Method for protein purification

Abstract

The present disclosure relates generally to methods for purifying protein preparations, e.g., antibody preparations, containing beta-glucuronidase. In some embodiments, the purified proteins, e.g., antibodies, are capable of conjugation to agents and / or for use in generating molecules comprising glucuronides, e.g., conjugation to agents via beta-glucuronidase-containing, glucuronide-cleavable linkers. Optionally in addition to a protein A purification step, the methods can involve the use of specific chromatography resins or size exclusion chromatography (SEC).

Description

[0001] Cross-references to related applications 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 herein by reference in its entirety. 1. Technical Field This disclosure generally relates to methods for purifying protein formulations (e.g., antibody formulations) containing β-glucuronidase. In some embodiments, the purified protein (e.g., antibody) can be coupled to a pharmaceutical agent and / or used to generate molecules containing glucuronide, for example, by coupling to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide. Optionally, in addition to the protein A purification step, the method may involve the use of specific chromatographic resins or size exclusion chromatography (SEC). 2. Background Technology Antibody-drug conjugates (ADCs) such as antibodies have been widely developed for therapeutic purposes. However, several challenges remain in manufacturing ADCs, potentially slowing their maturation into approved drugs. One of the biggest technical difficulties is providing a consistently effective drug.

[0004] Therefore, there is still a need to develop improved methods for the efficient and effective generation of conjugated products such as ADCs, for example by providing suitable protein (e.g., antibody) intermediates for conjugation. 3. Summary of the Invention This disclosure is based, at least in part, on the unexpected discovery that certain specific chromatographic resins can effectively remove β-glucuronidase from protein formulations during protein purification. This disclosure is also based, at least in part, on the unexpected discovery that size exclusion chromatography (SEC) can also effectively remove β-glucuronidase from protein formulations during protein purification.

[0006] Protein A was initially found in the bacteria Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus The 42 kDa surface protein found in the cell wall of antibodies is known for its ability to bind to many proteins, especially antibodies. Therefore, protein A chromatography is currently the most commonly used purification method in the industrial-scale downstream processing of antibodies. However, as described in this disclosure, surprisingly, the combination of protein A chromatography with commonly used ion-exchange chromatography does not adequately remove β-glucuronidase from antibodies. The presence of β-glucuronidase can release glucuronic acid from the substrate (e.g., a β-glucuronidase-cleavable linker containing glucuronide), thereby removing the protection of the linker provided by said glucuronic acid (especially linkers containing cleavable peptide motifs), for example, against proteolysis.

[0007] This disclosure provides an improved method for purifying proteins (e.g., antibodies) intended to produce molecules containing glucuronide, such as proteins (e.g., antibodies) to be coupled to an agent (e.g., cytotoxic drugs, immunomodulators, kinase inhibitors, protein degraders, peptides, oligonucleotides, or other bioactive moieties) via a β-glucuronidase-cleavable linker containing glucuronide, by removing β-glucuronidase from the protein (e.g., antibody) formulation. In some embodiments, the protein (e.g., antibody) formulation has been purified by contacting a protein A affinity chromatography resin. Alternatively or additionally, the method includes subjecting the protein (e.g., antibody) formulation to anion exchange (AEX) chromatography as described herein. Alternatively or additionally, the method includes subjecting the protein (e.g., antibody) formulation to multimode chromatography as described herein. Alternatively or additionally, the method includes subjecting the protein (e.g., antibody) formulation to hydrophobic interaction chromatography (HIC) as described herein. Alternatively or alternatively, the method includes subjecting the protein (e.g., antibody) formulation to cation exchange (CEX) chromatography as described herein. Alternatively or alternatively, the method includes subjecting the protein (e.g., antibody) formulation to size exclusion chromatography (SEC) as described herein.

[0008] This disclosure provides a method for purifying a protein, wherein the method includes subjecting a protein formulation containing β-glucuronidase to anion exchange (AEX) chromatography or multimode chromatography using a resin. In some embodiments, the ligands of the resin contain any one or more of the following functional groups (e.g., any two or all three): phenyl, hydroxyl, and quaternary ammonium functional groups. Alternatively or additionally, the purified protein can be coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a reagent via said linker.

[0009] This disclosure also provides a method for generating a coupled protein, wherein the method comprises: (1) purifying the protein by subjecting a protein formulation containing β-glucuronidase to AEX chromatography or multimode chromatography using a resin; and (2) coupling the purified protein to the pharmaceutical preparation via a β-glucuronidase-cleavable linker containing glucuronide. In some embodiments, the ligands of the resin contain any one or more of the following functional groups (e.g., any two or all three): phenyl functional group, hydroxyl functional group, and quaternary ammonium functional group.

[0010] In some embodiments, the ligand comprises a phenyl functional group and a hydroxyl 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 hydroxyl functional group and a quaternary ammonium cationic functional group. In some embodiments, the ligand comprises a phenyl functional group, a hydroxyl functional group, and a quaternary ammonium functional group. In some embodiments, the ligand comprises an N-benzyl-2-hydroxy-N-methylethane-1-ammonium moiety. In some embodiments, the ligand is... .

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

[0012] This disclosure further provides a method for purifying a protein, wherein the method includes subjecting a protein formulation containing β-glucuronidase to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin. In some embodiments, the ligands of the resin contain phenyl functional groups. Alternatively or additionally, the purified protein can be coupled to and / or used for coupling to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide.

[0013] This disclosure also provides a method for generating a coupled protein, wherein the method comprises: (1) purifying the protein by subjecting a protein formulation containing β-glucuronidase to AEX chromatography or HIC using a resin; and (2) coupling the purified protein to the pharmaceutical preparation via a β-glucuronidase-cleavable linker containing glucuronide. In some embodiments, the ligand of the resin comprises a phenyl functional group.

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

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

[0016] This disclosure further provides a method for purifying proteins, wherein the method includes subjecting a protein formulation containing β-glucuronidase to multimode chromatography using a resin. Alternatively or additionally, the purified protein can be coupled to and / or used for coupling to a drug via a β-glucuronidase-cleavable linker containing glucuronide. In some embodiments, the ligands of the resin comprise phosphate ester groups, calcium (e.g., Ca²⁺), etc. 2+The resin contains phosphate esters, hydroxyl groups, or any combination thereof. In a further embodiment, the ligands of the resin comprise phosphate esters. In a further embodiment, the ligands of the resin comprise phosphate ester groups and calcium. In a further embodiment, the ligands of the resin comprise phosphate ester groups, calcium, and hydroxyl groups. In some embodiments, the multimode chromatography is cation exchange (CEX) chromatography. In a further embodiment, the ligands of the resin comprise calcium. In a further embodiment, the ligands of the resin comprise calcium and hydroxyl groups. In some embodiments, the multimode chromatography is anion exchange (AEX) chromatography. In some embodiments, the resin is EconoFit CHT XT.

[0017] This disclosure also provides a method for generating a conjugated protein, wherein the method comprises: (1) purifying a protein formulation containing β-glucuronidase by subjecting it to multimode chromatography using a resin; and (2) conjugating the purified protein to the pharmaceutical preparation via a β-glucuronidase-cleavable linker containing glucuronide. In some embodiments, the ligand of the resin comprises a phosphate ester group, calcium (e.g., Ca2+), etc. 2+ ), hydroxyl groups, or any combination thereof. In a further embodiment, the ligands of the resin comprise phosphate ester groups. In a further embodiment, the ligands of the resin comprise phosphate ester groups and calcium. In a further embodiment, the ligands of the resin comprise phosphate ester groups, calcium, and hydroxyl groups. In some embodiments, the multimode chromatography is cation exchange (CEX) chromatography. In some embodiments, the resin is EconoFit CHT XT.

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

[0019] In various embodiments, the methods described herein also include a protein A purification step prior to an AEX, multimode, or HIC chromatography step, wherein the protein A purification step optionally produces the protein formulation.

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

[0021] In various embodiments, the methods described herein further include a cation exchange (CEX) chromatography step following an AEX, multimode, or HIC chromatography step, wherein the purified protein formulation obtained from the AEX, multimode, or HIC chromatography step is optionally subjected to a CEX chromatography step. In some embodiments, the CEX chromatography uses a resin, and the ligand of the resin comprises a sulfonate functional group. In some embodiments, the ligand comprises a sulfopropyl moiety. In some embodiments, the ligand is... In some embodiments, the resin is Praesto. ® SP65. In some embodiments, the resin is SP Sepharose™ Fast Flow ion exchange chromatography medium.

[0022] In various embodiments, the methods described herein result in the absence or undetectability of β-glucuronidase activity in the purified protein formulation. In some embodiments, the methods described herein result in no loss of glucuronide on the protein antibody conjugate.

[0023] This disclosure further provides a method for purifying a protein, wherein the method includes subjecting a protein formulation containing β-glucuronidase to size exclusion chromatography (SEC). In some embodiments, the purified protein can be coupled to and / or used for coupling to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide, and wherein the purification removes elution fractions larger than the protein molecules.

[0024] This disclosure also provides a method for generating a conjugated protein, wherein the method comprises: (1) purifying the protein by subjecting a protein preparation containing β-glucuronidase to SEC, wherein the purification removes elution fractions larger than said protein molecules; and (2) conjugating said purified protein to the pharmaceutical preparation via a β-glucuronidase-cleavable linker containing glucuronide.

[0025] In some implementations, the methods described herein result in a loss of less than 1% of glucuronide on the conjugate 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, scFv, VHH, a VHH-Fc fusion protein (also referred to herein as VHH-Fc), or a protein ligand (e.g., for targeting cell-associated receptors such as cell surface receptors). In various embodiments, the protein is an antibody.

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

[0029] In various embodiments, the β-glucuronidase cleavable linker containing glucuronide comprises a cleavable peptide motif. In some embodiments, the cleavable peptide motif is protected from proteolysis by glucuronic acid. In some embodiments, the β-glucuronidase cleavable linker containing glucuronide comprises a dipeptide unit protected by an adjacent glucuronic acid molecule.

[0030] In various embodiments, the method further includes controlling the concentration and / or activity level of β-glucuronidase in the purified protein formulation.

[0031] In some implementations, the control steps include controlling the retention time in (1) AEX chromatography or multimode chromatography, (2) AEX chromatography or HIC, (3) multimode chromatography, or (4) SEC.

[0032] In some implementations, the control step includes controlling the retention time in CEX chromatography.

[0033] In some implementations, the control step includes controlling the retention time in the protein A purification step.

[0034] In some embodiments, the control steps include adjusting the pH of (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the SEC.

[0035] In some implementations, the control step includes adjusting the pH when performing the CEX chromatography.

[0036] In some implementations, the control step includes adjusting the pH during the protein A purification step. 4. Description of the attached drawings Figure 1 Example calculation of glucuronide loss using hydrophobic interaction chromatography (HIC) method.

[0038] Figure 2 Multiple peaks corresponding to a drug / antibody ratio (DAR) of 2 were observed in the ADC containing the tandem cleavage linker-payload. Mass spectrometry analysis of the various DAR2 peaks indicated a loss of 176 Da (DAR2 #2) and approximately 2 x 176 Da (DAR2 #3), corresponding to the molecular weight of one or two glucuronic acids.

[0039] Figure 3 : Coupled to tandem pyrolysis joint - payload or tandem pyrolysis joint lacking glucuronic acid - payload ( HIC chromatogram of monoclonal antibody (mAb) of ).

[0040] Figure 4A The structure of Ac-glucuronide-PABA. Figure 4B HIC chromatogram of an ADC containing a tandem cleavage linker-load coupled in the presence of Ac-glucuronide-PABA.

[0041] Figure 5 : A flowchart of the research process of the four teams.

[0042] Figure 6 Antibody SEC chromatogram. The highlighted eluates were pooled and analyzed. 5. Detailed Implementation This disclosure provides an improved method for purifying proteins (e.g., antibodies) to produce molecules containing glucuronide, such as proteins (e.g., antibodies) to be coupled to an agent (e.g., a cytotoxic drug, immunomodulator, kinase inhibitor, protein degrader, peptide, oligonucleotide, or other bioactive moiety) via a β-glucuronidase-cleavable linker containing glucuronide. Such methods may involve the use of specific chromatographic resins or size exclusion chromatography (SEC), optionally in addition to a protein A purification step.

[0044] As understood, the chapter or subsection headings used in this document are for organizational purposes only and should not be construed as limiting and / or separating the topics described.

[0045] 5.1. Definition The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional methods (e.g., those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, 2001); *Current Protocols in Molecular Biology* (edited by Ausubel et al., 2003); *Therapeutic Monoclonal Antibodies: From Bench to Clinic* (edited by An, 2009); *Monoclonal Antibodies: Methods and Protocols* (edited by Albitar, 2010); and *Antibody Engineering, Volumes 1 and 2* (edited by Kontermann and Dübel, 2nd edition, 2010)). Unless otherwise defined herein, the technical and scientific terms used in this description have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terminology description will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. If any description of the terms set forth herein conflicts with any document incorporated herein by reference, the description of the terms set forth below shall prevail.

[0046] As used herein, the term "resin" refers to the solid phase in chromatographic methods such as column chromatography. Resins can be packed in column or non-column containers.

[0047] The term "column" refers to any housing or enclosure adapted for use in a chromatographic process, suitable for retaining all or part of the chromatographic resin and for contacting the sample and / or buffer solution with the chromatographic resin. A wide range of materials are suitable for use in columns and are known in the art.

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

[0049] "Multimode chromatography" (also known as mixed-mode chromatography (MMC)) refers to a chromatographic method used to separate one analyte from another by utilizing more than one form of interaction between the stationary phase and the analyte (e.g., cation exchange bonding and / or metal affinity).

[0050] The term "depth filtration" refers to the removal of particles from a solution using a series of sequentially arranged filters with reduced pore sizes. In some embodiments, the three-dimensional matrix of the depth filter establishes a labyrinthine path through which the sample traverses. The retention mechanism of the depth filter relies on random adsorption and mechanical embedding throughout the matrix depth. In various embodiments, the filter membrane or sheet may be wound cotton, polypropylene, rayon cellulose, glass fiber, sintered metal, porcelain, diatomaceous earth, or other known components.

[0051] The term "protein formulation" refers to a composition containing a protein of interest prepared for downstream purification. In some embodiments, the protein formulation is a composition obtained by filtering a culture of cells secreting the protein (also referred to herein as a clarified harvest), for example, by removing cells and cell debris and / or by deep filtration. In some embodiments, where it is not desirable to be bound by theory, the protein formulation harvested by filtering a culture of cells secreting the protein (e.g., by deep filtration) contains contaminants released by cells in the same culture, and such contaminants include β-glucuronidase. In a further embodiment, the protein formulation is a protein A affinity chromatography resin or has been purified by contacting a protein A affinity chromatography resin. Alternatively or alternatively, the protein formulation is an antibody formulation.

[0052] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein and, in the broadest sense and specifically, encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy and / or light chains. As used herein, “VHH” refers to a domain antibody derived from the variable region of a heavy-chain-only antibody.

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

[0054] The term "ADC" refers to antibody-drug conjugates, in which the antibody is conjugated to another part of a drug (also known as a payload), such as a cytotoxic agent or another bioactive compound.

[0055] As used herein, “drug” means a biologically active compound, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.), an immunomodulator, a kinase inhibitor, a protein degrader, a peptide, an oligonucleotide, or another biologically active fraction.

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

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

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

[0059] As used herein, comparative terms such as reduction, decrease, increase, or any grammatical variation thereof may refer to certain changes relative to the reference. In some embodiments, such a change may 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, or about 2, or about 3, or about 4, or about 5, or about 10, or about 20, or about 30, or about 40, or about 100 times or more than the reference. In some implementations, such variation may refer to the aforementioned reference as approximately 1%, or approximately 2%, or approximately 3%, or approximately 4%, or approximately 5%, or approximately 6%, or approximately 7%, or approximately 8%, or approximately 9%, or approximately 10%, or approximately 20%, or approximately 30%, or approximately 40%, or approximately 50%, or approximately 60%, or approximately 70%, or approximately 80%, or approximately 90%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

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

[0061] It should be understood that wherever the term "comprising" is used to describe an embodiment herein, other similar embodiments described as "consisting of" and / or "substantially consisting of" are also provided. It should also be understood that whenever an embodiment is described herein with the phrase "substantially consisting of," other similar embodiments described as "consisting of" are also provided.

[0062] The term “between” as used in phrases such as “between A and B” or “between A and B” refers to a range that includes both A and B or any subrange thereof.

[0063] The term "and / or" as used herein, as in phrases such as "A and / or B," is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover 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 terms “optional” or “optionally” mean that the situation described below may or may not occur, such that the description includes cases in which the situation occurs and cases in which the situation does not occur.

[0065] 5.2. Protein purification methods This disclosure provides a method for purifying a protein (e.g., antibody) formulation containing β-glucuronidase, the method involving the use of specific anion exchange (AEX) chromatography, multimode chromatography, or hydrophobic interaction chromatography (HIC) resin, or size exclusion chromatography (SEC), optionally in addition to a protein A purification step.

[0066] β-Glucuronidase (also known as GUSB, GUS, βGlu, or β-D-glucuronide glucuronide hydrolase) (International Union of Biochemistry and Molecular Biology no. EC3.2.1.31) is a glycoprotein acid hydrolase belonging to the glycosidase family 2. β-Glucuronidase exists as a tetrameric glycoprotein with four identical subunits. It is primarily located in lysosomes and is found in virtually all mammalian cells. β-Glucuronidase acts as an exoglycosidase that cleaves glucuronic acid residues from the non-reducing ends of glycosaminoglycans. Therefore, β-Glucuronidase catalyzes the deconjugation of β-D-glucuronide and the degradation of glucuronic acid-containing glycosaminoglycans, thus playing a necessary role in the degradation of macromolecules. Furthermore, β-Glucuronidase is produced by bacteria such as *Escherichia coli* (…). E. coli ), Peptostreptococcus ( Peptostreptococcus ), Bacteroides ( Bacteroides ) and Clostridium spp. ( Clostridium Excessive production. β-Glucuronidase can prematurely dissociate drug molecules from their glucuronide conjugates by uncoupling glucuronide.

[0067] In some embodiments, the resin described herein is packed into a column such that the corresponding chromatographic method is column chromatography. Suitable columns may be in the form of, for example, conduits, tubes, or other structures having a relatively large flow length to flow cross-section ratio. The columns may be in sizes such as 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 supporting components, such as agarose, beads, gels, monoliths, membranes, and / or non-woven carriers. In a particular embodiment, the resin described herein comprises an agarose matrix. In a preferred embodiment, the resin described herein comprises a highly cross-linked agarose matrix, particularly a highly cross-linked rigid agarose matrix.

[0068] In one aspect, this disclosure provides a method for purifying a protein (e.g., an antibody), wherein the method comprises subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained via filtration of a culture of cells secreting the protein (e.g., by deep filtration) and optionally by contact purification with a protein A affinity chromatography resin) to anion exchange (AEX) chromatography or multimode chromatography using the resin. In some embodiments, the ligands of the resin contain any one or more of the following functional groups (e.g., any two or all three): phenyl, hydroxyl, and quaternary ammonium functional groups. Additionally or alternatively, the purified protein (e.g., the antibody) is capable of being coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or is used for coupling to a pharmaceutical agent via said linker.

[0069] In another aspect, this disclosure also provides a method for generating a conjugated protein (e.g., an antibody), wherein the method comprises: (1) purifying the protein (e.g., the antibody) by subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained via filtration of a culture of cells secreting the protein (e.g., by deep filtration) and optionally by contact purification with a protein A affinity chromatography resin) to AEX chromatography or multimode chromatography using the resin; and (2) conjugating the purified protein (e.g., the antibody) to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide. In some embodiments, the ligand of the resin comprises any one or more of the following functional groups (e.g., any two or all three): phenyl functional group, hydroxyl functional group, and quaternary ammonium functional group.

[0070] In some embodiments, the ligand comprises a phenyl functional group and a hydroxyl 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 hydroxyl functional group and a quaternary ammonium cationic functional group. In some embodiments, the ligand comprises a phenyl functional group, a hydroxyl functional group, and a quaternary ammonium functional group. In some embodiments, the ligand comprises an N-benzyl-2-hydroxy-N-methylethane-1-ammonium moiety. In some embodiments, the ligand comprises or is .

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

[0072] In some embodiments, the resin described herein comprises a high-flow-rate agarose matrix. In some embodiments, the resin described herein comprises a highly cross-linked agarose matrix (particularly, a highly cross-linked rigid agarose matrix). In some embodiments, the resin described herein has a d50v particle size between 25 μm and 100 μm (d50v is the average particle size of the cumulative volume distribution). In some embodiments, the resin described herein has a d50v particle size of about 95 μm. In some embodiments, the resin described herein has a d50v particle size of about 90 μm. In some embodiments, the resin described herein has a d50v particle size of about 85 μm. In some embodiments, the resin described herein has a d50v particle size of about 80 μm. In some embodiments, the resin described herein has a d50v particle size of about 75 μm. In some embodiments, the resin described herein has a d50v particle size of about 70 μm. In some embodiments, the resin described herein has a d50v particle size of about 65 μm. In some embodiments, the resin described herein has a d50v particle size of about 60 μm. In some embodiments, the resin described herein has a d50v particle size of about 55 μm. In some embodiments, the resin described herein has a d50v particle size of about 50 μm. In some embodiments, the resin described herein has a d50v particle size of about 45 μm. In some embodiments, the resin described herein has a d50v particle size of about 40 μm. In some embodiments, the resin described herein has a d50v particle size of about 35 μm. In some embodiments, the resin described herein has a d50v particle size of about 30 μm. In some embodiments, the resin described herein has a total ionic capacity of 0.09 to 0.12 mmol Cl- / mL medium. In some embodiments, the resin described herein has a flow rate of at least 600 cm / h in a 1 m diameter column with a bed height of 20 cm using a process buffer having the same viscosity as water at < 3 bar (0.3 MPa) at 20 °C. In some embodiments, the resins described herein have a short-term clean-in-place (CIP) stability pH range of 2 to 14, which is the pH range within which the media can withstand clean-in-place or sterilization. Clean-in-place is a procedure to remove impurities (e.g., lipids, endotoxins, nucleic acids, and precipitated or denatured proteins) that remain in the packed column after regeneration. In some embodiments, the resins described herein have an operational stability pH range of 3 to 12, which is the pH range within which the media can be operated without significant functional alteration. In some embodiments, the resins described herein have an operating temperature range of 4°C to 30°C. In some embodiments, the resins described herein are chemically stable to all commonly used aqueous buffers, 1M acetic acid, and / or 1M sodium hydroxide.In some embodiments, the ion-binding capacity and carbon content of the resins described herein do not change significantly after storage at 40°C in 1M NaOH for one week. In some embodiments, oxidizing agents and anionic cleaning agents should be avoided when using the resins described herein. In some embodiments, suitable storage conditions for the resins described herein are 4°C to 30°C in 20% ethanol.

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

[0074] In another aspect, this disclosure further provides a method for purifying a protein (e.g., an antibody), wherein the method comprises subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained via filtration of a culture of cells secreting the protein (e.g., by deep filtration) and optionally purified by contact with a protein A affinity chromatography resin) to AEX chromatography or hydrophobic interaction chromatography (HIC) using the resin. In some embodiments, the ligands of the resin contain phenyl functional groups. Additionally or alternatively, the purified protein (e.g., the antibody) is capable of being coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or is used for coupling to a pharmaceutical agent via the linker.

[0075] In another aspect, this disclosure also provides a method for generating a conjugated protein (e.g., an antibody), wherein the method comprises: (1) subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained via filtration of a culture of cells secreting the protein (e.g., by deep filtration) and optionally purified by contact with a protein A affinity chromatography resin) to AEX chromatography or HIC using a resin, wherein the ligands of the resin contain phenyl functional groups; and (2) conjugating the purified protein (e.g., the antibody) to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide.

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

[0077] In some embodiments, the resin described herein comprises a high-flow-rate agarose matrix. In some embodiments, the resin described herein comprises a highly cross-linked agarose matrix (particularly, a highly cross-linked rigid agarose matrix). In some embodiments, the resin described herein has a d50v particle size between 25 μm and 100 μm. In some embodiments, the resin described herein has a d50v particle size of about 95 μm. In some embodiments, the resin described herein has a d50v particle size of about 90 μm. In some embodiments, the resin described herein has a d50v particle size of about 85 μm. In some embodiments, the resin described herein has a d50v particle size of about 80 μm. In some embodiments, the resin described herein has a d50v particle size of about 75 μm. In some embodiments, the resin described herein has a d50v particle size of about 70 μm. In some embodiments, the resin described herein has a d50v particle size of about 65 μm. In some embodiments, the resin described herein has a d50v particle size of about 60 μm. In some embodiments, the resin described herein has a d50v particle size of about 55 μm. In some embodiments, the resin described herein has a d50v particle size of about 50 μm. In some embodiments, the resin described herein has a d50v particle size of about 45 μm. In some embodiments, the resin described herein has a d50v particle size of about 40 μm. In some embodiments, the resin described herein has a d50v particle size of about 35 μm. In some embodiments, the resin described herein has a d50v particle size of about 30 μm. In some embodiments, the resin described herein has a ligand density of about 27 μmol / mL medium. In some embodiments, the resin described herein has a dynamic binding capacity of about 27 mg bovine serum albumin (BSA) / mL medium at QB10% (10% crossflow). In some embodiments, the resin described herein exhibits a dynamic binding capacity of approximately 19 mg BSA / mL at a retention time of 4 min (150 cm / h) using a Tricorn™ 5 / 100 column (with a 10 cm bed height, 0.1 M sodium phosphate buffer, and 1.2 M ammonium sulfate (pH 7)) under 10% crossflow. In some embodiments, the resin described herein exhibits 45 to 50 min of lysozyme retention hydrophobicity. In some embodiments, the resin described herein uses water at a suitable liquid velocity of 150 to 350 cm / h at room temperature. In some embodiments, the resin described herein uses a 1 m column (with a 20 cm bed height) at 20°C with a process buffer of the same viscosity as water (corresponding to a 2 min retention time) at a liquid velocity of at least 600 cm / h.In some embodiments, the resin described herein exhibits a flow rate of at least 220 cm / h in a 1 m diameter column with a bed height of 20 cm, measured at 20°C using a process buffer having the same viscosity as water at 300 kPa. In some embodiments, the resin described herein has an operating pH range of 3 to 13. In some embodiments, the resin described herein has a short-term stability pH range of 2 to 14, which is the pH range in which the medium can withstand in-situ cleaning or sterilization without significant functional change. In some embodiments, the resin described herein has a long-term stability pH range of 3 to 13, which is the pH range in which the medium can be operated on without significant functional change. In some embodiments, the resin described herein is chemically stable in the following 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 some embodiments, the resin described herein shows no significant functional change after one month of storage in 1 M NaOH at ambient temperature. In some embodiments, the resin described herein is suitable for storage at 4°C to 30°C in 20% ethanol.

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

[0079] In another aspect, this disclosure further provides a method for purifying a protein (e.g., an antibody), wherein the method comprises subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained via filtration of a culture of cells secreting the protein (e.g., by deep filtration) and optionally purified by contact with a protein A affinity chromatography resin) to multimode chromatography using the resin. Additionally or alternatively, the purified protein (e.g., the antibody) can be coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a pharmaceutical agent via said linker.

[0080] In another aspect, this disclosure also provides a method for generating a conjugated protein (e.g., an antibody), wherein the method comprises: (1) purifying the protein (e.g., the antibody) by subjecting a protein (e.g., antibody) preparation containing β-glucuronidase (e.g., a composition obtained by filtering a culture of cells secreting the protein (e.g., by deep filtration) and optionally by contacting a protein A affinity chromatography resin for purification) to a multimode chromatography using the resin; and (2) conjugating the purified protein (e.g., the antibody) to a pharmaceutical agent via a β-glucuronidase cleavable linker containing glucuronide.

[0081] In some embodiments, the ligands of the resin comprise phosphate ester groups, calcium (e.g., Ca2+) 2+ ), hydroxyl groups, or any combination thereof. In some embodiments, the ligands of the resin comprise phosphate ester groups. In some embodiments, the ligands of the resin comprise calcium (e.g., Ca). 2+ In some embodiments, the ligands of the resin comprise phosphate ester groups and calcium (e.g., Ca). 2 + In some embodiments, the ligands of the resin comprise phosphate ester groups and hydroxyl groups. In some embodiments, the ligands of the resin comprise calcium (e.g., Ca). 2+ ) and hydroxyl groups. In some embodiments, the ligands of the resin contain phosphate ester groups, calcium (e.g., Ca) 2+ ) and hydroxyl groups.

[0082] In some embodiments, the multimode chromatography comprises any one or more of the following functional groups: Ca 2 + Phosphate ester group (PO4) and / or hydroxyl group (-OH). In some embodiments, the multimode chromatography includes the following functional groups: Ca 2+ Phosphate ester group (PO4) and hydroxyl group (-OH).

[0083] In some embodiments, the multimode chromatography is cation exchange (CEX) multimode chromatography. In a further embodiment, the ligands of the resin contain phosphate ester groups. In some embodiments, the multimode chromatography is anion exchange (AEX) multimode chromatography. In a further embodiment, the ligands of the resin contain calcium (e.g., Ca). 2+ In a further embodiment, the ligand of the resin comprises a hydroxyl group (-OH).

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

[0085] In some 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. Alternatively or concurrently, the matrix is ​​represented by the following formula: Ca 10(PO4)6(OH)2. In some embodiments, the multimode chromatography provides cation exchange and calcium affinity interactions. In some embodiments, the resins described herein have a particle size between 10 μm and 100 μm. In some embodiments, the resins described herein have a particle size of about 100 μm. In some embodiments, the resins described herein have a particle size of about 90 μm. In some embodiments, the resins described herein have a particle size of about 80 μm. In some embodiments, the resins described herein have a particle size of about 75 μm. In some embodiments, the resins described herein have a particle size of about 70 μm. In some embodiments, the resins described herein have a particle size of about 65 μm. In some embodiments, the resins described herein have a particle size of about 60 μm. In some embodiments, the resins described herein have a particle size of about 55 μm. In some embodiments, the resins described herein have a particle size of about 50 μm. In some embodiments, the resins described herein have a particle size of about 45 μm. In some embodiments, the resins described herein have a particle size of about 40 μm. In some embodiments, the resin described herein has a particle size of about 35 μm. In some embodiments, the resin described herein has a particle size of about 30 μm. In some embodiments, the resin described herein has a particle size of about 25 μm. In some embodiments, the resin described herein has a particle size of about 20 μm. In some embodiments, the resin described herein has a particle size of about 15 μm. In some embodiments, the resin described herein has a particle size of about 10 μm. In some embodiments, the resin described herein has a nominal average particle size of 40 ± 4 μm. In some embodiments, the resin binds to molecules of neutral and basic nature. In some embodiments, the resin described herein has an observed dynamic binding capacity of about 17.4–24.8 mg lysozyme / g resin. In some embodiments, the resin described herein has an observed dynamic binding capacity of about ≥60 mg / mL IgG in a 0.5 x 9.5 cm column at 100 cm / hr with 5 mM sodium phosphate and 25 mM sodium chloride (pH 7). In some embodiments, the resin described herein has a tap settling density of about 0.67 g / mL in a tap settling bed. In some embodiments, the resin described herein has a typical linear flow rate range of about 50 to about 400 cm / hr. In some embodiments, the resin described herein has a pH stability range of 6.5 to 14. In some embodiments, the resin described herein has alkali stability in 1N NaOH for at least 1 year. In some embodiments, suitable regeneration conditions for the resin described herein are 0.4M sodium phosphate (pH 7-7.5) and / or 1M trisodium phosphate (pH 11-12).In some embodiments where a higher concentration of sodium phosphate is required to regenerate the resin described herein, suitable regeneration conditions are approximately 0.4 to approximately 1 M potassium phosphate. In some embodiments, the sterilization conditions for the resin described herein are approximately 1 to approximately 2 N NaOH. In some embodiments, the recommended column storage for the resin described herein is approximately 0.1 N NaOH.

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

[0087] In various embodiments, the methods described herein further include a protein A purification step prior to the AEX, multimode, or HIC chromatography step, wherein optionally the protein A purification step produces a protein (e.g., antibody) formulation. In some embodiments, the protein A purification step uses a resin. In some embodiments, the ligand of the resin is Repligen. ® NGL-Impact™ A ligand. In some 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 µm. In some embodiments, the resin described herein has a particle size range of >95% between 35 and 90 µm. 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 some embodiments, the resin described herein is suitable for storage at 2°C to 8°C in 20% ethanol. In some embodiments, the resin is Praesto® Jetted A50.

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

[0089] In various embodiments, the methods described herein result in the absence or undetectability of β-glucuronidase activity in the purified protein (e.g., antibody) formulation. In various embodiments, the methods described herein result in a reduction of β-glucuronidase activity in the purified protein (e.g., antibody) formulation by 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%, compared to an unpurified protein (e.g., antibody) formulation or compared to a protein (e.g., antibody) formulation purified using a control method (e.g., without a corresponding AEX, multimode, or HIC chromatographic step).

[0090] In various embodiments, the methods described herein result in no glucuronide loss on the conjugate of the purified protein (e.g., antibody). In various embodiments, the methods described herein result in glucuronide loss on the conjugate of the purified protein (e.g., antibody) of 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%, for example, compared to an unpurified protein (e.g., antibody) formulation or compared to a protein (e.g., antibody) formulation purified using a control method (e.g., without its respective AEX, multimode, or HIC chromatographic steps).

[0091] In another aspect, this disclosure further provides a method for purifying a protein (e.g., an antibody), wherein the method includes subjecting a protein (e.g., antibody) formulation containing β-glucuronidase to size exclusion chromatography (SEC). In some embodiments, the purified protein (e.g., the antibody) is capable of being coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a pharmaceutical agent via said linker. Alternatively or additionally, purification removes the eluent containing molecules larger than said protein (e.g., the antibody).

[0092] In another aspect, this disclosure also provides a method for generating a conjugated protein (e.g., an antibody), wherein the method comprises: (1) purifying the protein (e.g., the antibody) by subjecting a protein (e.g., antibody) formulation containing β-glucuronidase (e.g., a composition obtained by filtering a culture of cells secreting the protein (e.g., by deep filtration) and optionally by contacting a protein A affinity chromatography resin for purification) to SEC, wherein the purification removes elution fractions eluted with molecules larger than said protein (e.g., the antibody); and (2) conjugating said purified protein (e.g., the antibody) to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide.

[0093] In some embodiments, the method described herein further includes a protein A purification step prior to the SEC step, wherein the protein A purification step optionally produces a protein (e.g., antibody) formulation.

[0094] In some embodiments, the methods described herein result in a reduction of β-glucuronidase activity in a purified protein (e.g., antibody) formulation by at least 99%, for example, compared to an unpurified protein (e.g., antibody) formulation or compared to a protein (e.g., antibody) formulation purified using a control method (e.g., without an SEC chromatography step). In some embodiments, the methods described herein result in a reduction of β-glucuronidase activity in a purified protein (e.g., antibody) formulation by 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%, for example, compared to an unpurified protein (e.g., antibody) formulation or compared to a protein (e.g., antibody) formulation purified using a control method (e.g., without an SEC chromatography step). In some embodiments, the methods described herein result in no or undetectable β-glucuronidase activity in the purified protein (e.g., antibody) formulation.

[0095] In some embodiments, the methods described herein result in a glucuronide loss of less than 1% on the conjugate of the purified protein (e.g., antibody), for example, compared to an unpurified protein (e.g., antibody) formulation or a protein (e.g., antibody) formulation purified using a control method (e.g., without an SEC chromatography step). In some embodiments, the methods described herein result in a glucuronide loss of 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% on the conjugate of the purified protein (e.g., antibody), for example, compared to an unpurified protein (e.g., antibody) formulation or a protein (e.g., antibody) formulation purified using a control method (e.g., without an SEC chromatography step). In some embodiments, the methods described herein result in no glucuronide loss on the conjugate of the purified protein (e.g., antibody).

[0096] In a preferred embodiment, the β-glucuronidase-cleavable linker containing glucuronide is a β-glucuronidase-cleavable linker containing β-glucuronide. In various embodiments, removal of glucuronide from the β-glucuronidase-cleavable linker directly triggers the release of an agent (e.g., payload). In various embodiments, the β-glucuronidase-cleavable linker containing glucuronide comprises a cleavable peptide motif (i.e., a peptide motif that can be cleaved by a protease). In some embodiments, the cleavable peptide motif is protected from proteolytic hydrolysis (e.g., by adjacent glucuronide) by glucuronic acid. In some embodiments, the β-glucuronidase-cleavable linker containing glucuronide comprises a dipeptide unit protected by an adjacent glucuronide. Additional suitable connectors and drug delivery connectors can be found in, for example, International Patent Application Publications Nos. 2020 / 154437, 2022 / 109335, 2022 / 155347, 2022 / 155362, 2022 / 187370 and 2023 / 009759, each of which is incorporated herein by reference in its entirety. Furthermore, suitable therapeutic conjugates and connector payloads can be found, for example, in International Patent Application Publications Nos. 2020 / 154437, 2022 / 109335, 2022 / 155347, 2022 / 155362, 2022 / 187370 and 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, scFv, VHH, VHH-Fc, or a protein ligand (e.g., for targeting cell-associated receptors such as cell surface receptors). In some embodiments, the protein comprises an Fc. Alternatively or additionally, contact with a protein A affinity chromatography resin is used to purify compositions containing proteins (e.g., antibody formulations) but without sufficiently removing β-glucuronidase.

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

[0100] In some embodiments, the proteins described herein (e.g., antibodies) target tumor antigens (e.g., cell surface tumor antigens). In some embodiments, the proteins described herein (e.g., antibodies) target antigens of pathogens (e.g., antigens of pathogens expressed at the cell surface).

[0101] In various embodiments, the agent (to be conjugated to the protein (e.g., antibody)) is a cytotoxic drug, immunomodulator, 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, such as a cytotoxic drug (e.g., auristatin or a 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 can be used in this invention include small molecule drugs, such as cancer chemotherapeutic agents. Cancer chemotherapeutic agents include non-peptide (i.e., non-protein) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell growth inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, phytoalkaloids (catharanthus vinca) and steroid hormones. Peptide compounds may also be used.

[0103] Suitable chemotherapeutic agents for cancer include dolastatin and its active analogs and derivatives; and olastatin and its active analogs and derivatives (e.g., monomethylolastatin D (MMAD), monomethylolastatin E (MMAE), monomethylolastatin F (MMAF), etc.). See, for example, WO 96 / 33212, WO 96 / 14856 and US 6,323,315. Suitable chemotherapeutic agents for cancer also include maytansine and its active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996)). Proc. Natl. Acad. Sci. USA93:8618-8623); duocarmycin and its active analogs and derivatives (e.g., including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBD)).

[0104] Agents used to reduce cell proliferation are known and widely used in the field. Such agents include alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazine, including (but not limited to) nitrogen mustard, cyclophosphamide (CYNOTAN™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracilmustard, chlormethine, ifosfamide, chlorambucil, pipebroman, triethylene melamine, triethylene thiophosphoric acid, 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), fluorouridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-didenifolinic acid (PDDF, CB3717), 5,8-didenifolinic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0106] Suitable natural products and their derivatives (such as vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxin) include (but are not limited to) Ara-C and paclitaxel (TAXOL). ®Docetaxel (TAXOTERE) ® Deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, such as etoposide, teniposide, etc.; antibiotics, such as anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin, and morpholino derivatives, etc.; phenoxizone. Biscyclopeptides, such as actinomycin; basic glycopeptides, such as bleomycin; anthraquinone glycosides, such as plicamycin (mithramycin); anthraquinones, such as mitoxantrone; azirinopyrrolo indoledione, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; etc.

[0107] Other anti-cytotoxic agents include navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.

[0108] Microtubule-influencing agents with 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), and paclitaxel (TAXOL). ® ), TAXOL ® Derivatives, Taxostatin ® Thiocolchicine (NSC 361792), trimethylcysteine, vincristine sulfate, vinblastine sulfate, natural and synthetic epothilones including (but not limited to) eopthilone A, eopthilone B, and discormolide; estramustine, nocodazole, etc.

[0109] Suitable hormone modulators and steroids (including synthetic analogs) include (but are not limited to) adrenocorticosteroids, such as prednisone and dexamethasone; estrogens and pregestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate. Acetate), estradiol, clomiphene, tamoxifen; etc.; adrenocortical inhibitors, such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testrolide, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estradiol, medroxyprogesterone acetate, leuprolide, flutamide (DROGENIL) ® ), toremifene (FARESTON) ® ) and goserelin (ZOLADEX) ® Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0110] Other suitable chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP), carboplatin, etc.; ureas, such as hydroxyurea; hydrazine, such as N-methylhydrazine; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; folate; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants, such as mycophenolic acid, thalidomide, deoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); gefitinib (IRESSA). ® , ZD 1839), 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazolin; etc.

[0111] Taxanes are suitable for use. "Taxanes" includes paclitaxel and any active taxane derivatives or prodrugs. "Pacific paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as docetaxel, TAXOL) ® TAXOTERE ® (Formulations of docetaxel), 10-deacetyl analogs of paclitaxel and 3'N-debenzoyl-3'N-tert-butoxycarbonyl analogs of paclitaxel) can be readily prepared using 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, WO93 / 10076; U.S. Patents 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 various commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (from short-leaved yew) Taxus brevifolia T7402 from Yunnan yew ( Taxus yannanensis (T-1912). Pacific paclitaxel should be understood not only to the common chemically available form of paclitaxel, but also to its analogues and derivatives (such as TAXOTERE as described herein). ®(such as paclitaxel) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-polydextrose, or paclitaxel-xylose).

[0112] The term "taxane" also includes a variety of known derivatives, including hydrophilic and hydrophobic derivatives. Taxane derivatives include (but are not limited to) galactose and mannose derivatives described in International Patent Application WO 99 / 18113; piperazine 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; sulfinamide derivatives described in U.S. Patent No. 5,821,263; and paclitaxel derivatives described in U.S. Patent No. 5,415,869. It also includes prodrugs 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] Suitable biological response modifiers 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 specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) angiogenesis inhibitors.

[0114] The present invention also envisions using the chromatographic resins, chromatographic steps and / or conditions described herein, or size exclusion chromatography methods described herein to control or adjust the concentration or activity level of β-glucuronidase in purified protein formulations (i.e., purified antibody formulations), for example, to achieve a desired concentration or level.

[0115] In various embodiments, the method further includes controlling or adjusting the concentration and / or activity level of β-glucuronidase in the purified protein formulation.

[0116] In some embodiments, the control or regulation steps include controlling (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the retention time in the SEC.

[0117] In some implementations, the control or regulation step includes controlling the retention time in the CEX chromatography.

[0118] In some implementations, the control or regulation step includes controlling the retention time in the protein A purification step.

[0119] In some embodiments, the control or regulation step includes adjusting the pH during (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the SEC.

[0120] In some implementations, the control or regulation step includes adjusting the pH when performing the CEX chromatography.

[0121] In some embodiments, the control or regulation step includes adjusting the pH during the protein A purification step.

[0122] 6. Explanatory Implementation Plan This disclosure covers the following non-restrictive implementations: 1. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to anion exchange (AEX) chromatography or multimode chromatography using a resin, wherein the ligands of said resin contain any one or more of the following functional groups (e.g., any two or all three): phenyl functional group, hydroxyl functional group, and quaternary ammonium functional group, and optionally wherein said purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or for coupling to said reagent via said linker.

[0123] 2. A method for preparing coupling proteins, the method comprising: (1) Purification of a protein formulation containing β-glucuronidase by subjecting it to AEX chromatography or multimode chromatography using a resin, wherein the ligands of said resin contain any one or more of the following functional groups (e.g., any two or all three): phenyl, hydroxyl, and quaternary ammonium functional groups; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

[0124] 3. The method as described in embodiment 1 or 2, wherein the ligand comprises a phenyl functional group and a hydroxyl functional group.

[0125] 4. The method as described in embodiment 1 or 2, wherein the ligand comprises a phenyl functional group and a quaternary ammonium functional group.

[0126] 5. The method as described in embodiment 1 or 2, wherein the ligand comprises a hydroxyl functional group and a quaternary ammonium cationic functional group.

[0127] 6. The method as described in embodiment 1 or 2, wherein the ligand comprises a phenyl functional group, a hydroxyl functional group, and a quaternary ammonium functional group.

[0128] 7. The method of embodiment 6, wherein the ligand comprises an N-benzyl-2-hydroxy-N-methylethane-1-ammonium moiety.

[0129] 8. The method as described in embodiment 7, wherein the ligand is .

[0130] 9. The method of any one of embodiments 1-8, wherein the resin is Capto™ Adhere.

[0131] 10. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin, wherein the ligands of the resin contain phenyl functional groups, and optionally wherein the purified protein is capable of being coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or for coupling to the pharmaceutical agent via the linker.

[0132] 11. A method for producing a coupling protein, the method comprising: (1) Purification of a protein formulation containing β-glucuronidase by subjecting it to AEX chromatography or HIC using a resin, wherein the ligands of the resin contain phenyl functional groups; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

[0133] 12. The method as described in embodiment 10 or 11, wherein the ligand comprises a phenoxy moiety.

[0134] 13. The method of embodiment 12, wherein the ligand is a phenoxy group.

[0135] 14. The method as described in embodiment 13, wherein the resin is Capto™ Phenyl.

[0136] 15. The method as described in embodiment 14, wherein the resin is Capto™ Phenyl (high sub).

[0137] 16. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to multimode chromatography using a resin, and optionally wherein the purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or for coupling to the reagent via the linker.

[0138] 17. A method for producing a coupling protein, the method comprising: (1) Purification of proteins by subjecting a protein formulation containing β-glucuronidase to multimode chromatography using a resin; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

[0139] 18. The method of embodiment 16 or 17, wherein the ligand of the resin comprises a phosphate group, calcium, hydroxyl group or any combination thereof.

[0140] 19. The method of any one of embodiments 16-18, wherein the ligand of the resin comprises a phosphate ester group.

[0141] 20. The method of any one of embodiments 16-19, wherein the ligand of the resin comprises a phosphate ester group and calcium.

[0142] 21. The method of any one of embodiments 16-20, wherein the ligand of the resin comprises a phosphate group, calcium, and a hydroxyl group.

[0143] 22. The method of any one of embodiments 19-21, wherein the chromatography is cation exchange (CEX) chromatography.

[0144] 23. The method of any one of embodiments 16-18, wherein the ligand of the resin comprises calcium.

[0145] 24. The method of any one of embodiments 16-18, wherein the ligand of the resin comprises calcium and hydroxyl groups.

[0146] 25. The method as described in any one of embodiments 16-18, 20-21 and 23-24, wherein the chromatography is anion exchange (AEX) chromatography.

[0147] 26. The method of any one of embodiments 16-25, wherein the resin is EconoFit CHT XT.

[0148] 27. The method of any one of embodiments 1-26, wherein the chromatography is performed at a pH between 5 and 8.

[0149] 28. The method of embodiment 27, wherein the chromatography is performed at a pH of about 5.5.

[0150] 29. The method of embodiment 27, wherein the chromatography is performed at a pH of about 6.5.

[0151] 30. The method of embodiment 27, wherein the chromatography is performed at a pH of about 7.5.

[0152] 31. The method of any one of embodiments 1-30, the method further comprising a protein A purification step prior to the chromatographic step, wherein optionally the protein A purification step produces the protein formulation.

[0153] 32. The method of any one of embodiments 1-31, the method further comprising subjecting the purified protein preparation obtained from the chromatographic step to a cation exchange (CEX) chromatographic step.

[0154] 33. The method of embodiment 32, wherein the CEX chromatography step uses a resin, and the ligands of the resin contain sulfonate functional groups.

[0155] 34. The method of embodiment 33, wherein the ligand comprises a sulfopropyl moiety.

[0156] 35. The method of embodiment 34, wherein the ligand is .

[0157] 36. The method of embodiment 35, wherein the resin is Praesto ® SP65.

[0158] 37. The method of embodiment 35, wherein the resin is SP Sepharose™ Fast Flow ion exchange chromatography medium.

[0159] 38. The method of any one of embodiments 1-37, wherein the method results in the absence or undetectability of β-glucuronidase activity in the purified protein formulation.

[0160] 39. The method of any one of embodiments 1-38, wherein the method results in no loss of glucuronide on the conjugate product of the purified protein.

[0161] 40. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to size exclusion chromatography (SEC), wherein purification removes elution fractions eluted with molecules larger than said protein, and optionally wherein said purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or is used for coupling to said reagent via said linker.

[0162] 41. A method for producing a coupling protein, the method comprising: (1) Purifying a protein by subjecting a protein formulation containing β-glucuronidase to SEC, wherein the purification removes elution fractions larger than said protein molecules; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

[0163] 42. The method as described in embodiment 40 or 41, wherein the method results in a loss of less than 1% of glucuronide on the conjugate product of the purified protein.

[0164] 43. The method of any one of embodiments 1-42, wherein the agent is a cytotoxic drug.

[0165] 44. The method of any one of embodiments 1-43, wherein the protein is a therapeutic protein.

[0166] 45. The method of any one of embodiments 1-44, wherein the protein is an antibody, an Fc-fusion protein, a bispecific binder, scFv, VHH, VHH-Fc, or a protein ligand.

[0167] 46. ​​The method of any one of embodiments 1-45, wherein the protein is an antibody.

[0168] 47. The method of embodiment 46, wherein the conjugate product of the purified protein is an antibody-drug conjugate (ADC).

[0169] 48. The method of any one of embodiments 1-47, wherein the β-glucuronidase cleavable linker containing glucuronide comprises a cleavable peptide motif.

[0170] 49. The method of embodiment 48, wherein the cleavable peptide motif is protected from proteolysis by glucuronic acid.

[0171] 50. The method of any one of embodiments 1-49, wherein the β-glucuronidase-cleavable linker containing glucuronide comprises a dipeptide unit protected by an adjacent glucuronic acid.

[0172] 51. The method of any one of embodiments 1-50, further comprising controlling the concentration of β-glucuronidase and / or the level of β-glucuronidase activity in the purified protein formulation.

[0173] 52. The method of embodiment 51, wherein the control step comprises controlling (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the retention time in the SEC.

[0174] 53. The method as described in embodiment 51 or 52, wherein the control step includes controlling the retention time in the CEX chromatography.

[0175] 54. The method of any one of embodiments 51-53, wherein the control step includes controlling the retention time in the protein A purification step.

[0176] 55. The method of any one of embodiments 51-54, wherein the control step comprises adjusting the pH when performing (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the SEC.

[0177] 56. The method of any one of embodiments 51-55, wherein the control step includes adjusting the pH when performing the CEX chromatography.

[0178] 57. The method of any one of embodiments 51-56, wherein the control step includes adjusting the pH during the protein A purification step.

[0179] It should be understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Therefore, the following examples are intended to illustrate but not limit this disclosure.

[0180] 7. Examples The antibody preparation is (1) harvested via filtration of a culture of cells secreting the antibody (e.g., by deep filtration), and (2) purified, for example using protein A chromatography, as described in the art. After the antibody is conjugated to the payload via a linker, the resulting antibody-drug conjugate (ADC) is then subjected to hydrophobic interaction chromatography (HIC) for purity analysis. As expected, a major HIC peak representing the ADC with the desired DAR is observed (e.g., as designed in site-specific conjugation, where one antibody is conjugated to a specific number of payloads) (see, for example...). Figures 2 to 3 The DAR2 peak in the image, and secondary peaks representing other possible DAR substances (e.g., antibodies not conjugated to a payload) (see example...). Figures 2 to 3 (DAR0 and DAR1 peaks in the image). Surprisingly, additional peaks were observed following the main peak (see, for example, in...). Figures 2 to 3 The peaks labeled DAR2 #2 and DAR2 #3 indicate certain systemic contamination, which in turn significantly reduces the stability of the coupling product (e.g., ADC). Therefore, several experiments were designed and conducted to investigate this problem (data not shown). As a result, β-glucuronidase contamination was identified as the primary cause of this impurity problem in the ADC product. See, for example, section 7.2.1. Furthermore, this paper investigates and provides solutions to this contamination problem. See, for example, sections 7.2.2 through 7.2.8.

[0181] The presence of process-related contaminants (β-glucuronidase) has been detected in some antibody formulations. When present, this contaminant can degrade chemical linkers used in the preparation of therapeutic conjugates. For example, linkers containing tandem cleavage elements paired with cytotoxic payloads (e.g., olistatin or topoisomerase I inhibitors) can be coupled to antibodies to prepare antibody-drug conjugates (ADCs). These tandem cleavage elements consist of dipeptide units protected by adjacent glucuronic acid. The dipeptide, in its monosaccharide-intact form, is resistant to proteolytic cleavage. Once the glucuronic acid has been removed by glucuronidase (a process that occurs in vivo within the lysosome after ADC internalization and transport), the dipeptide becomes unprotected and can be cleaved by proteases (e.g., cathepsin B), subsequently releasing the payload.

[0182] When process-related contaminants (β-glucuronidase) are present in purified antibodies (API intermediates) intended for conjugation and ADC production, the enzymatic activity of these contaminants can affect the removal of glucuronic acid from the chemical linker during and / or after ADC production and in the formulation buffer. This degradation process is facilitated by the fact that glucuronidase is active under acidic conditions, which mimic the acidic conjugation conditions required by some conjugation methods and the acidic formulation (storage) conditions preferred by most proteins, antibodies, and ADCs.

[0183] 7.1. Methods 7.1.1. Determination of glucuronidase activity in quantitative monoclonal antibody (mAb) formulations Residual β-glucuronidase was monitored throughout the downstream process of antibody formulation using two orthogonal techniques. To determine GUSB (β-glucuronidase) concentration by activity assay, samples were incubated at 37°C with 4-methylumbelliferyl β-D-glucuronide, which was cleaved into 4-methylumbelliferyl, and then measured fluorescence at Ex / Em wavelengths of 360 / 450 nm or 330 / 450 nm. Calibration was performed using prepared recombinant human GUSB at concentrations ranging from 10 to 50 ng / mL.

[0184] 7.1.2. HIC Method for Quantitative Calculation of Glucuronide Loss on ADCs (as a Proximity to β-Glucuronidase Levels in mAb Formulations) Purified antibodies (3-15 mg / mL) produced from various purification processes in the absence of ( Figure 1 ) or exist ( Figure 4B Excessive Ac-glucuronide-PABA (e.g.) Figure 4A The adapter-payload, which includes a tandem cleavage linker, is coupled to 0.85 mM at 0.85 mM to produce an ADC with a drug / antibody ratio (DAR) of 2.

[0185] The reaction was carried out at 37°C for 72 hours in a buffer containing 0.85% dimethylacetamide (DMA) in 20 mM sodium citrate and 50 mM NaCl (pH 5.5) or 20 mM histidine (pH 5.5) and 6% trehalose. After coupling, the ADC was exchanged with buffer to remove residual free drug. To determine the percentage of glucuronide loss, the ADC was quantified by analytical chromatography corresponding to the peak at DAR 2 (DAR2) using hydrophobic interaction chromatography (HIC) (Tosoh #14947). Examples of glucuronide loss calculations using the HIC method are shown in Table 1 and Figure 1 middle.

[0186] Table 1. Calculation of glucuronide loss 7.1.3. Mass spectrometry determination of β-glucuronidase protein levels in quantitative mAb preparations To determine GUSB concentration by LC-MS, the sample was incorporated with internal standards, exchanged into Tris buffer, digested with trypsin overnight, reduced with DTT at 95°C, separated by reversed-phase LC, and detected by MS. The first two or three peptides of each incorporated protein were used for quantification, and the average value was reported as the measured quantity.

[0187] 7.1.4. Capto™ Adhere FPLC method Clean the AKTA Pure instrument and necessary buffer lines with 0.1N NaOH, wash with DI / MilliQ, and equilibrate with 20 mM sodium citrate pH 5.5 buffer. Connect the HiTrap Capto™ Adhere column to the AKTA Pure and wash with 5 column volumes (CV) of 0.1N NaOH, followed by equilibration with 5–10 CV of 20 mM citrate pH 5.5 until the pH reaches 5.5 ± 0.1. Load the antibody purified using the protein-A affinity method onto the column at a flow rate of 1 mL / min (1 mL column, Cytvia #28405844) or 5 mL / min (5 mL column, Cytvia #28405846) and wash the column with 6 CV of 20 mM citrate pH 5.5 buffer. Collect unbound samples, exchange with appropriate buffer, and sterilely filter using a 0.22 µm membrane filter. After use, sterilize the column with 5 CV of 0.1N NaOH, then equilibrate with 5–10 CV of 20 mM citrate pH 5.5 buffer. After rinsing with 5 CV of DI / MilliQ water, followed by 5 CV of 20% ethanol, store the column in 20% ethanol.

[0188] 7.1.5. Capto™ Adhere Batch Combination Method Capto™ Adhere resin slurry was used to remove glucuronidase from antibody formulations <100 mg. An appropriate volume of the 50 / 50 slurry mixture was washed with 10 x volumes of 20 mM citrate pH 5.5 buffer (see Table 2 for more details). All contents were centrifuged at 500 x g for 10 min and the solution was removed without disturbing the resin settling at the bottom of the centrifuge tube. The washing step was repeated twice, followed by the addition of protein A affinity-purified antibody. After overnight binding (with 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 settling at the bottom. The resin was then washed with 5 CV of 20 mM citrate pH 5.5. Unbound samples were collected, buffer-exchanged to an appropriate buffer, and sterilely filtered using a 0.22 µm membrane filter.

[0189] Table 2. Amount of protein / antibody formulation and Capto™ Adhere used 7.1.6. SEC Removal Method The AKTA pure instrument connected to the 500 µL loop for loading the sample was cleaned with 0.1 N NaOH and DI / MilliQ water, followed by system equilibration with 20 mM histidine at pH 5.5 + 150 mM NaCl. A Sepax SRT-10 SEC 3002 1.3x300 column (part number 225300-21230) was connected to the instrument and cleaned with 2 column volumes (CV) of DI / MilliQ water at a flow rate of 4 mL / min. The column was then equilibrated with 3 CV of 20 mM histidine at pH 5.5 + 150 mM NaCl at pH 5.5. The mAb sample was manually injected into the 500 µL loop to load onto the column and the SEC assay was run at 4 mL / min for 1.5 CV, and 2 mL of the eluent was collected into a 96-well plate. The eluent containing the molecule of interest was collected, avoiding high or low molecular weight substances as indicated by the chromatogram. Figure 6 The combined eluent fractions were exchanged with buffer for appropriate buffer and then aseptically filtered using a 0.22 µm membrane filter.

[0190] 7.2. Results 7.2.1. Detection of the presence of β-glucuronidase The presence of β-glucuronidase in the antibody formulation was confirmed by β-glucuronidase activity at different stages of the purification process (Table 3). The loss of glucuronic acid from the coupled tandem cleavage linker – a characteristic illustrated by… Figure 2Furthermore, three additional data points supported this finding: 1) Mass spectrometry data showed losses of 176 Da and 2 x 176 Da, corresponding to the molecular weight of glucuronic acid (data not shown); 2) The tandem cleavage linker-loador was synthesized without glucuronic acid and, when coupled, overlapped with the "DAR2 #3" peak on the chromatogram. Figure 3 ) and 3) When excess Ac-glucuronide-PABA is added as a substrate for β-glucuronidase to the coupling reaction, the DAR2 #2 and DAR2 #3 peaks are significantly reduced / eliminated. Figure 4A and Figure 4B ).

[0191] Table 3. Enzyme activity data of β-glucuronidase in 250 L batches 7.2.2. Evaluation of purification studies for the removal of β-glucuronidase from protein formulations To mitigate the impact of process-related contaminants on the stability of conjugates during preparation and storage, a series of purification studies were conducted to evaluate the removal of β-glucuronidase from protein formulations. To assess whether alternative purification methods remove β-glucuronidase, three analytical methods were established as described in Section 7.1: 1) a β-glucuronidase activity assay to quantify β-glucuronidase activity, 2) a hydrophobic interaction chromatography (HIC) method to quantify the amount of glucuronide lost when conjugated to an antibody containing a glucuronide linker-loador, and 3) a mass spectrometry method to quantify β-glucuronidase protein.

[0192] The following sections present the various purification conditions and column tests used to remove β-glucuronidase at each step of the mAb purification process.

[0193] A. Protein A Step Initially, 10 μL of culture was purified by adding various wash buffers to the protein A capture step, and the samples were then evaluated to determine whether the inclusion of the wash step reduced β-glucuronidase levels. Different wash buffers were tested. None of the washes significantly and adequately removed β-glucuronidase. See Table 4 below.

[0194] Table 4. Results of Protein A Screening Next, β-glucuronidase removal was tested on two additional protein A resins (both on a 1 mL scale, in addition to the resin used in the usual protein A capture step) with clarified harvests treated with pH 3.2 elution, high pH washing, octanoate washing, and / or detergent treatment. None of the washes adequately removed β-glucuronidase. Approximately 10% to approximately 18% glucuronide loss was still observed.

[0195] B. Anion exchange chromatography procedure To reduce β-glucuronidase levels, three anion exchange (AEX) columns (all at 1 mL resin) were tested at different pH values. Table 5 shows the percentage of glucuronide loss on ADCs prepared with antibodies produced using different anion exchange resins / conditions. All three conditions showed the optimal clearance of β-glucuronidase with the lowest percentage of ADC glucuronide loss, both using Capto™ Adhere resin.

[0196] Table 5. AEX Filtering Results C. Cation exchange chromatography procedure To reduce β-glucuronidase levels, various cation exchange (CEX) conditions were tested (all at 1 mL resin scale): intermediate pH washes (20 mM sodium phosphate, pH 7.0) were explored before elution (through a 30 CV gradient from 0 to 400 mM NaCl), and various CEX resins were loaded at pH 6.5 (50 mM MES at pH 6.0, followed by 50 mM MES at pH 6.5). Table 6 shows the percentage of glucuronide loss on ADCs prepared with antibodies produced using different resins / conditions. None of the different CEX conditions / resins adequately removed β-glucuronidase.

[0197] Table 6. CEX Step Filtering Results This study shows that the combination of protein A purification and ion exchange (IEX) chromatography, commonly used for monoclonal antibody purification, is insufficient to remove β-glucuronidase, although commonly used IEX columns do reduce HCP (host cell proteins, e.g., contaminating proteins released by antibody-secreting cells). Surprisingly, this study shows that only certain columns adequately remove β-glucuronidase, and that β-glucuronidase levels are not entirely correlated with HCP levels.

[0198] 7.2.3. Confirmatory Experiment for Reducing β-Glucuronidase Based on the results of purification using Protein A, AEX, and CEX, further investigation was conducted to select the final purification step for removing β-glucuronidase. Four cohort studies were performed to compare the performance of the second and third fine purification steps and their impact on β-glucuronidase removal. The starting material for 5.5 g mAb was a clarified harvest material purified by Protein A chromatography (106 mL resin; pH 3.2 elution) followed by virus inactivation (NVI). Figure 5 A comparative graph of the processes studied by the four teams is shown, with each column at a scale of 15–20 mL. Results are summarized in Table 7. pH washing refers to a washing step using a pH buffer solution of 0.02 M sodium phosphate (pH 7.0). Capto™ Adhere resin followed by SP65 CEX (team #2) was selected as the final process (team #4 used the original process). The ligands of Capto™ Adhere resin are... The ligand for Capto™ Phenyl (high sub) resin is phenoxy. The ligand for Praesto® SP65 resin is... .

[0199] Table 7. Overview data from the four team studies 7.2.4. Confirm Operation (1000 L) To confirm the effectiveness of the selected purification protocol on a manufacturing scale, materials from 1000 L of culture were purified using the final method: Protein A (Praesto® Jetted A50; 31.2 L), AEX (Capto™ Adhere resin; 13.8 L), and CEX (Praesto® SP65; 13.8 L; no pH wash). The results are summarized in Table 8. The process successfully and adequately removed β-glucuronidase, with no glucuronide loss observed on the ADC.

[0200] Table 8. 1000 L Confirmation Operation 7.2.5. Testing Capto™ Adhere resin on various antibodies and at various scales To evaluate the effectiveness of Capto™ Adhere resin in removing β-glucuronidase from formulations of different antibody backbones, four different antibodies were passed through Capto™ Adhere resin, and the loss of glucuronidase on the tandem cleavage linker payload (ADC) was analyzed using a HIC quantitative method. For the four antibodies tested, passing the antibody through Capto™ Adhere resin reduced or eliminated glucuronidase (Table 9).

[0201] Table 9. Capto™ Adhere Antibody Screening Results The type and amount of Capto™ Adhere resin used for the efficient removal of β-glucuronidase vary depending on the amount of protein. For research-scale purification, multiple 1 mL or 5 mL HiTrap Capto™ Adhere columns can be used in tandem to remove glucuronidase from protein / antibody formulations ranging from 200–4000 mg (see Table 10 for more details). For protein amounts >4000 mg, a 25 mL Capto™ Adhere column can be used.

[0202] Table 10. Capto™ Adhere column volume 7.2.6. Multimode Chromatography Procedure Additional chromatographic resin, EconoFit CHT XT, was tested to reduce β-glucuronidase levels. Three antibodies (antibody P, antibody Q, and antibody E, with isoelectric points (pI) ranging from 7.6 to 8.0) were used. Briefly, cell culture supernatants were harvested, initially purified with protein A affinity chromatography resin, and then buffer-exchanged, centrifuged, and diluted to 10 mM NaPO4 pH 6.5, 6 ppm CaCl2 buffer.

[0203] Connect the EconoFit CHT XT column to the AKTA system. Load the obtained sample onto the column and wash it with 10 mM NaPO4 pH 6.5, 6 ppm CaCl2 buffer. Apply gradient elution (10 mM NaPO4 pH 6.5, 2 M NaCl, 15 mM CaCl2 buffer) from 0 to 100% using 20 CV of stripping buffer (400 mM NaPO4 pH 6.5) and other appropriate clean-in-place (CIP) steps. Without being bound by theory, it is believed that acidic proteins (e.g., with a pI below 7) bind primarily to the calcium sites of the EconoFit CHT XT resin via metal affinity, while basic proteins (e.g., with a pI greater than 7) bind primarily to the phosphate sites via cation exchange. It is also believed that, in some contexts, calcium affinity plays a role in addition to cation exchange by basic proteins. For further details, see BIO-RAD Bulletin 7404 ver A, Mixed-Mode Chromatography: Ceramic Apatite Media, 2020, which is incorporated herein by reference in its entirety.

[0204] The purified sample was coupled and then analyzed for glucuronide loss, for example, by following the HIC method as described above. HIC overlap showed that (1) the control sample without the EconoFit CHT XT purification step presented a peak corresponding to the removal of its glucuronide group from the ADC, but (2) this peak disappeared in the tested sample with the EconoFit CHT XT step (data not shown). A representative summary is presented in Table 11 below, also indicating good removal of β-glucuronidase after CHT XL purification.

[0205] Table 11. EconoFit CHT XT Antibody Screening Results 7.2.7. Determination of β-glucuronidase removal using size exclusion chromatography. Size exclusion chromatography (SEC) was tested as an alternative to Capto™ Adhere resin for the removal of β-glucuronidase. β-glucuronidase is synthesized as a monomer but tends to form polymers. Therefore, purification is performed to remove eluents larger than the antibody eluent (see, for example...). Figure 6The use of SEC columns to purify antibodies was successful in removing β-glucuronidase (see Table 12).

[0206] Table 12. SEC Antibody Screening Results 7.2.8. Validation procedures for compliance with Good Manufacturing Practices (GMP) standards for pharmaceuticals To further confirm the effectiveness of the selected purification protocol on a manufacturing scale, two additional antibodies (antibody G and antibody F) and their formulations were tested. Briefly, the mAb formulation was harvested by filtering 2000 μL of cell culture secreting mAb (e.g., via deep filtration). The resulting formulation was purified by protein A chromatography (Praesto® Jetted A50), followed by virus inactivation (NVI, e.g., at pH 3.5), and further subjected to AEX (Capto™ Adhere resin) and then CEX (Praesto® SP65). The purified sample was diluted and subjected to virus filtration followed by tangential flow filtration (TFF) to obtain the final mAb.

[0207] Results for activity and residual β-glucuronidase by LC-MS are summarized in Tables 13 through 16. Given the significant analytical differences between the two methods and the semi-quantitative properties determined by LC-MS, the results for activity and LC-MS methods are highly consistent. Both confirm the presence of β-glucuronidase in the clarified harvest, measurable β-glucuronidase in the protein A eluate, and β-glucuronidase levels below the detection limit in all samples after AEX purification. This confirms that removal in the manufacture of ADCs containing glucuronide reaches a level expected to have a negligible impact on glucuronide loss. Overall, these results support the successful development of a process to remove β-glucuronidase from several antibody formulations, such as clarified harvest.

[0208] Table 13. 2000 L GMP Validation Procedure - Antibody G - Repeat 1 Table 14. 2000 L GMP Validation Procedure - Antibody G - Repeat 2 Table 15. 2000 L GMP Validation Procedure - Antibody F - Repeat 1 Table 16. 2000 L GMP Validation Procedure - Antibody F - Repeat 2 Throughout this application, references have been made to various publications, patents, patent applications, and other documents. The disclosures of these publications, patents, patent applications, and other documents are incorporated herein by reference in their entirety for all purposes, including to provide a more comprehensive description of the level of technical development to which the subject matter disclosed herein pertains. Although the disclosed subject matter has been described with reference to the embodiments provided above, it should be understood that various modifications may 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

1. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to anion exchange (AEX) chromatography or multimode chromatography using a resin, wherein the ligands of said resin contain any one or more of the following functional groups: phenyl functional group, hydroxyl functional group, and quaternary ammonium functional group, and optionally wherein said purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a reagent via said linker.

2. A method for producing a coupling protein, the method comprising: (1) Purification of a protein formulation containing β-glucuronidase by subjecting it to AEX chromatography or multimode chromatography using a resin, wherein the ligands of the resin contain one or more of the following functional groups: phenyl, hydroxyl, and quaternary ammonium functional groups; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

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

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

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

8. The method of claim 7, wherein the ligand is 。 9. The method of any one of claims 1-8, wherein the resin is Capto™ Adhere.

10. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to AEX chromatography or hydrophobic interaction chromatography (HIC) using a resin, wherein the ligands of the resin contain phenyl functional groups, and optionally wherein the purified protein is capable of being coupled to a pharmaceutical agent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a pharmaceutical agent via said linker.

11. A method for producing a coupling protein, the method comprising: (1) Purification of a protein formulation containing β-glucuronidase by subjecting it to AEX chromatography or HIC using a resin, wherein the ligands of the resin contain phenyl functional groups; and (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

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 a phenoxy group.

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 for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to multimode chromatography using a resin, and optionally wherein the purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a reagent via said linker.

17. A method for producing a coupling protein, the method comprising: (1) Proteins are purified by subjecting a protein formulation containing β-glucuronidase to multimode chromatography using a resin. as well as (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

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

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

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

21. The method of any one of claims 16-20, wherein the 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 cation exchange (CEX) chromatography.

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

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

25. The method of any one of claims 16-18, 20-21 and 23-24, wherein the chromatography is 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 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, further comprising a protein A purification step prior to the chromatographic step, wherein optionally the protein A purification step produces the protein formulation.

32. The method of any one of claims 1-31, further comprising subjecting the purified protein formulation obtained from the chromatographic step to a cation exchange (CEX) chromatographic step.

33. The method of claim 32, wherein the CEX chromatography step uses a resin, and the ligands of the resin contain sulfonate functional groups.

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

35. The method of claim 34, wherein the ligand is .

36. 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 medium.

38. The method of any one of claims 1-37, wherein the method results in the absence or undetectability of β-glucuronidase activity in the purified protein formulation.

39. The method of any one of claims 1-38, wherein the method results in no loss of glucuronide in the conjugate of the purified protein.

40. A method for purifying a protein, the method comprising subjecting a protein formulation containing β-glucuronidase to size exclusion chromatography (SEC), wherein purification removes elution fractions eluted with molecules larger than said protein, and optionally wherein said purified protein is capable of being coupled to a reagent via a β-glucuronidase-cleavable linker containing glucuronide and / or used for coupling to a reagent via said linker.

41. A method for producing a coupling protein, the method comprising: (1) The protein is purified by subjecting a protein preparation containing β-glucuronidase to SEC, wherein the purification removes the elution fraction that is larger than the protein molecules. as well as (2) The purified protein is coupled to the pharmaceutical via a β-glucuronidase-cleavable linker containing glucuronide.

42. The method of claim 40 or 41, wherein the method results in a loss of less than 1% of glucuronide on the conjugate 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, scFv, VHH, 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 conjugate product of the purified protein is an antibody-drug conjugate (ADC).

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

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

50. The method of any one of claims 1-49, wherein the β-glucuronidase-cleavable linker containing glucuronide comprises a dipeptide unit protected by an adjacent glucuronic acid.

51. The method according to any one of claims 1-50, further comprising controlling the concentration of β-glucuronidase and / or the level of β-glucuronidase activity in the purified protein formulation.

52. The method of claim 51, wherein the control step comprises controlling the retention time of: (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the SEC.

53. The method of claim 51 or 52, wherein the control step includes controlling the retention time in the CEX chromatography.

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

55. The method of any one of claims 51-54, wherein the control step comprises adjusting pH, wherein (1) the AEX chromatography or multimode chromatography, (2) the AEX chromatography or HIC, (3) the multimode chromatography, or (4) the SEC are performed at said pH.

56. The method of any one of claims 51-55, wherein the control step comprises adjusting the pH when performing the CEX chromatography.

57. The method of any one of claims 51-56, wherein the control step comprises adjusting the pH during the protein A purification step.

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