide which was cleaved into 4- methylumbelliferone and in turn measured fluorescently with Ex / Em wavelengths of 360 / 450 nm or 330 / 450 nm. Calibration was performed with recombinant human GUSB prepared from 10 to 50 ng / mL.7.1.2. HIC method to quantify glucuronide loss on ADCs (as a proxy for |J- glucuronidase levels in mAb preparation)

[0185] Purified antibodies generated from various purification processes (3-15 mg / mL) were conjugated to linker-payloads comprising a tandem-cleavage linker at 0.85 mM in the absence (FIG. 1) or presence (FIG. 4B) of excess Ac-Glucoronide-PABA as illustrated in FIG.4A at 0.85 mM, in order to produce ADCs having a drug-to-antibody ratio (DAR) of 2.

[0186] Reactions proceeded for 72 h at 37 °C in 20 mM sodium citrate, 50 mM NaCl pH 5.5 buffer or 20 mM histidine, pH 5.5, 6% trehalose containing 0.85% dimethylacetamide (DMA). After conjugation, ADCs were buffer exchanged to remove residual free drug. To determine the percent glucuronide loss, the peaks corresponding to a DAR of 2 (DAR2) of the ADC were quantified by analytical chromatography using hydrophobic interaction chromatography (HIC) (Tosoh #14947). An example calculation of glucuronide loss using the HIC method is shown in Table 1 and FIG. 1.

[0187] Table 1. Calculation of Glucuronide Loss7.1.3. Mass spectrometry assay to quantify P-glucuronidase protein levels in mAb preparations

[0188] For the determination of GUSB concentration by LC-MS, samples were spiked with internal standards, exchanged into a Tris buffer, digested overnight with trypsin, reduced with DTT at 95 °C, separated by reversed-phase LC, and detected with MS. The top two or three peptides of each spiked protein were used for quantitation with the average reported as the measured quantity.7.1.4. Capto™ Adhere FPLC method

[0189] An AKTA Pure instrument and the necessary buffer lines were cleaned with 0.1 N NaOH, washed with DI / MilliQ and equilibrated with 20 mM Sodium Citrate pH 5.5 buffer. HiTrap Capto™ Adhere column was attached to AKTA Pure and was washed with 5 column volumes (CVs) of 0.1 NNaOH, followed by equilibration with 5-10 CVs 20 mM citrate pH 5.5 until pH reached 5.5 ± 0.1. Antibodies purified using Protein-A affinity method were loaded on to the column at a flow rate of 1 mL / min (1 mL columns, Cytvia #28405844), or 5 mL / min (5 mL columns, Cytvia #28405846) and the column was washed using 6 CVs of 20 mM Citrate pH 5.5 buffer. The unbound sample was collected, buffer exchanged into appropriate buffer and sterile filtered using 0.22 pm membrane filter. After use, the column was sanitized with 5 CVs0.1 N NaOH, followed by equilibration with 5-10 CVs 20 mM citrate pH 5.5 buffer. The column was stored in 20% ethanol after rinsing with 5 CVs DI water / MilliQ water, followed by 5 CVs of 20% ethanol.7.1.5. Capto™ Adhere batch binding method

[0190] Capto™ Adhere resin slurry was used to remove glucuronidase from antibody preps of <100 mg. An appropriate amount of 50 / 50 slurry mixture (refer to Table 2 for more details) was washed with 10X volume of 20 mM citrate pH 5.5 buffer. The whole content was centrifuged at 500 x g for 10 min and solution was removed without disturbing the resin settled at the bottom of the centrifuge tube. The wash step was repeated twice before adding the Protein-A affinity purified antibodies. After overnight binding (by gentle agitation), the protein / resin mixture was centrifuged at 500 x g for 10 min and the protein solution was removed without disturbing the resin settled at the bottom. The resin was washed with another 5 CVs of 20 mM citrate pH 5.5. The unbound sample was collected, buffer exchanged into an appropriate buffer, and sterile filtered using a 0.22 pm membrane filter.

[0191] Table 2. Amounts of Protein / Antibody Preps and Capto™ Adhere Used7.1.6. SEC Removal Method

[0192] An AKTA pure instrument attached with 500 pL loop for loading sample was cleaned with 0.1 N NaOH and DI / MilliQ water followed by system equilibration with 20 mM histidine pH 5.5 + 150 mM NaCl. A Sepax SRT-10 SEC 300 21.3x300 column (Part number 225300- 21230) was attached to the instrument and cleaned with 2 column volumes (CV) of DI / MilliQ water at the flow rate of 4 mL / min. Then the column was equilibrated with 3 CVs of 20 mM histidine pH 5.5 + 150 mM NaCl pH 5.5. The mAb sample was manually injected into the 500 pL loop to be loaded onto the column and the SEC method was run at 4 mL / min for 1.5 CVs and 2 mb fractions were collected into 96-deep well plates. The fractions containing the molecule of interest were collected, avoiding high or low molecular weight species as guided by chromatogram (FIG. 6). The pooled fraction was buffer exchanged into an appropriate buffer and sterile filtered using 0.22 pm membrane filter.7.2. Results7.2.1. Detection of the Presence of p-Glucuronidase

[0193] The presence of P-glucuronidase in antibody preparations was demonstrated by P- glucuronidase activity at various stages of the purification process (Table 3). The loss of glucuronic acid from the conjugated tandem-cleavage linker-payload was illustrated in FIG. 2 and supported by three additional pieces of data: 1) mass spectrometry data showed the loss of 176 Da and 2x 176 Da, corresponding to the molecular weight of glucuronic acid (data not shown); 2) the tandem-cleavage linker-payload was synthesized without the glucuronic acid and, when conjugated, overlayed chromatographically with the “DAR2 #3” peak (FIG. 3) and 3) the DAR2 #2 and DAR2 #3 peaks were greatly reduced / eliminated when excess Ac-glucuronide- PABA was added to conjugation reactions as a substrate for P-glucuronidase (FIGs. 4A and 4B).

[0194] Table 3. P-Glucuronidase Enzyme Activity Data from 250 L Lot7.2.2. Purification Studies to Evaluate Removal of P-Glucuronidase from Protein Preparations

[0195] To mitigate the impact of process-related contaminants on conjugate stability during preparation and storage, a series of purification studies were performed to evaluate the removal of P-glucuronidase from protein preparations. To assess whether alternative purification methods removed P-glucuronidase, three analytical methods were established as described in Section 7.1 : 1) a P-glucuronidase activity assay to quantify P-glucuronidase activity, 2) a hydrophobic interaction chromatography (HIC) method to quantify the amount of glucuronide loss on the antibody conjugated to a glucuronide-containing linker-payload, and 3) a mass spectrometry method to quantify P-glucuronidase protein.

[0196] The sections below present the testing of various purification conditions and columns for removal of P-glucuronidase at each step of the mAb purification process.

[0197] A. Protein A Step

[0198] Initially, a 10 L culture purification was carried out with various wash buffers were added to Protein A capture step and then samples were evaluated to determine if inclusion of the wash step reduced P-glucuronidase levels. Different wash buffers were tested. None of the washes significantly removed P-glucuronidase sufficiently. See Table 4 below.

[0199] Table 4 Protein A Screening Results

[0200] Next, two additional Protein A resins (in addition to the resin used in the usual Protein A capture step, all at 1 mL scale) with pH 3.2 elution, high pH wash, caprylic salt wash, and / or detergent-treated clarified harvest were tested for P-glucuronidase removal. None of the washes removed P-glucuronidase sufficiently. About 10% to about 18% of glucuronide loss were still observed.

[0201] B. Anion Exchange Chromatography Step

[0202] In order to reduce P-glucuronidase levels, three anion exchange (AEX) columns (all at 1 mL resin) were tested at different pHs. Table 5 shows the % glucuronide loss on ADCs made with antibodies produced using different anion exchange resins / conditions. Three conditions showed the best clearance for the P-glucuronidase enzyme with the lowest percent ADC glucuronide loss, two being the Capto™ Adhere resin.

[0203] Table 5. AEX Screening Results

[0204] C. Cation Exchange Chromatography Step

[0205] In order to reduce P-glucuronidase levels, various cation exchange (CEX) conditions were tested (all at 1 mL resin scale): exploring intermediate pH washes (20 mM Sodium Phosphate, pH 7.0) before the elution (0 to 400 mM NaCl gradient over 30 CVs), loading at pH 6.5 (50 mM MES pH 6.0 followed by 50 mM MES pH 6.5) for various CEX resins. Table 6 shows the % glucuronide loss on ADCs made with antibodies produced using different resins / conditions. None of the different CEX conditions / resins removed P-glucuronidase sufficiently.

[0206] Table 6. CEX Step Screening Results

[0207] The study showed that Protein A purification in combination with the ion exchange (IEX) chromatography commonly used for monoclonal antibody purification were not enough to remove P-glucuronidase even though the commonly used IEX columns did lower HCPs (host cell proteins, for example, contaminant proteins released by the cells secreting an antibody).Surprisingly, the study showed that only certain chromatography columns sufficiently removed P -glucuronidase and that the P-glucuronidase levels did not fully correlate with HCP levels.7.2.3. Confirmation Experiments for Reduction of P-Glucuronidase

[0208] Based on the results above with Protein A, AEX, and CEX purifications, select chromatography steps were further investigated to determine a final purification step for removal of P-glucuronidase. A four-arm study was performed to compare the performance of second and third polish steps and their impact on the clearance of P-glucuronidase. The starting material of 5.5 g mAb was clarified harvest material that was purified by Protein A chromatography (106 mb resin; pH 3.2 elution) followed by viral inactivation (NVI). FIG. 5 shows the process comparison diagram from the four-arm study with each column at a 15-20 mL scale. Results are summarized in Table 7. The pH wash refers to a washing step with a pH buffer of 0.02 M sodium phosphate, pH 7.0. Capto™ Adhere resin followed by SP65 CEX (Arm #2) was selected as the final process (Arm #4 was the original process). The ligand of the Capto™ Adhere resin is. The ligand of the Capto™ Phenyl (high sub) resin is phenoxy. The ligand of the Praesto® SP65 resin is.

[0209] Table 7. Summary Data from Four- Arm Study7.2.4. Confirmation Run (1000 L)

[0210] To confirm that the selected purification scheme would be effective at manufacturing scale, material from a 1000 L culture was purified using the finalized method: Protein A (Praesto® Jetted A50; 31.2 L), AEX (Capto™ Adhere resin; 13.8 L), and CEX (Praesto® SP65;13.8 L; without pH washing). Results are summarized in Table 8. The process worked to successfully remove P-glucuronidase sufficiently to see no glucuronide loss on the ADC.

[0211] Table 8. 1000 L Confirmation Run7.2.5. Testing Capto™ Adhere Resin on Various Antibodies and Various Scales

[0212] To evaluate the effectiveness of Capto™ Adhere resin to remove P-glucuronidase from preparations of different antibody backbones, four different antibodies were passed through Capto™ Adhere resin and analyzed for glucuronide loss on a tandem-cleavage linker payload ADC using the HIC quantification method. Passing antibodies through Capto™ Adhere resin reduced or eliminated glucuronidase for the four antibodies tested (Table 9).

[0213] Table 9. Capto™ Adhere Antibody Screening Results

[0214] The column type and amount of Capto™ Adhere resin used to effectively remove P- glucuronidase varied depending on the amount of protein. For research lab scale purifications, multiple 1 mL or 5 mL HiTrap Capto™ Adhere columns could be used in tandem to remove glucuronidase from protein / antibody preps of 200-4000 mg (see Table 10 for more details). For protein amounts >4000 mg, a 25 mL Capto™ Adhere column could be used.

[0215] Table 10. Capto™ Adhere Column Volumes7.2.6. Multimodal Chromatography Step

[0216] Additional chromatography resin, EconoFit CHT XT, was tested in order to reduce P- glucuronidase levels. Three antibodies (Antibody P, Antibody Q, and Antibody E, having an isoelectric point (pl) ranging from 7.6 to 8.0) were used. Briefly, the cell culture supernatant was harvested, initially purified with a protein A affinity chromatography resin, and buffer exchanged, centrifuged and diluted into a 10 mM NaPCh pH 6.5, 6 ppm CaCb buffer.

[0217] The EconoFit CHT XT column was attached to the AKTA system. The obtained samples were loaded onto the column and the column was washed using 10 mM NaPCh pH 6.5,6 ppm CaCk buffer. Gradient elution (10 mM NaPCh pH 6.5, 6 ppm CaCE buffer + 10 mM NaPC pH 6.5, 2 M NaCl, 15 mM CaCk buffer) was applied over 20 CVs from 0 to 100% 10 mM NaPCh pH 6.5, 2 M NaCl, 15 mM CaCh buffer, followed by 10 CVs of stripping buffer (400 mM NaPO4 pH 6.5) and other appropriate cleaning-in-place (CIP) steps. Without wishing to be bound by the theory, an acid protein (e.g., having a pl lower than 7) was believed to bind primarily to calcium sites of the EconoFit CHT XT resin via metal affinity, while a basic protein (e.g., having a pl greater than 7) was believed to bind primarily to phosphate sites via cation exchange. Calcium affinity was also believed to play a role in addition to the cation exchange for the basic proteins in some situations. See more details at BIO-RAD Bulletin 7404 ver A, Mixed- Mode Chromatography: Ceramic Apatite Media, 2020, which is incorporated herein by reference in its entirety.

[0218] The purified samples were conjugated and then analyzed for glucuronide loss, for example, by following the HIC method as described above. HIC overlays showed that (1) the control sample without the EconoFit CHT XT purification step presented a peak corresponding to the ADC having its glucuronide group removed, but (2) such a peak disappeared in the tested sample with the EconoFit CHT XT step (data not shown). A representative summary is presented below in Table 11, also indicating good removal of P-glucuronidase post-CHT XL purification.

[0219] Table 11. EconoFit CHT XT Antibody Screening Results7.2.7. Testing Removal of P-Glucuronidase Using Size Exclusion Chromatography

[0220] Size exclusion chromatography (SEC) was tested as an alternative to Capto™ Adhere resin for removal of P-glucuronidase. P-glucuronidase is synthesized as a monomer but tends to form multimers. Therefore, fractions eluting larger than the antibody were purified away (see,e.g., FIG. 6). Purifying antibody using an SEC column was successful at removing 0- glucuronidase (see Table 12).

[0221] Table 12. SEC Antibody Screening Results7.2.8. Confirmation Runs Following Good Manufacturing Practice (GMP) Standards

[0222] To further confirm that the selected purification scheme would be effective at the manufacturing scale, two additional antibodies (Antibody G and Antibody F) and their preparations were tested. Briefly, mAb preparation was harvested via filtering 2000 L cultures of cells secreting the mAb (for example, by depth filtration). The obtained preparation was purified by Protein A chromatography (Praesto® Jetted A50) followed by viral inactivation (NVI, for example under pH 3.5), and further subjected to: AEX (Capto™ Adhere resin) and then CEX (Praesto® SP65). The purified sample was diluted, and viral filtration followed by tangential flow filtration (TFF) were performed to arrive at the final mAb.

[0223] Results for residual 0-glucuronidase by activity and by LC-MS are summarized in Tables 13-16. Considering significant analytical differences between the two methods and the semi-quantitative nature of LC-MS assays, results agreed well between activity and LC-MS methods. Both demonstrated 0-glucuronidase was present in the clarified harvest, measurable 0- glucuronidase was present in Protein A eluate, and 0-glucuronidase was below the limit of detection for all samples after AEX purification. This demonstrated clearance to a level expected to have negligible impact on glucuronide loss in manufacturing glucuronide-containing ADCs. Overall, these results all supported that the developed process successfully cleared 0- glucuronidase from several antibody preparations, such as clarified harvest.

[0224] Table 13. 2000 L GMP Confirmation Run - Antibody G - Repeat 1

[0225] Table 14. 2000 L GMP Confirmation Run - Antibody G - Repeat 2

[0226] Table 15. 2000 L GMP Confirmation Run - Antibody F - Repeat 1

[0227] Table 16. 2000 L GMP Confirmation Run - Antibody F - Repeat 2

[0228] Throughout this application various publications, patents, patent applications and other documents have been referenced. The disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference in this application for all purposes, including in order to more fully describe the state of the art to whichthis the subject matter disclosed herein pertains. Although the disclosed subject matter has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the disclosed subject matter. Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to anion exchange (AEX) chromatography or multimodal chromatography using a resin, wherein a ligand of the resin comprises any one or more of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

2. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to AEX chromatography or multimodal chromatography using a resin, wherein a ligand of the resin comprises any one or more of the following functional groups: a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

3. The method of claim 1 or 2, wherein the ligand comprises a phenyl functional group and a hydroxy functional group.

4. The method of claim 1 or 2, wherein the ligand comprises a phenyl functional group and a quaternary ammonium functional group.

5. The method of claim 1 or 2, wherein the ligand comprises a hydroxy functional group and a quaternary ammonium cation functional group.

6. The method of claim 1 or 2, wherein the ligand comprises a phenyl functional group, a hydroxy functional group, and a quaternary ammonium functional group.

7. The method of claim 6, wherein the ligand comprises an N-benzyl-2-hydroxy-N- methylethan-1 -ammoniumyl moiety.

8. The method of claim 7, wherein the ligand is9. The method of any one of claims 1-8, wherein the resin is Capto™ Adhere.

10. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin, wherein a ligand of the resin comprises a phenyl functional group, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

11. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to AEX chromatography or HIC using a resin, wherein a ligand of the resin comprises a phenyl functional group; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

12. The method of claim 10 or 11, wherein the ligand comprises a phenoxy moiety.

13. The method of claim 12, wherein the ligand is phenoxy.

14. The method of claim 13, wherein the resin is Capto™ Phenyl.

15. The method of claim 14, wherein the resin is Capto™ Phenyl (high sub).

16. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to a multimodal chromatography using a resin, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

17. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to a multimodal chromatography using a resin; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

18. The method of claim 16 or 17, wherein a ligand of the resin comprises a phosphate group, calcium, a hydroxyl group, or any combination thereof.

19. The method of any one of claims 16-18, wherein a ligand of the resin comprises a phosphate group.

20. The method of any one of claims 16-19, wherein a ligand of the resin comprises a phosphate group and calcium.

21. The method of any one of claims 16-20, wherein a ligand of the resin comprises a phosphate group, calcium, and a hydroxyl group.

22. The method of any one of claims 16-21, wherein the chromatography is a cation exchange (CEX) chromatography.

23. The method of any one of claims 16-18, wherein a ligand of the resin comprises calcium.

24. The method of any one of claims 16-18, wherein a ligand of the resin comprises calcium and a hydroxyl group.

25. The method of any one of claims 16-18, 20-21 and 23-24, wherein the chromatography is an anion exchange (AEX) chromatography.

26. The method of any one of claims 16-25, wherein the resin is EconoFit CHT XT.

27. The method of any one of claims 1-26, wherein the chromatography is performed at a pH of between 5 and 8.

28. The method of claim 27, wherein the chromatography is performed at a pH of about 5.5.

29. The method of claim 27, wherein the chromatography is performed at a pH of about 6.5.

30. The method of claim 27, wherein the chromatography is performed at a pH of about 7.5.

31. The method of any one of claims 1-30, which further comprises a protein A purification step before the chromatography step, wherein optionally the protein A purification step produces the protein preparation.

32. The method of any one of claims 1-31, which further comprises subjecting the purified protein preparation obtained from the chromatography step to a cation exchange (CEX) chromatography step.

33. The method of claim 32, wherein the CEX chromatography step uses a resin, and a ligand of the resin comprises a sulfonate functional group.

34. The method of claim 33, where the ligand comprises a sulfopropyl moiety.

35. The method of claim 34, wherein the ligand is36. The method of claim 35, wherein the resin is Praesto® SP65.

37. The method of claim 35, wherein the resin is SP Sepharose™ Fast Flow ion exchange chromatography media.

38. The method of any one of claims 1-37, which results in no or undetectable P- glucuronidase activity in the purified protein preparation.

39. The method of any one of claims 1-38, which results in no loss of glucuronide on the conjugation product of the purified protein.

40. A method of purifying a protein, comprising subjecting a P-glucuronidase- containing protein preparation to size exclusion chromatography (SEC), wherein fractionseluting molecules larger than the protein are purified away, and optionally wherein the purified protein is capable of being conjugated to and / or for use in conjugation to an agent via a glucuronide-containing P-glucuronidase cleavable linker.

41. A method of producing a conjugated protein, comprising:(1) purifying a protein by subjecting a P-glucuronidase-containing protein preparation to SEC, wherein fractions eluting molecules larger than the protein are purified away; and(2) conjugating the purified protein to an agent via a glucuronide-containing P- glucuronidase cleavable linker.

42. The method of claim 40 or 41, which results in less than 1% loss of glucuronide on the conjugation product of the purified protein.

43. The method of any one of claims 1-42, wherein the agent is a cytotoxic drug.

44. The method of any one of claims 1-43, wherein the protein is a therapeutic protein.

45. The method of any one of claims 1-44, wherein the protein is an antibody, an Fc- fusion protein, a bispecific binder, an scFv, a VHH, a VHH-Fc, or a protein ligand.

46. The method of any one of claims 1-45, wherein the protein is an antibody.

47. The method of claim 46, wherein the conjugation product of the purified protein is an antibody-drug conjugate (ADC).

48. The method of any one of claims 1-47, wherein the glucuronide-containing P~ glucuronidase cleavable linker comprises a cleavable peptide motif.

49. The method of claim 48, wherein the cleavable peptide motif is protected from proteolysis by a glucuronic acid.

50. The method of any one of claims 1-49, wherein the glucuronide-containing P- glucuronidase cleavable linker comprises a dipeptide unit shielded by a neighboring glucuronic acid.51 . The method of any one of claims 1-50, which further comprises controlling 0- glucuronidase concentration and / or 0-glucuronidase activity level in the purified protein preparation.

52. The method of claim 51, wherein the controlling step comprises controlling residence time in (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC.

53. The method of claim 51 or 52, wherein the controlling step comprises controlling residence time in the CEX chromatography.

54. The method of any one of claims 51-53, wherein the controlling step comprises controlling residence time in the protein A purification step.

55. The method of any one of claims 51-54, wherein the controlling step comprises modulating pH at which (1) the AEX chromatography or multimodal chromatography, (2) the AEX chromatography or HIC, (3) the multimodal chromatography, or (4) the SEC, is performed.

56. The method of any one of claims 51-55, wherein the controlling step comprises modulating pH at which the CEX chromatography is performed.

57. The method of any one of claims 51-56, wherein the controlling step comprises modulating pH at which the protein A purification step is performed.

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