Antibody Refining

Abstract

To provide methods for purification of antibodies.SOLUTION: Purification methods provided involve the use of hydroxyapatite resin (HA) to separate an antibody of interest from one or more impurities.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 635,943, filed February 27, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to methods for purifying antibodies from impurities such as antibody degradation products. The purification methods disclosed herein involve the use of hydroxyapatite resin. [Background technology]

[0003] Antibodies are important biological molecules for use in medicine, diagnostics, industry, etc. For example, due to the size and molecular complexity of antibodies, although many methods and reagents are available for the recombinant production of antibodies, it often remains challenging to efficiently produce and purify recombinant antibodies of interest, especially at large-scale / industrial production levels.

[0004] For example, during the production of a recombinant antibody of interest, degradation products associated with the antibody of interest may occasionally occur; these degradation products are undesirable impurities. These degradation products may have some molecular properties very similar to those of the antibody of interest (e.g., identical or nearly identical amino acid sequence or mass). Due to the molecular similarity between the intact antibody of interest and the degraded version of the antibody, it may be very difficult to efficiently separate the intact antibody from the degraded antibody. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Provisional Patent Application No. 62 / 635,943 [Patent Document 2] International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545) [Patent Document 3] International Patent Application Number PCT / IB2011 / 054899 (WO2012 / 059882) [Patent Document 4] U.S. Patent Application No. 15 / 085,644 (U.S. Publication No. 20160297885) [Patent Document 5] U.S. Patent Application No. 15 / 993,874 (U.S. Publication No. 20180346601) [Non-patent literature]

[0006] [Non-Patent Document 1] Suresh et al., Methods in Enzymology 121:210, 1986 [Non-patent document 2] Millstein and Cuello, Nature 305, 537-539, 1983 [Non-patent document 3] Giese et al., Biotechnology Progress, “Bispecific Antibody Process Development: Assembly and Purification of Knob and Hole Bispecific Antibodies,” January 17, 2018. [Non-patent document 4] Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press [Non-Patent Document 5] Oligonucleotide Synthesis (M.J. Gait, ed., 1984) [Non-patent document 6] Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. JECellis, 1998) Academic Press [Non-Patent Document 7] Animal Cell Culture (ed. RIFreshney, 1987) [Non-patent document 8] Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press [Non-Patent Document 9] Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J.B. Griffiths, and D.G. Newell, 1993-1998) J. Wiley and Sons [Non-Patent Document 10] Methods in Enzymology (Academic Press, Inc.) [Non-Patent Document 11] Handbook of Experimental Immunology (eds. D.M. Weir and C.C. Blackwell) [Non-Patent Document 12] Gene Transfer Vectors for Mammalian Cells (eds. J.M. Miller and M.P. Calos, 1987) [Non-Patent Document 13] Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987) [Non-Patent Document 14] PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994) [Non-Patent Document 15] Current Protocols in Immunology (eds. JE Coligan et al., 1991) [Non-Patent Document 16] Short Protocols in Molecular Biology (Wiley and Sons, 1999) [Non-Patent Document 17] Immunobiology (C.A. Janeway and P. Travers, 1997) [Non-Patent Document 18] Antibodies (P. Finch, 1997) [Non-Patent Document 19] Antibodies: a practical approach (ed. D. Catty., IRL Press, 1988~1989) [Non-Patent Document 20] Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000) [Non-Patent Document 21] Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999) [Non-Patent Document 22] The Antibodies (eds. M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995) Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need for new and improved methods for the purification of antibodies from impurities. [Means for solving the problem]

[0008] Provided herein are methods for purifying an antibody of interest from one or more impurities.

[0009] In some embodiments, provided herein are methods for purifying an antibody, the methods comprising: A) loading an antibody preparation in a load buffer onto a hydroxyapatite (HA) resin, wherein the antibody preparation comprises I) an intact antibody of interest, and II) clipped versions of the antibody of interest that are degradation products from the intact antibody of interest and have a mass that differs from the mass of the intact antibody of interest by less than 10%; and B) eluting the intact antibody of interest from the HA resin with an elution buffer.

[0010] In some embodiments, provided herein are methods for purifying a bispecific antibody, the method comprising: A) loading an antibody preparation in a load buffer onto a hydroxyapatite (HA) resin, wherein the antibody preparation comprises I) the intact bispecific antibody of interest; and II) at least one impurity species, the impurity species being selected from the group consisting of: a) a clipped version of the bispecific antibody of interest that is a degradation product from the intact bispecific antibody of interest and has a mass that differs by less than 10% from the mass of the intact bispecific antibody of interest; b) a first parent antibody that is a monospecific antibody with the same antigen specificity as the first arm of the intact bispecific antibody; c) a second parent antibody that is a monospecific antibody with the same antigen specificity as the second arm of the intact bispecific antibody; and d) a high molecular mass species (HMMS); and B) eluting the intact bispecific antibody of interest from the HA resin with an elution buffer.

[0011]

[0010] In some embodiments, provided herein are methods for purifying bispecific antibodies, the methods comprising: A) loading an antibody preparation in a load buffer onto a hydroxyapatite (HA) resin, wherein I) the antibody preparation comprises a) the intact bispecific antibody of interest and b) a clipped version of the bispecific antibody of interest that is a degradation product from the intact bispecific antibody of interest and has a mass that differs by less than 10% from the mass of the intact bispecific antibody of interest; II) the ratio of clipped bispecific antibody molecules to intact bispecific antibody molecules in the antibody preparation is between at least 1:50 and no more than 1:5; B) eluting the intact bispecific antibody from the HA resin with an elution buffer. In some embodiments, the methods further comprise C) collecting a purified fraction eluted from the HA resin, wherein the purified fraction comprises the intact bispecific antibody.

[0012] In some embodiments, a method of purifying an antibody comprises the steps of: A) loading an antibody preparation in a load buffer onto a hydroxyapatite (HA) resin, wherein I) the antibody preparation comprises a) an intact antibody of interest and b) a clipped version of the antibody of interest that is a degradation product from the intact antibody of interest and has a mass that differs by less than 10% from the mass of the intact antibody of interest; II) the clipped version of the antibody comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 1 Provided herein is a method comprising the steps of: A) eluting the intact antibody from the HA resin with an elution buffer, the HA resin comprising 15% or 20% by weight of the clipped antibody; B) eluting the intact antibody from the HA resin with an elution buffer; and C) collecting a purified fraction eluted from the HA resin, wherein the purified fraction comprises intact antibody and less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by weight of the clipped antibody, wherein the purified fraction comprises a lower weight percent of the clipped antibody than the antibody preparation.

[0013] In some embodiments, in the methods provided herein involving eluting the antibody of interest from the HA resin with an elution buffer, the elution buffer comprises an ion. The concentration of the ion in the buffer may increase during elution. The concentration of the ion in the buffer surrounding the HA resin may increase during elution.

[0014] In some embodiments, in the methods provided herein involving an antibody, the antibody is a heterodimeric bispecific antibody.

[0015] In some embodiments, in the methods provided herein that involve collecting a purified fraction eluted from an HA resin, wherein the purified fraction comprises an intact antibody of interest, the purified fraction comprises at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the intact antibody of interest.

[0016] In some embodiments, in the methods provided herein involving a purified fraction comprising an intact bispecific antibody and a clipped bispecific antibody, the ratio of clipped bispecific antibody molecules to intact bispecific antibody molecules in the purified fraction is less than or equal to 1:400, 1:200, 1:100, or 1:50.

[0017] In some embodiments, in the methods provided herein involving an antibody of interest that is an anti-CD3 antibody or a bispecific antibody that comprises an anti-CD3 arm, the antibody comprises at least one of: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO:1; ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2; iii) a VH region comprising the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:3; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:4.

[0018] In some embodiments, in methods provided herein involving a bispecific antibody, the bispecific antibody is i) an anti-BCMA / anti-CD3 bispecific antibody comprising an anti-BCMA arm and an anti-CD3 arm, or ii) an anti-FLT3 / anti-CD3 bispecific antibody comprising an anti-FLT3 arm and an anti-CD3 arm.

[0019] In some embodiments, in methods provided herein involving a bispecific antibody comprising an anti-BCMA arm, the anti-BCMA arm comprises at least one of: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO:5; ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6; iii) a VH region comprising the amino acid sequence set forth in SEQ ID NO:5 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:7; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:8.

[0020] In some embodiments, in methods provided herein involving a bispecific antibody comprising an anti-FLT3 arm, the anti-FLT3 arm comprises at least one of: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 9; ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10; iii) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 11; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0021] In some embodiments, in methods provided herein involving loading an antibody preparation onto an HA resin, the antibody preparation is loaded onto the HA resin to an on-resin density of between 2, 3, 4, or 5 g / L and 8, 9, 10, 12, 15, or 20 g / L.

[0022] In some embodiments, in methods provided herein involving loading an antibody preparation onto an HA resin, at least 1, 5, 10, 50, 100, 500, 1000, or 5000 grams of the antibody preparation are loaded onto the HA resin.

[0023] In some embodiments, in methods provided herein involving an antibody preparation, the antibody preparation comprises at least 50%, 60%, 70%, or 80% by weight, but less than 90%, 95%, 97%, 98%, or 99% by weight, intact antibody of interest.

[0024] In some embodiments, in methods provided herein involving a clipped antibody, the clipped antibody has a mass that differs from the mass of the intact antibody by less than 0.1%, 0.5%, 1%, or 2%. In some embodiments, in methods provided herein involving a clipped antibody, the clipped antibody has a mass that is between about 5 and 100 daltons greater than the mass of the intact antibody. In some embodiments, in methods provided herein involving a clipped antibody, the clipped antibody has a mass that is about 18 daltons greater than the mass of the intact antibody.

[0025] In some embodiments, the methods provided herein involving a clipped antibody, the clipped antibody has a cleaved peptide bond in the polypeptide chain of the antibody, wherein the cleaved peptide bond is in the heavy chain of the antibody. In some embodiments, the methods provided herein involving a clipped antibody, the clipped antibody has a cleaved peptide bond in the polypeptide chain of the antibody, wherein the cleaved peptide bond is in the light chain of the antibody.

[0026] In some embodiments, in methods provided herein involving a clipped antibody, the clipped antibody contains the same number of amino acids and the same amino acid sequence as an intact antibody, or in some embodiments, the clipped antibody contains a different number of amino acids than an intact antibody.

[0027] In some embodiments, in the methods provided herein involving an antibody of interest comprising a VH and VL domain that specifically binds CD3, the corresponding clipped antibody comprises a cleaved peptide bond in the VH domain that specifically binds CD3.

[0028] In some embodiments, in the methods provided herein involving an HA resin, the HA resin is a ceramic hydroxyapatite (cHA) resin.

[0029] In some embodiments, in the methods provided herein, after loading the antibody preparation onto the HA resin but before eluting the intact bispecific antibody from the resin, the HA resin is washed with a wash buffer comprising phosphate ions, which may contain phosphate ions at a concentration of between about 5, 10, 15, 20 mM and 30, 40, or 50 mM.

[0030] In some embodiments, in methods provided herein involving an elution buffer comprising an ion, the ion is phosphate. In some embodiments, the concentration of phosphate ions during elution can be increased from about 30, 40, or 50 mM to about 60, 70, 80, 100, 150, or 200 mM.

[0031] In some embodiments, in the methods provided herein, the pH of at least one of the load buffer, wash buffer, and elution buffer is about pH 7.0 and 8.0, or is between about pH 7.0 and 8.0.

[0032] In some embodiments, in methods provided herein involving an antibody preparation, the antibody preparation comprises proteins previously loaded onto and eluted from at least one of i) a Protein A resin and ii) an ion exchange resin. The antibody preparation may comprise proteins previously loaded onto and eluted from both i) a Protein A resin and ii) an ion exchange resin.

[0033] In some embodiments, antibodies prepared using the methods described herein are isolated and / or purified for use as a medicament or for use in the preparation of a medicament.

[0034] In some embodiments, antibodies prepared using the methods described herein are provided. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows a schematic diagram of a method for preparing a bispecific antibody that can be purified according to the methods provided herein. [Figure 2] Schematic representation of an exemplary i) intact bispecific antibody (left panel) and ii) clipped version of the bispecific antibody (right panel), which is an impurity that can be present in antibody preparations along with the intact bispecific antibody. [Figure 3] 1 shows a chromatogram demonstrating the separation of the anti-BCMA / anti-CD3 bispecific antibody of interest ("POI") from several different impurities via elution from an HA resin. [Figure 4] FIG. 4 shows a graph depicting the relative amounts of different protein species (including the antibody of interest and various impurities) in different fractions eluted from the HA resin according to the HA chromatography run shown in the chromatogram of FIG. 3. [Figure 5] 1 shows a chromatogram demonstrating the separation of the anti-BCMA / anti-CD3 bispecific antibody of interest ("POI") from several different impurities via elution from an HA resin. [Figure 6] 6 shows a graph depicting the relative amounts of different protein species (including the antibody of interest and various impurities) in different fractions eluted from the HA resin according to the HA chromatography run shown in the chromatogram of FIG. 5, where the anti-BCMA / anti-CD3 bispecific antibody of interest is separated from several different impurities upon elution from the HA resin. [Figure 7]1 shows a graph depicting the relative amounts of different protein species (including the antibody of interest and various impurities) in different fractions eluted from an HA resin following an HA chromatography run, where the anti-FLT3 / anti-CD3 bispecific antibody of interest is separated from several different impurities upon elution from the HA resin. DETAILED DESCRIPTION OF THE INVENTION

[0036] Provided herein are methods for purifying an antibody of interest from one or more impurities. The methods provided herein involve the use of a hydroxyapatite resin to separate the antibody of interest from the impurities. In some embodiments, the antibody of interest is a bispecific antibody. In some embodiments, the impurity is an antibody related to the antibody of interest (i.e., having a similar or identical amino acid sequence(s) as the antibody of interest), but is modified in one or more ways compared to the antibody of interest and has a different mass than the antibody of interest. The mass of the antibody impurity species may be very similar to the mass of the antibody of interest. For example, in some embodiments, the mass of the antibody impurity species differs from the mass of the antibody of interest by less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, or 0.01%. The methods provided herein may be used for large-scale purification of an antibody of interest from one or more impurities.

[0037] definition Unless otherwise defined, all terms, notations, and other scientific terms or terminology of the art used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from the definition commonly understood in the art.

[0038] The following terms, unless otherwise indicated, shall be understood to have the following meanings: An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, e.g., a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (ScFv) and domain antibodies (including, e.g., shark and camelid antibodies), diabodies, and fusion proteins containing antibodies, as well as any other modified configuration of an immunoglobulin molecule containing an antigen recognition site. The term "antibody" includes monospecific, bispecific, and multispecific antibodies. Antibodies include antibodies of any class, e.g., IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0039] As used herein, the terms "heavy chain," "light chain," "variable region," "variable domain," "framework region," "constant domain," and the like have their usual meaning in the field of immunology and refer to the corresponding domains in naturally occurring immunoglobulins and recombinant binding proteins (e.g., humanized antibodies, bispecific antibodies, single-chain antibodies, chimeric antibodies, etc.). The basic structural unit of naturally occurring immunoglobulins is a tetramer with two light chains and two heavy chains, typically expressed as a glycoprotein of approximately 150,000 Da. The amino-terminal (N-terminal) portion of each chain contains a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region having CH1, hinge, CH2, and CH3 domains. The variable region of an IgG molecule comprises regions of hypervariability called complementarity-determining regions (CDRs), which contain residues that contact antigen, and non-CDR segments called framework regions (FRs), which largely maintain the structure and determine the positioning of the CDR loops (although certain framework residues may also contact antigen). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following structure: n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-c. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0040] A "bispecific" or "bi-specific" is a hybrid antibody having two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes that may reside on the same or different protein targets.

[0041] An "intact" antibody refers to a recombinant antibody that includes all of the predicted peptide bonds and amino acids of the recombinant antibody (i.e., as predicted based on the nucleic acid sequence(s) encoding the antibody polypeptide(s). In contrast, a "clipped" antibody refers to a version of the corresponding "intact" antibody that lacks at least one peptide bond compared to the corresponding "intact" antibody. References herein to an "antibody of interest" generally refer to the intact antibody of interest, unless the context clearly indicates otherwise.

[0042] Reference herein to "about" a value or parameter includes embodiments that relate to the value or parameter itself, as well as values ​​or parameters that may be 10% below or above the stated numerical value for that parameter. For example, reference to "about 5 mg" includes 5 mg, and also includes any value between 4.5 mg and 5.5 mg.

[0043] method The methods provided herein can be used to purify an antibody of interest from one or more impurities. In the methods provided herein, an antibody preparation containing the antibody of interest and one or more impurity molecules (also referred to herein as a "starting sample") is loaded onto a hydroxyapatite (HA) resin, which binds the antibody of interest and, optionally, one or more impurity molecules. The HA resin is then washed to remove any loosely bound impurities (in some embodiments, all impurity molecules may flow through the HA resin and not bind to it). The antibody of interest is then eluted from the HA resin using a phosphate elution buffer, typically introduced over the resin, through a gradient of increasing phosphate ion concentration. Elution of the antibody of interest from the HA resin produces a purified sample containing the antibody of interest and containing little (or no) impurities that were present with the antibody of interest in the starting sample. During elution of the antibody of interest from the HA resin, any impurity molecules bound to the HA resin may also elute at some point during the gradient of increasing phosphate ion concentration. However, the impurity molecules are eluted from the HA resin under conditions sufficiently different from those for elution of the antibody of interest so that the antibody of interest can be efficiently separated from the impurity molecules during the elution process. Further details about the above method steps and related materials and steps are provided below.

[0044] Hydroxyapatite Resin Various hydroxyapatite resins are commercially available, and any available form of the material can be used in the methods provided herein. Hydroxyapatite can be in crystalline form. Hydroxyapatite can be agglomerated to form particles and sintered at high temperatures into a stable, porous ceramic mass.

[0045] In some embodiments, the HA resins provided herein are ceramic hydroxyapatite (cHA) resins. "Ceramic hydroxyapatite" / "cHA" refers to a hydroxyapatite resin of formula Ca that has been sintered at high temperature into a spherical macroporous ceramic. 10"Ceramic hydroxyapatite" refers to an insoluble hydroxylated calcium phosphate of (PO4)6(OH)2. As used herein, "ceramic hydroxyapatite" / "cHA" includes, but is not limited to, ceramic hydroxyapatite types I and II, and also includes any suitable particle size unless otherwise specified. Typical cHA particle sizes that can be used in the methods provided herein include, for example, particle sizes between 1 and 100 μm or between 1 and 1000 μm in diameter, e.g., 20 μm, 40 μm, or 80 μm. Exemplary cHA resins that can be used in the methods provided herein include CHT™ Type I and Type II resins (Bio-Rad). Any reference herein to "HA resin" or the like includes cHA resin.

[0046] Typically, in the methods provided herein, the HA resin is provided in one or more chromatography columns. Column characteristics, such as the diameter, length, and packing density, can be selected based on various factors, including the requirements of a particular purification scheme (i.e., the amount of protein to be purified), as well as factors related to the HA resin used in the column, such as its pore size, particle size, compressibility, loading capacity, and dynamic binding capacity. Furthermore, the methods provided herein are frequently described in terms of the HA resin in a chromatography column; however, other suitable related configurations for the resin are not excluded. Also, references herein to an "HA column" or the like refer to a chromatography column packed with HA resin.

[0047] Equilibrate the HA column before protein loading In some embodiments, prior to loading a sample containing the antibody of interest onto the HA column, the methods provided herein may include pre-equilibrating the column with one or more equilibration buffer(s). These equilibration buffers may be introduced onto the column, for example, to ensure that the HA resin is clean at the start of the method (i.e., to ensure that the resin does not have impurities already bound to it) and / or to ensure that the solution surrounding the HA resin is compatible with the sample to be loaded onto the resin.

[0048] In some embodiments, the equilibration buffer is a phosphate buffer containing, for example, sodium phosphate, and the phosphate ion concentration in the buffer is about 100-500 mM. Such equilibration buffers may also be referred to herein as "high-phosphate equilibration buffers." For example, in certain embodiments, the high-phosphate equilibration buffer may contain about 250-450 mM phosphate ions; in other embodiments, it may contain about 200, 250, 300, 350, 400, or 450 mM phosphate ions. This equilibration buffer contains a relatively high concentration of phosphate ions in order to elute any contaminants / impurities already present on the HA resin (i.e., those present before a sample containing the protein of interest is loaded onto the resin; such impurities may be present, for example, if the HA resin has previously been used for a purification method and the resin was not completely cleaned after the previous use). The high-phosphate equilibration buffer may have a pH of about 6.0-9.0. For example, in certain embodiments, the high phosphate equilibrated buffer may have a pH of about 7.0-8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the high phosphate equilibrated buffer contains about 400 mM phosphate ions and has a pH of about 7.5. In some embodiments, the high phosphate equilibrated buffer may also be referred to herein as "Equilibration Buffer 1."

[0049] In some embodiments, the equilibration buffer is a phosphate buffer containing, for example, sodium phosphate, and the phosphate ion concentration in the buffer is about 1 to 20 mM. Such equilibration buffers may also be referred to herein as "low-phosphate equilibration buffers." For example, in certain embodiments, the low-phosphate equilibration buffer may contain about 1 to 10 mM phosphate ions; in other embodiments, about 1, 2, 3, 4, 5, or 10 mM phosphate ions. This equilibration buffer contains a relatively low concentration of phosphate ions in the buffer to create conditions around the HA resin that are conducive to binding of the protein of interest to the resin. The low-phosphate equilibration buffer may further contain HEPES at a concentration of about 1 to 50 mM. For example, in certain embodiments, the low-phosphate equilibration buffer may contain about 2 to 30 mM HEPES; in other embodiments, about 5, 10, 15, 20, or 25 mM HEPES. The low-phosphate equilibration buffer may have a pH of about 6.0 to 9.0. For example, in certain embodiments, the low-phosphate equilibration buffer may have a pH of about 7.0-8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the low-phosphate equilibration buffer contains about 2 mM phosphate ions, 20 mM HEPES, and has a pH of about 7.5. In some embodiments, the low-phosphate equilibration buffer may also be referred to herein as "Equilibration Buffer 2." Importantly, however, when using the methods provided herein, the low-phosphate equilibration buffer may be used to pre-equilibrate the resin without the prior use of a high-phosphate equilibration buffer during the method.

[0050] Load the sample containing the antibody of interest onto the HA column Once the HA column is ready for protein loading, a sample containing the antibody of interest and impurities is loaded onto the HA column. The buffer in which the sample is loaded onto the HA column may be referred to herein as a "load buffer." In some embodiments, when a sample containing the antibody of interest is initially obtained for use in the methods provided herein, the sample is already in a load buffer appropriate for loading the sample onto the HA column. However, in other embodiments, the sample may be treated (e.g., diluted, concentrated, or buffer-exchanged) before loading onto the HA column to modify the buffer conditions of the sample so that it is in an appropriate buffer for loading onto the column. For example, when using the methods provided herein, the load buffer cannot have a high concentration of phosphate ions that would interfere with binding of the antibody of interest to the HA resin. Thus, if the initial sample containing the antibody of interest contains a high concentration of phosphate ions, the sample must be diluted or buffer-exchanged, for example, until the concentration of phosphate ions in the sample is reduced to an appropriately low concentration that allows binding of the antibody of interest to the HA resin.

[0051] In some embodiments, the loading buffer contains about 10 mM or less phosphate ions. For example, in some embodiments, the loading buffer contains less than about 10 mM, 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM phosphate ions. In some embodiments, the loading buffer contains 0 mM phosphate ions. The loading buffer may contain various other salts or buffer components (e.g., Tris, glycine). The loading buffer may have a pH of about 6.0 to 9.0. For example, in certain embodiments, the loading buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0.

[0052] In some embodiments, a sample containing an antibody of interest may be loaded onto an HA resin to an on-resin density of at least 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L. In some embodiments, a sample containing an antibody of interest may be loaded onto an HA resin to an on-resin density of at least 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 15 g / L, 20 g / L, or 25 g / L and not more than 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L, where the second value is greater than the first value.

[0053] Washing the HA column After loading the sample containing the antibody of interest and impurities onto the HA column, but before eluting the antibody of interest, the methods provided herein may include the additional step of washing the loaded column with one or more wash buffer(s), e.g., to remove nonspecifically immobilized impurities or to otherwise prepare or equilibrate the column for the elution step. The properties of any wash buffer can be determined by one of skill in the art. In one embodiment, the wash buffer is a phosphate buffer containing, for example, sodium phosphate, and the concentration of phosphate ions in the buffer is about 5-50 mM. For example, in certain embodiments, the wash buffer may contain about 10-40 mM phosphate ions; in other embodiments, it may contain about 10, 20, 30, 40, or 50 mM phosphate ions. The wash buffer may further contain HEPES at a concentration of about 1-50 mM. For example, in certain embodiments, the wash buffer may contain about 2-30 mM HEPES; in other embodiments, it may contain about 5, 10, 15, 20, or 25 mM HEPES. The wash buffer may have a pH of about 6.0 to 9.0. For example, in certain embodiments, the wash buffer may have a pH of about 7.0 to 8.0; in other embodiments, the wash buffer may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the wash buffer contains about 40 mM phosphate ions, 20 mM HEPES, and has a pH of about 7.5.

[0054] Eluting the antibody of interest from the HA column The methods provided herein include eluting the bound antibody of interest from the HA resin. The bound antibody of interest is eluted with one or more elution buffers. Typically, the elution buffer contains one or more salts or ions, and the concentration of the salts or ions increases during elution.

[0055] In some embodiments, the elution buffer provided herein contains phosphate ions. The concentration of phosphate ions in the elution buffer may be increased during elution from an initial concentration of about 20 mM to about 200 mM. For example, in certain embodiments, the concentration of phosphate ions in the elution buffer is increased during elution from an initial concentration of about 40 mM to about 80 mM, or from about 40 mM to about 100 mM. The specific manner and rate of increasing the concentration of phosphate ions in the elution buffer can be determined as appropriate for the antibody of interest, also taking into account the type of impurity molecules bound to the HA resin. For example, the concentration of phosphate ions in the elution buffer can be increased in a gradual / shallow linear gradient. The use of a shallow gradient can enable efficient separation of one or more molecules eluting from the HA resin under similar but different conditions. Alternatively, in some embodiments, the concentration of phosphate ions in the elution buffer can be increased in a steep gradient or in steps. The elution buffer may further contain HEPES at a concentration of about 1-50 mM. For example, in certain embodiments, the elution buffer may contain about 2-30 mM HEPES; in other embodiments, about 5, 10, 15, 20, or 25 mM HEPES. The elution buffer may have a pH of about 6.0-9.0. For example, in certain embodiments, the elution buffer may have a pH of about 7.0-8.0; in other embodiments, about 7.0, 7.5, or 8.0. In one embodiment, the elution buffer contains about 40-80 mM phosphate ions (increasing gradually over the gradient), 20 mM HEPES, and has a pH of about 7.5.

[0056] Elution conditions, including but not limited to, the characteristics of the elution buffer appropriate for use with the HA resin (e.g., buffer composition, pH, concentration, ionic strength, etc.); any necessary steps or gradient changes in the properties of the elution buffer; the number of column volumes of elution buffer used; flow rate, etc., can be determined to optimize the elution of the antibody of interest from the HA column as well as the separation of the antibody of interest from impurity molecules.

[0057] After elution, one or more peak fractions containing the antibody of interest are optionally collected individually or separately, optionally pooled, optionally pH adjusted, optionally filtered, and then optionally stored before further processing as desired. Peak fractions for collection can be identified by any suitable means, such as using ultraviolet light at A280 and beginning collection when the ultraviolet signal rises above a desired amount and / or at a desired point in the elution conditions.

[0058] The material eluted from the HA resin containing the antibody of interest may optionally be referred to herein as a "purified fraction." This purified fraction may contain material from a single fraction eluted from the HA resin, or may be a combination of multiple fractions eluted from the HA resin pooled together. Typically, this purified fraction is prepared to strike a balance between collecting high amounts of the antibody of interest and collecting low amounts of impurity molecules. For example, there may be at least partial overlap between the conditions under which the antibody of interest elutes from the HA resin and the conditions under which impurity molecular species elute from the HA resin, and these competing goals often must be reconciled.

[0059] Any of the buffers (e.g., equilibration buffers, load buffers, wash buffers, or elution buffers) for the methods provided herein may also contain additional or alternative suitable components, such as acetate, succinate, MES, ACES, MOPSO, PIPES, BES, TAPSO, AMPSO, TRICINE, EPPS, bicine, DIPSO, HEPPSO, imidazole, Tris, Bis-tris, TAPS, arginine, glycine, acetonitrile, ethanol, methanol, 1% sodium dodecyl sulfate (SDS) or other detergents.

[0060] In some embodiments, any of the buffers provided herein can have a pH of about 6.0 to 9.0. In other embodiments, any of the buffers provided herein can have a pH of about 5.0 to 9.0, 5.5 to 9.0, 6.5 to 9.0, 7.0 to 9.0, 7.5 to 9.0, 7.0 to 8.0, or 6.5 to 8.5.

[0061] In buffers provided herein that are described as containing "phosphate ions," the phosphate ions can be generated in the buffer from any suitable phosphate salt, e.g., sodium phosphate or potassium phosphate. Additionally, solutions provided herein that are described as being prepared with "sodium phosphate" can be prepared with any suitable sodium phosphate salt (e.g., monobasic or dibasic).

[0062] After the antibody of interest has been eluted from the HA column, the HA column may be purified to remove impurities and other components that may degrade the column resin and prepare it for storage following use. In one embodiment, the column is first regenerated using a buffer, e.g., containing sodium phosphate at a concentration of about 0.4 M and a pH of about 7.5, followed by an optional purification step using a purification solution, e.g., about 1 M NaOH and about 0.5 M potassium phosphate, and then prepared for storage using a storage solution, e.g., about 0.1 M NaOH.

[0063] antibody The methods provided herein can be used to purify an antibody of interest from one or more impurities. For example, the purified antibody can be used as a pharmaceutical or in the preparation of a pharmaceutical.

[0064] In some embodiments, the antibody purified according to the methods provided herein is any type of antibody provided herein.For example, the antibody purified according to the methods provided herein can be a full-length antibody or an antibody fragment (e.g., scFv or Fab), and can be monospecific or bispecific.Typically, the antibody of interest purified according to the methods provided herein is a recombinant antibody.

[0065] IgG antibodies In some embodiments, the antibody that can be purified according to the methods provided herein is an immunoglobulin G (IgG) antibody. As known in the art, IgG antibodies contain two heavy chains and two light chains, roughly shaped like a "Y." In a standard IgG molecule, the two heavy chains have the same amino acid sequence, and the two light chains have the same amino acid sequence. An IgG antibody can be described as having two "arms" (i.e., a "first arm" and a "second arm"), where each arm contains one heavy chain and one light chain linked together by a disulfide bond. In a standard IgG molecule, the first arm of the antibody is identical to the second arm of the antibody (due to each arm containing a heavy chain and a light chain that have the same amino acid sequence as the heavy chain and light chain in the other arm, respectively). The N-terminal region of the heavy chain contains the heavy chain variable region (VH), and the N-terminal region of the light chain contains the light chain variable region (VL). The VH and VL regions contain the portion of the antibody that specifically binds to an antigen. Thus, each arm of an IgG antibody can specifically bind to an antigen. In a standard IgG molecule, both the first and second arms of the IgG antibody bind to the same antigen (due to the fact that both arms contain heavy and light chains with the same respective amino acid sequences). Based on having the same two arms, a standard IgG antibody can be said to be "homodimeric." The IgG antibody purified according to the methods provided herein can be of the subclass IgG1, IgG2, IgG3, or IgG4.

[0066] Bispecific IgG antibody In some embodiments, antibodies that can be purified according to the methods provided herein are bispecific IgG antibodies. In bispecific IgG antibodies, each of the two arms of the antibody specifically binds to a different antigen. Furthermore, the amino acid sequence of the heavy chain in the first arm of a bispecific IgG antibody is different from the amino acid sequence of the heavy chain in the second arm of the same bispecific IgG antibody; similarly, the amino acid sequence of the light chain in the first arm of a bispecific IgG antibody is typically different from the amino acid sequence of the light chain in the second arm of the same bispecific IgG antibody. Therefore, bispecific IgG antibodies can be said to be "heterodimeric" based on having two different arms. The first arm of a bispecific IgG antibody can be described as specific for a "first antigen," and the second arm of a bispecific IgG antibody can be described as specific for a "second antigen." In some embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody comprises an immunologically inert Fc region.

[0067] Bispecific IgG antibodies - how to make them Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al., Methods in Enzymology 121:210, 1986). Traditionally, recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

[0068] More recently, methods have been developed for preparing bispecific heterodimeric antibodies in which the following general steps are taken: 1) A first homodimeric antibody (also referred to herein as the "first parent antibody") and a second homodimeric antibody (also referred to herein as the "second parent antibody") are expressed and purified individually. The first homodimeric antibody is specific for a first target antigen of the bispecific antibody being prepared, and the second homodimeric antibody is specific for a second target antigen of the bispecific antibody being prepared. Thus, for example, if the goal is to prepare a bispecific antibody with specificity for BCMA and CD3, a monoclonal anti-BCMA antibody (the "first parent antibody") and a monoclonal anti-CD3 antibody (the "second parent antibody") are expressed and purified separately.

[0069] 2) The purified first homodimeric / parent antibody and the purified second homodimeric / parent antibody are then mixed and incubated together under conditions that promote antibody arm exchange to form a heterodimeric bispecific antibody comprising a first arm derived from the first parent antibody and a second arm derived from the second parent antibody. These conditions typically involve a series of reducing conditions followed by oxidizing conditions. The reducing conditions promote cleavage of the disulfide bonds holding the two heavy chains of the homodimeric antibody together, thereby allowing antibody arm exchange between the first parent antibody and the second parent antibody. Subsequent oxidizing conditions then form new disulfide bridges that stabilize the newly formed bispecific antibody. This general approach to generating bispecific antibodies is outlined in Figure 1. FIG. 1 shows a first parent antibody ("Parent Antibody A"; white) and a second parent antibody ("Parent Antibody B"; black), each of which is a monospecific homodimer and comprises a first arm and a second arm. Typically, the first parent antibody and the second parent antibody are specific for different antigens. The first parent antibody and the second parent antibody are then mixed together and subjected to reduction and oxidation steps that result in the formation of the desired bispecific antibody comprising the first arm from Parent Antibody A and the second arm from Parent Antibody B, and the respective specificities of both arms.

[0070] The amino acid sequence of an antibody heavy chain may be modified in one or more ways to facilitate the formation of a bispecific antibody. The heavy chain of one arm of a bispecific antibody may contain an amino acid modification in the first hinge region, for example, such that the substituted / replaced amino acid in the first hinge region has the opposite charge to the corresponding amino acid in the hinge region of the other arm of the formed bispecific antibody. This is described, for example, in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545). In another approach, the formation of a desired heteromultimeric or heterodimeric protein (e.g., a bispecific antibody) is enhanced by altering or manipulating the interface between the first immunoglobulin-like Fc region and the second immunoglobulin-like Fc region (e.g., the hinge region and / or CH3 region). In this approach, the bispecific antibody can comprise a CH3 region comprising a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 interface, wherein one or more amino acids within the CH3 interface destabilize or electrostatically disfavor homodimer formation. This approach is also described in International Patent Application No. PCT / US2011 / 036419 (WO2011 / 143545).

[0071] The above and other methods for preparing bispecific antibodies are further described, for example, in International Patent Application Nos. PCT / IB2011 / 054899 (WO2012 / 059882), PCT / US2011 / 036419 (WO2011 / 143545), and Giese et al., Biotechnology Progress, "Bispecific Antibody Process Development: Assembly and Purification of Knob and Hole Bispecific Antibodies," January 17, 2018, and the references cited therein, each of which is incorporated by reference herein for all purposes. The methods provided herein for purifying antibodies can be used to purify bispecific antibodies prepared by any suitable method.

[0072] Bispecific IgG antibodies - specificity In some embodiments, antibodies that can be purified according to the methods provided herein are full-length human bispecific IgG antibodies, wherein a first antibody variable domain of a first arm of the bispecific antibody is capable of binding to a first antigen, and a second antibody variable domain of a second arm of the bispecific antibody is capable of binding to a second antigen. The first and second antigens can have any of the characteristics of the antigens described herein. In some embodiments, the first antigen is present on a first cell type, and the second antigen is present on a second cell type.

[0073] In some embodiments, antibodies that can be purified according to the methods provided herein are full-length human bispecific IgG antibodies, wherein a first antibody variable domain of the antibody is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells, and a second antibody variable domain of the antibody is capable of specifically binding to a target antigen.

[0074] The human immune effector cells to which the antibodies provided herein can bind can be any of a variety of immune effector cells known in the art. For example, the immune effector cells can be members of the human lymphoid lineage, including, but not limited to, T cells (e.g., cytotoxic T cells), B cells, and natural killer (NK) cells. The immune effector cells can also be members of the human myeloid lineage, including, but not limited to, monocytes, neutrophilic granulocytes, and dendritic cells. Such immune effector cells can have either a cytotoxic or apoptotic effect on target cells or other desired effects upon activation by binding an effector antigen. The effector antigen is an antigen (e.g., a protein or polypeptide) expressed on human immune effector cells. Examples of effector antigens to which the antibodies provided herein can bind include, but are not limited to, human CD3 (or the CD3 (cluster of differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

[0075] The target antigen is expressed on target cells in a disease state (e.g., an inflammatory disease, a proliferative disease (e.g., cancer), an immunological disorder, a neurological disease, a neurodegenerative disease, an autoimmune disease, an infectious disease (e.g., a viral or parasitic infection), an allergic reaction, a graft-versus-host disease, or a host-versus-graft disease). The target antigen is not an effector antigen. Examples of target antigens include BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor)-2 / neu, HER-3, HER-4, EGFR (epidermal growth factor receptor), FLT3 (Fms-like tyrosine kinase 3), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD3, GM2, globo H, fucosyl GM1, poly SA, GD2, carbonic anhydrase IX (MN / CA) IX), CD44v6, Shh (sonic hedgehog), Wue-1, plasma cell antigen, (membrane-bound) IgE, MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, and MIS (Mullerian inhibitory substance) receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, and CD63.

[0076] In some embodiments, the antibodies purified according to the methods provided herein can be any antibody described in U.S. Patent Application No. 15 / 085,644, filed March 30, 2016 (U.S. Publication No. 20160297885), or U.S. Patent Application No. 15 / 993,874, filed May 31, 2018 (U.S. Publication No. 20180346601), which are incorporated by reference in their entirety for all purposes.

[0077] In some embodiments, the antibodies purified according to the methods provided herein can be bispecific IgG antibodies in which one arm of the antibody specifically binds to cluster of differentiation 3 (CD3). Information about CD3 is provided, for example, via UniProtKB ID number P07766.

[0078] In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VH region of the heavy chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS (SEQ ID NO: 1). In some embodiments, in a bispecific IgG antibody where one arm of the antibody specifically binds to CD3, the heavy chain of the CD3 binding arm has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VH region of the heavy chain of the CD3-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 1.

[0079] In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIK (SEQ ID NO: 3). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the light chain of the CD3-binding arm has an amino acid sequence comprising the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VL region of the light chain of the CD3-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 3.

[0080] In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to CD3, the VH region of the heavy chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 1, and the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to CD3, the heavy chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, and the light chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to CD3, the VH region of the heavy chain of the CD3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 1, and the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 3.

[0081] In some embodiments, the antibodies purified according to the methods provided herein can be bispecific IgG antibodies in which one arm of the antibody specifically binds to B-cell maturation antigen (BCMA). Information about BCMA is provided, for example, via UniProtKB ID number Q02223.

[0082] In some embodiments, in a bispecific IgG antibody where one arm of the antibody specifically binds BCMA, the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS (SEQ ID NO: 5). In some embodiments, in a bispecific IgG antibody where one arm of the antibody specifically binds to BCMA, the heavy chain of the BCMA binding arm has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT CNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6). In some embodiments, in a bispecific IgG antibody where one arm of the antibody specifically binds BCMA, the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising CDR1, CDR2 and CDR3 of the VH sequence set forth in SEQ ID NO: 5.

[0083] In some embodiments, in a bispecific IgG antibody where one arm of the antibody specifically binds BCMA, the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK (SEQ ID NO: 7). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the light chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence:EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds BCMA, the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising CDR1, CDR2 and CDR3 of the VL sequence set forth in SEQ ID NO:7.

[0084] In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to BCMA, the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 5, and the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to BCMA, the heavy chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 6, and the light chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to BCMA, the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising CDR1, CDR2 and CDR3 of the VH sequence set forth in SEQ ID NO: 5, and the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising CDR1, CDR2 and CDR3 of the VL sequence set forth in SEQ ID NO: 7.

[0085] In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS (SEQ ID NO: 13). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQEWPLTFGQGTKVEIK (SEQ ID NO: 14). In some embodiments, for any reference herein to an antibody comprising a VH region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 5, the antibody may alternatively comprise a VH region comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, for any reference herein to an antibody comprising a VL region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 7, the antibody may alternatively comprise a VL region comprising the amino acid sequence set forth in SEQ ID NO: 14. Similarly, anti-BCMA heavy and light chains comprising the VH and VL sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively, are also included herein.

[0086] In some embodiments, the antibodies purified according to the methods provided herein can be bispecific IgG antibodies in which one arm of the antibody specifically binds to fms-like tyrosine kinase 3 (FLT3). Information about FLT3 is provided, for example, via UniProtKB ID number P36888.

[0087] In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VH region of the heavy chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSS (SEQ ID NO: 9). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the heavy chain of the FLT3 binding arm has the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VH region of the heavy chain of the FLT3-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 9.

[0088] In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VL region of the light chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIK (SEQ ID NO: 11). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the light chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence:EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VL region of the light chain of the FLT3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 11.

[0089] In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to FLT3, the VH region of the heavy chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 9, and the VL region of the light chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to FLT3, the heavy chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 10, and the light chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, in a bispecific IgG antibody, one arm of the antibody specifically binds to FLT3, the VH region of the heavy chain of the FLT3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 9, and the VL region of the light chain of the FLT3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 11.

[0090] In some embodiments, provided herein are bispecific anti-BCMA / anti-CD3 antibodies, wherein the anti-BCMA arm of the antibody has any of the characteristics described above for the anti-BCMA arm, and the anti-CD3 arm of the antibody has any of the characteristics described above for the anti-CD3 arm. In some embodiments, provided herein are bispecific anti-FLT3 / anti-CD3 antibodies, wherein the anti-FLT3 arm of the antibody has any of the characteristics described above for the anti-FLT3 arm, and the anti-CD3 arm of the antibody has any of the characteristics described above for the anti-CD3 arm.

[0091] Also provided herein are methods for purifying monospecific antibodies having affinity for any of the above antigens and / or comprising any of the above amino acid sequences, e.g., the purification of a monospecific homodimeric anti-CD3 antibody comprising the VH amino acid sequence set forth in SEQ ID NO: 1.

[0092] impurities The methods provided herein can be used to purify an antibody of interest from one or more impurities.

[0093] Impurities include, for example, clipped versions of the antibody of interest, protein aggregates, and, in the case of a bispecific antibody of interest, the parent monospecific antibody that is involved in the formation of the bispecific antibody of interest. These different impurities may also be referred to herein as different "species of impurity," "impurity molecules," etc.

[0094] Clip-on version of the antibody of interest A "clipped version of the antibody of interest," "clipped antibody," and the like refer to a recombinant antibody in which one or more polypeptide bonds in the antibody have been truncated compared to the corresponding intact antibody of interest. In contrast, an "intact" antibody refers to a recombinant antibody that includes all of the predicted peptide bonds and amino acids of the recombinant antibody (i.e., as predicted based on the nucleic acid sequence(s) encoding the antibody polypeptide(s).

[0095] As such, clipped antibodies can be considered degradation products associated with the antibody of interest. Cleavage of peptide bonds in antibodies can occur, for example, through enzymatic (e.g., protease-mediated) or non-enzymatic activity.

[0096] In some embodiments, when a peptide bond in an antibody polypeptide is cleaved, the cleaved portion of the polypeptide chain may no longer be covalently linked to the remainder of the antibody after cleavage; in this case, the cleaved portion of the polypeptide chain may dissociate from the remainder of the antibody. This most commonly occurs when the cleavage occurs at a peptide bond near the N- or C-terminus of the polypeptide chain, resulting in a clipped antibody that has lost one or more amino acids compared to the corresponding intact antibody. These clipped antibodies have a smaller mass than the corresponding intact antibody due to the loss of one or more amino acids from the antibody.

[0097] Alternatively, in some other embodiments, when a peptide bond in an antibody polypeptide is cleaved, after cleavage, the cleaved portion of the polypeptide chain may still remain covalently linked to the remainder of the antibody (e.g., via an intra- or inter-chain disulfide bond). In this case, even in the presence of cleavage of an antibody peptide bond, the cleaved portion of the polypeptide chain remains tethered to the remainder of the antibody via the remaining intact covalent bond(s) linking the cleaved portion of the polypeptide chain to the remainder of the antibody. In this situation, the clipped antibody still has the same number of amino acids and amino acid sequence as the intact antibody. Furthermore, in at least some embodiments, this type of clipped antibody may have a slightly larger mass than the corresponding intact antibody. This gain in mass may be the result of, for example, one or more chemical reactions that occur upon cleavage of the peptide bond. During such a reaction, one or more atoms (e.g., H, O) may react with atoms of the antibody polypeptide chain and become covalently linked to the antibody chain, resulting in a gain in mass by the clipped antibody compared to the corresponding intact antibody.

[0098] Because a "clipped" antibody is generated from a corresponding "intact" antibody, the "clipped" version of the antibody has the same amino acid sequence (if there are no amino acids lost from the antibody as a result of peptide bond cleavage) or nearly the same amino acid sequence (if there are one or more amino acid sequences lost from the antibody as a result of peptide bond cleavage) as the corresponding "intact" version of the antibody.

[0099] Typically, a clipped version of an antibody has a mass similar to that of the corresponding intact antibody. As noted above, in some embodiments, a clipped antibody may have a smaller mass than the corresponding intact antibody (e.g., if the clipping results in the loss of one or more amino acids from the antibody). Alternatively, in some embodiments, a clipped antibody may have a larger mass than the corresponding intact antibody (if the clipping does not result in the loss of any amino acids from the antibody, but instead results in the antibody gaining at least one atom via one or more reactions resulting from the cleavage of a peptide bond).

[0100] In some embodiments, the clipped version of an antibody has a mass that differs by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or less from the mass of the corresponding intact antibody of interest. In other words, in some embodiments, the clipped version of an antibody of interest has a mass that differs by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% from the mass of the corresponding intact antibody of interest.

[0101] As noted above, in some embodiments, the clipped version of an antibody of interest has a mass that is less than the mass of the corresponding intact antibody of interest. For example, in some embodiments, the clipped version of an antibody has a mass that is 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or less than the mass of the corresponding intact antibody of interest. In other words, the clipped version of the antibody has a mass that is no more than 10% less than the mass of the corresponding intact antibody of interest; for example, if the intact antibody of interest has a mass of 100,000 Da, the clipped version of the antibody has a mass that is no more than 10,000 Da less (10% of 100,000 is 10,000) - i.e., has a mass between 90,000 Da and 100,000 Da.

[0102] As also noted above, in some embodiments, the clipped version of an antibody of interest has a mass greater than the corresponding intact antibody of interest, e.g., in some embodiments, the clipped version of an antibody has a mass that is no more than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% greater than the mass of the corresponding intact antibody of interest. In other words, the clipped version of the antibody has a mass that is no more than 1% greater than the mass of the corresponding intact antibody of interest; for example, if the intact antibody of interest has a mass of 100,000 Da, the clipped version of the antibody has a mass that is no more than 1,000 Da greater (1% of 100,000 is 1,000) - i.e., has a mass between 100,000 Da and 101,000 Da.

[0103] High molecular mass species (HMMS) / protein aggregates In some embodiments, impurities suitable for the methods provided herein are referred to as "high molecular mass species" (HMMS). HMMS refers to any high molecular mass contaminant or impurity, but is typically an association of at least two proteins that form aggregates. By way of example, HMMS can include multiple molecules of an antibody of interest aggregated together and / or aggregates of proteins from host cells used to produce the antibody of interest. Aggregates can arise, for example, by any process involving covalent or non-covalent binding of molecules.

[0104] Parent antibody In some embodiments, impurities suitable for the methods provided herein include "parent antibodies" or "parental antibodies." This type of impurity molecule is suitable for the methods provided herein, where the antibody of interest is a bispecific antibody generated from two different parent antibodies, e.g., as outlined in FIG. 1 . These parent antibodies are monospecific homodimers. Parent antibodies may be present in the antibody preparation along with the bispecific antibody of interest due to several possible mechanisms, such as: i) in some circumstances, some parent antibody molecules do not separate into a first arm and a second arm during the reduction step to separate the parent antibody into separate first and second arms (thus, the antibody remains a monospecific homodimer); or ii) in some circumstances, separated first and second arms from parent antibodies of the same type link together, thus forming a monospecific homodimer (rather than participating in the formation of a heterodimeric bispecific antibody). As used herein, "parent antibody" refers to a homodimeric molecule generated by any of the above mechanisms. Additionally, the antibody preparations provided herein may contain, as impurities, parent antibodies from one or both parent species.

[0105] Purification of the desired antibody from impurities Provided herein are methods for purifying an antibody of interest from one or more impurities.

[0106] In some embodiments, in the methods provided herein, the antibody of interest is present in an antibody preparation (also referred to herein as a "starting sample") that contains the antibody of interest as well as one or more species of impurity molecules. In this starting material for the methods provided herein, the antibody of interest may, for example, constitute at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% by weight of the protein in the antibody preparation. Then, in some embodiments of the methods provided herein, a purified fraction (also referred to herein as a "purified sample") is collected as an eluate from the HA resin. This purified fraction contains the antibody of interest and, in some embodiments, still contains one or more impurity molecules. In some embodiments, in the purified fractions provided herein, the antibody of interest may constitute, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the protein in the purified fraction.

[0107] In some embodiments, in the methods provided herein, a starting sample is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% by weight of the antibody of interest, and a subsequent purified sample in the same method is at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the antibody of interest, where the second value is greater than the first value.

[0108] In some embodiments, in the methods provided herein, the starting sample comprises at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% by weight of the clipped version of the antibody of interest. In some embodiments, in the methods provided herein, the purified sample comprises no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by weight of the clipped version of the antibody of interest. In some embodiments, in the methods provided herein, a starting sample comprises at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% by weight of the clipped version of the antibody of interest, and a subsequent purified sample in the same method comprises no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by weight of the clipped version of the antibody of interest, wherein the second value is less than the first value.

[0109] In some embodiments, in the methods provided herein, the starting sample comprises an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody is at least about 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1:3. In some embodiments, in the methods provided herein, the purified sample comprises an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody is no more than about 1:100, 1:50, 1:25, 1:20, or 1:10. In some embodiments, in the methods provided herein, a starting sample comprises an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody in the starting sample is at least about 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1:3, and the ratio of clipped antibody to intact antibody in a subsequent purified sample in the same method is no more than about 1:200, 1:100, 1:50, 1:25, 1:20, or 1:10, wherein the second ratio is less than the first ratio. In some embodiments, in the methods provided herein, a starting sample comprises an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody in the starting sample is between about one of the ratios in Group A (Group A ratio: 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, or 1:4) and one of the ratios in Group B (Group B ratio: 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or 1:3), and the ratio of clipped antibody to intact antibody in a subsequently purified sample in the same method is about 1:200, 1:100, 1:50, 1:25, 1:20, or 1:10 or less, wherein the ratio in the purified sample is less than the ratio in the starting sample.

[0110] In some embodiments, the starting sample provided according to the methods provided herein contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest. In some embodiments, the purified sample provided according to the methods provided herein contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest. In some embodiments, a starting sample provided according to the methods provided herein contains at least 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest, and a subsequent purified sample in the same method contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, or 5000 grams of intact antibody of interest, where the first value is greater than the second value.

[0111] Furthermore, any of the above statements regarding a) the amount or b) the purity of the antibody of interest in the starting sample or purified sample can be combined with respect to the same sample. For example, as described above, the starting sample can contain at least about 80% by mass of the antibody of interest; further, as also described above, the starting sample can contain at least about 10 grams of the antibody of interest. Thus, starting samples containing at least about 80% by mass of the antibody of interest and at least 10 grams of the antibody of interest, etc., are also provided herein.

[0112] General technology The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (eds. JM Miller and MPCalos, 1987); Current Protocols in Molecular Biology (eds. FMA Musubel et al., 1987); PCR: The Polymerase Chain Reaction, (eds. Mullis et al., 1994); Current Protocols in Immunology (eds. JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P.Antibodies are fully described in literature such as *Travers*, 1997; *Antibodies* (P. Finch, 1997); *Antibodies: a practical approach* (D. Catty, ed., IRL Press, 1988-1989); *Monoclonal antibodies: a practical approach* (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); *Using antibodies: a laboratory manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and, where applicable, in subsequent editions of the above references and their corresponding websites.

[0113] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [Example]

[0114] Example 1 Purification of anti-BCMA / anti-CD3 bispecific antibodies by cHA resin chromatography the purpose: In this example, a method for separating a full-length bispecific human IgG of interest from various impurities was tested. The antibody of interest was a heterodimeric bispecific anti-BCMA / anti-CD3 antibody (i.e., one arm of the bispecific antibody was specific for BCMA and the other arm was specific for CD3). Impurities present with the bispecific antibody of interest at the start of purification included: i) a clipped version of the intact anti-BCMA / anti-CD3 bispecific antibody of interest; ii) a homodimeric monospecific anti-BCMA parent antibody; iii) a homodimeric monospecific anti-CD3 antibody; and iv) protein aggregates / high molecular mass species (HMMS).

[0115] Purification of the intact bispecific antibody of interest from the clipped version of the bispecific antibody presented particular challenges because the clipped version of the bispecific antibody contains the same number of amino acids and the same amino acid sequence as the intact bispecific antibody of interest, but differs in mass by 18 daltons (Da). Specifically, the mass of the intact anti-BCMA / CD3 bispecific antibody is 148095.5 Da, while the mass of the corresponding clipped version of the bispecific antibody is 148113.5 Da (as determined by mass spectrometry). Thus, the clipped bispecific antibody has a difference in mass of less than 0.1% (or even less than 0.02%) compared to the mass of the intact bispecific antibody (i.e., 0.1% of 148095 Da is 148 Da; 0.02% of 148095 Da is 29.6 Da). In other words, the mass of the clipped bispecific antibody is approximately 100.01% of the mass of the intact bispecific antibody of interest. Thus, the mass of the clipped bispecific antibody is very similar to the mass of the intact bispecific antibody of interest.

[0116] The clipped version of the bispecific antibody contains a cleavage in the peptide bond of the anti-CD3 heavy chain between amino acids 56 and 57 in the heavy chain. The anti-CD3 heavy chain has the amino acid sequence set forth in SEQ ID NO:2; therefore, the cleavage occurs between amino acids R and G in the "RG" sequence of SEQ ID NO:2 ("RG" appears only once in SEQ ID NO:2). This cleavage is believed to result in the gain of one oxygen atom and two hydrogen atoms in the clipped bispecific antibody (corresponding to a gain of 18 Da in mass) compared to the intact bispecific antibody. Additionally, although there is a cleaved peptide bond in the anti-CD3 heavy chain, the cleaved 56-amino acid portion of the heavy chain (i.e., the first 56 amino acids of the chain) remains connected to the rest of the antibody via an intact intrachain disulfide bond. The intrachain disulfide bond is between C22 and C98 of the anti-CD3 heavy chain (i.e., between C22 and C98 in the sequence set forth in SEQ ID NO:2). The surviving tethering of the cleaved 56 amino acid portion to the remainder of the antibody by an intrachain peptide bond is shown schematically in FIG.

[0117] In Figure 2, the schematic on the left shows an intact bispecific antibody of interest, comprising intact heavy and light chains for both the anti-CD3 and anti-BCMA arms of the bispecific antibody (in the figure, the longer chains represent the heavy chains of the antibody, and the shorter chains represent the light chains). In addition, Figure 2 also shows various intra-antibody disulfide bonds, including intra-chain disulfide bonds (e.g., linking different amino acids in the same light or heavy chain), and also inter-chain disulfide bonds (e.g., linking the heavy chain of an anti-BCMA arm to the heavy chain of an anti-CD3 arm, or linking the light chain of an anti-CD3 arm to the heavy chain of an anti-CD3 arm).

[0118] Another challenge presented in developing a method for purifying an intact bispecific antibody of interest from various impurities was the objective of developing a method for efficiently separating the bispecific antibody of interest from impurities when relatively large quantities of the bispecific antibody of interest are being purified. For example, one objective of the work related to this Example was to develop a method for purifying a bispecific antibody that is effective for processes in which at least 1 gram of bispecific antibody is purified. As is known in the art, purifying a protein on a large scale often presents numerous difficulties that are not present (or are not significantly problematic) during the purification of the same protein on a small scale, due, for example, to the difficulty in obtaining sharp chromatographic resolution between different proteins when chromatography is performed on a large scale.

[0119] material and method: The starting material for this work was an antibody preparation containing the desired bispecific IgG anti-BCMA / CD3 antibody as well as various impurities, such as those listed above. The amino acid sequences of the polypeptides of this bispecific antibody are set forth in the following SEQ ID NOs: BCMA heavy chain: SEQ ID NO:6; BCMA light chain: SEQ ID NO:8; CD3 heavy chain: SEQ ID NO:2; CD3 light chain: SEQ ID NO:4. The desired bispecific antibody was prepared from two separate parent antibodies, as previously described herein. More specifically, the parent monospecific anti-CD3 and anti-BCMA antibodies were purified via Protein A chromatography, and these purified parent monospecific anti-CD3 and anti-BCMA antibodies were used to generate the desired bispecific anti-BCMA / CD3 antibody. The generated bispecific anti-BCMA / CD3 antibody was then purified via ion exchange chromatography. The antibody preparation used as the starting material for the purification work in this example was the eluate from an ion exchange column containing the bispecific antibody of interest and various remaining impurities, with buffer / salt at an approximate concentration of 50 mM Tris and 60 mM glycine, pH 7.5. This antibody preparation contained greater than 85% by mass of the intact bispecific antibody of interest; it also contained approximately 8% clipped bispecific antibody, approximately 1% monospecific anti-BCMA parent antibody, approximately 1% monospecific anti-CD3 parent antibody, and approximately 2% protein aggregates / high molecular mass species (HMMS). Thus, while the antibody preparation used as the starting material in this method already contained a relatively pure intact bispecific antibody of interest, the goal of this method was to develop a method for increasing the purity of this intact bispecific antibody.

[0120] result: Several different chromatographic resins and conditions were tested to attempt to identify suitable resins and buffer conditions that would allow for efficient purification of the intact anti-BCMA / CD3 bispecific antibody of interest from the clipped bispecific antibody, the monospecific parent antibody, and protein aggregates. During this process, for example, several different ion exchange and hydrophobic interaction resins, as well as various buffers and pH conditions, were tested. After extensive testing, hydroxyapatite resin was the only resin identified that was able to allow for efficient purification of the intact anti-BCMA / CD3 bispecific antibody from various impurities, including the clipped bispecific antibody.

[0121] Figure 3 shows the chromatographic profile of the elution of a bispecific antibody of interest from a ceramic hydroxyapatite ("cHA") resin column, as well as the concurrent separation of the anti-BCMA / CD3 bispecific antibody of interest from multiple different impurities, including clipped versions of the bispecific antibody, both parent antibody species, and high-molecular-mass protein species. In the chromatographic run shown in Figure 3, the antibody preparation loaded onto the cHA resin was spiked with extra parent anti-BCMA and parent anti-CD3 antibodies (but no extra intact or clipped bispecific antibody) to more clearly identify the elution position of these molecules from the cHA column. In Figure 3, the X-axis, from left to right, indicates the order of elution from the cHA column (i.e., material on the left elutes earlier / at lower salt than material on the right). Typically, the eluate is collected in sequential fractions from the column; therefore, the X-axis can also be considered to indicate the order of eluate fractions from the cHA resin column. The Y-axis shows both UV absorbance (at 280 nM) and conductivity, which are shown separately in the graph. UV absorbance corresponds to the presence of eluted protein, and conductivity corresponds to the salt concentration in the eluted material. Thus, Figure 3 shows the elution profile of different proteins from a cHA resin column as the salt concentration in the elution buffer flowing through the cHA resin increases. Traversing the UV graph in Figure 3 from left to right, the graph shows various peaks and shoulders corresponding to either the intact bispecific anti-BCMA / CD3 antibody of interest or various impurities. Specifically, from left to right, the first UV peak corresponds to the elution of the first parent antibody ("Parent 1"; monospecific anti-BCMA homodimer). The early portion of the next major peak (the largest peak in the graph) corresponds to the intact bispecific antibody protein of interest ("POI"). The later portion / tail end of that same major peak corresponds to the clipped version of the bispecific antibody ("Clip").While there is some overlap in the elution profiles between the intact and clipped bispecific antibodies, these two antibody types elute from the cHA column under sufficiently different salt conditions and fractions to significantly separate the intact bispecific antibody from the clipped version of the bispecific antibody. Finally, after the clipped version of the bispecific antibody elutes, the last major peak / shoulder from the cHA column corresponds to the elution of the second parent antibody (monospecific anti-CD3 homodimer) as well as high molecular mass species ("HMMS") (also known as protein aggregates) from the cHA column. Thus, the graph in Figure 3 demonstrates that intact anti-BCMA / CD3 antibodies can be efficiently purified from multiple impurities, including related clipped and parent antibody species, by cHA resin chromatography.

[0122] Figure 4 provides a graph showing more detailed information about the different molecular species present in fractions sequentially eluted from the cHA resin column according to the cHA elution profile shown in Figure 3. Specifically, Figure 4 provides detailed information about the relative amounts of: i) the intact anti-BCMA / CD3 bispecific antibody protein of interest ("POI"); ii) the clipped version of the bispecific antibody; iii) the first parent antibody / monospecific anti-BCMA antibody; iv) the second parent antibody / monospecific anti-CD3 antibody; and v) high molecular mass species ("HMMS") in the different fractions eluted from the cHA column. The x-axis of Figure 4, from left to right, indicates the order of elution from the cHA column (i.e., from low salt to high salt; each data point represents a fraction eluted from the column). The Y-axis in Figure 4 shows, on the left, the percentage of each of: i) intact anti-BCMA / CD3 bispecific antibody protein of interest ("POI"); ii) clipped version of the bispecific antibody; iii) first parent antibody / monospecific anti-BCMA antibody; and iv) second parent antibody / monospecific anti-CD3 antibody in each fraction. The Y-axis in Figure 4 shows, on the right, the % HMMS in each fraction.

[0123] The method used to generate the data in Figure 3 was also used with an antibody preparation that was not spiked with any additional antibodies; data from this chromatography run is shown in Figure 5. Thus, the data in Figure 5 reflects the purification via cHA resin of a typical antibody preparation eluted from an ion exchange column during the preparation of an anti-BCMA / CD3 bispecific antibody, which primarily contains the intact anti-BCMA / CD3 bispecific antibody of interest as well as various impurities, including clipped versions of the bispecific antibody. In Figure 5, the X-axis, from left to right, indicates the order of material eluted from the cHA column. The Y-axis indicates both UV absorbance (at 280 nM) and conductivity, which are shown separately in the graph. The Parent 1 and Parent 2 peaks are smaller in Figure 5 than in Figure 3 because the sample loaded onto the cHA column in Figure 5 was not spiked with additional Parent 1 and Parent 2 antibodies (whereas for Figure 3, the sample was spiked with additional Parent 1 and Parent 2 antibodies).

[0124] Figure 6 provides a graph showing the recovery of the intact anti-BCMA / CD3 bispecific antibody of interest from the cHA resin, as well as further information about the relative amounts of clipped bispecific antibody in various eluted fractions from the cHA resin. The X-axis, from left to right, shows the order of elution from the cHA column (i.e., low salt to high salt; each data point represents a fraction eluted from the column). Along the vertical / Y-axis, three different variables are plotted for each fraction: Variable 1) (diamonds): Cumulative intact bispecific antibody recovery ("% POI"), which is the total amount of intact anti-BCMA / CD3 bispecific antibody (i.e., protein of interest) recovered from the cHA column up to that fraction in the chromatography run (in other words, as if all material eluted from the cHA column during the run up to and including that fraction were collected and pooled, the total amount of intact anti-BCMA / CD3 bispecific antibody in that pooled material would be measured); and the "% POI" value is shown as a percentage of the total amount of intact anti-BCMA / CD3 bispecific antibody / protein of interest loaded onto the cHA column that was recovered (i.e., rather than shown as a gram value). Variable 2) (squares): The % of protein in each respective fraction that is clipped bispecific antibody ("% Clipped"). Variable 3) (triangle): Cumulative Clipped Bispecific Antibody Recovery ("Cumulative Clip"), which is the total amount of clipped bispecific antibody recovered from the cHA column up to that fraction in the chromatography run. Additionally, in the graph of Figure 6, the left Y-axis lists values ​​for % POI, and the right Y-axis lists values ​​for % Clip.

[0125] FIG. 6 also contains information showing how certain different fractions in FIG. 6 correspond to different points in UV absorbance in the chromatographic profile shown in FIG. 5. Thus, for example, FIG. 5 shows that the largest peak of UV absorbance / protein elution from the cHA resin corresponds to the protein of interest / intact anti-BCMA / CD3 bispecific antibody. The apex of this large peak of UV absorbance / protein elution from the cHA column is also referred to as the "Apex" of the run; the chromatographic fraction peak corresponding to this Apex point is then shown on FIG. 6. Various points after the Apex peak are also shown in FIG. 6. These points are "90%, " "80%, " "70%, " "60%, and "50%" and are calculated as follows: The UV absorbance value at Apex is set as the starting point for further calculations. The UV value that is 90% of the Apex UV value is then determined. The 90% Apex UV value, occurring after the Apex UV value is reached during the chromatographic run (i.e., toward the end of the tail of the peak), is designated the "90%" fraction; this may also be referred to herein as the "90% post-peak Apex" fraction, etc. This process is repeated for 80%, 70%, 60%, and 50% values ​​(i.e., each of these values ​​is further down the tail from the peak and thus corresponds to increasingly larger amounts of collected material). As Figure 6 shows, as the % Apex value decreases, the % clipped in the fraction increases. This is consistent with the process by which the clipped bispecific antibody elutes from the cHA column at later time points / higher salt conditions than the intact anti-BCMA / CD3 bispecific antibody. Thus, when larger fractions of the Apex peak are collected (i.e., to a lower % post-peak Apex fraction), more clipped bispecific antibody is also collected due to the overlap between the elution profiles of the intact and clipped bispecific antibodies.

[0126] Various values ​​from Figure 6 are also provided in Table 1 below. As shown in Table 1, according to the cHA purification method provided herein, for example, when a 90% post-peak Apex eluate is collected from the cHA column (i.e., POI % recovery up to the "90% post-peak Apex" fraction), 54% of the intact bispecific antibody / protein of interest loaded onto the cHA column is recovered, and this recovered protein pool contains 0% clipped bispecific antibody. In another example, when a 50% post-peak Apex eluate is collected from the cHA column (i.e., POI % recovery up to the "50% post-peak Apex" fraction), 74% of the intact bispecific antibody / protein of interest loaded onto the cHA column is recovered, and this recovered protein pool contains 0.2% clipped bispecific antibody. Thus, as shown in Figure 6 and Table 1, robust purification of the intact bispecific antibody of interest from the clipped bispecific antibody can be efficiently achieved via the cHA resin.

[0127] [Table 1]

[0128] For the cHA chromatography results shown in Figures 3-6, cHA chromatography was performed as follows. The bispecific anti-BCMA / CD3 antibody of interest and an antibody preparation containing various impurities were loaded onto a chromatography column containing cHA resin. The cHA resin was cHA type 1, 40 μM bead size (Bio-Rad). For the chromatography runs shown in Figures 3 and 4, the sample was loaded onto the cHA resin to a protein density of 30 g / L. For the chromatography runs shown in Figures 5 and 6, the sample was loaded onto the cHA resin to a protein density of 10 g / L. Prior to loading the sample onto the cHA resin, the resin was pre-equilibrated with 5 column volumes of equilibration buffer 1, followed by 5 column volumes of equilibration buffer 2. The compositions of the various buffers described in this method are listed in Table 2 below. An antibody preparation containing a partially purified bispecific anti-BCMA / CD3 antibody of interest was loaded onto a cHA resin column in a load buffer containing approximately 50 mM Tris and 60 mM glycine, pH 7.5. After loading the antibody preparation onto the cHA resin column, the column was washed with three column volumes of wash buffer. The bispecific antibody of interest (i.e., the intact bispecific antibody) was then eluted from the cHA resin using 20 column volumes of elution buffer, in which the sodium phosphate concentration in the elution buffer was increased from 40 mM to 80 mM over the course of the elution. As shown in Figures 3-6, various impurities present in the antibody preparation along with the bispecific antibody of interest also eluted from the cHA column depending on the elution buffer, but these eluted at sufficiently different salt concentrations than the intact anti-BCMA / CD3 bispecific antibody of interest so that the intact bispecific antibody could be efficiently separated from various impurities, including clipped versions of the intact bispecific antibody.

[0129] After the protein of interest is eluted from the cHA resin column with elution buffer according to the above protocol, the cHA resin can then be stripped with 5 column volumes of strip buffer, followed by 5 column volumes of cleaning buffer, followed by 5 column volumes of storage buffer.

[0130] For each of the above analyses of different materials eluted from the cHA column, the types and amounts of different molecular species in the various fractions / pools were determined by analytical cation exchange (CEX) analysis.

[0131] [Table 2]

[0132] Example 2 Purification of anti-BCMA / anti-CD3 bispecific antibodies by cHA resin chromatography - Mass loading challenge the purpose: The objective in this example was to determine whether the bispecific antibody purification method described in Example 1 could be used efficiently with various cHA resin column mass loading challenges. For example, a specific objective was to determine whether it was possible to consistently recover greater than 50% of the input bispecific antibody for various mass loading challenges on a cHA resin column, while simultaneously having 1% or less of clipped bispecific antibody as an impurity in the recovered purified bispecific antibody product.

[0133] material and method: The materials and methods for this example were the same as in Example 1, except that the cHA resin was loaded with antibody preparation samples to a protein density on the cHA resin of 8 g / L, 10 g / L, or 12 g / L (in different chromatography runs).

[0134] result: The results from these chromatography runs are summarized below in Table 3. For all three chromatography runs, 90% post-peak Apex material (as described in Example 1) was collected and analyzed. As shown in Table 3, for each of the 8 g / L, 10 g / L, and 12 g / L mass challenges, more than 50% of the loaded intact bispecific antibody was recovered as purified bispecific antibody, and the recovered purified bispecific antibody product contained less than 1% clipped bispecific antibody as an impurity.

[0135] Thus, these experiments demonstrate that intact bispecific antibodies of interest can be consistently and efficiently separated from clipped bispecific antibodies by cHA resin chromatography at a variety of cHA resin mass loading challenges.

[0136] [Table 3]

[0137] Example 3 Purification of anti-BCMA / anti-CD3 bispecific antibodies by cHA resin chromatography - Different pH challenges the purpose: The objective in this example was to determine whether the bispecific antibody purification method described in Example 1 could function efficiently under different pH conditions.

[0138] material and method: The materials and methods for this example were the same as in Example 1, except that the pH of the buffer used throughout the method was (in different chromatographic runs) pH 7.0, pH 7.5, or pH 8.0. For these chromatographic runs, the cHA resin was loaded with a sample of the antibody preparation to a protein density on the cHA resin of 30 g / L.

[0139] result: The results from these chromatography runs are summarized below in Table 4. For all three chromatography runs, peak Apex material (as described in Example 1) was collected and analyzed. As shown in Table 4, the intact bispecific antibody protein of interest was successfully recovered for each of the different pH conditions tested, with pH 7.5 resulting in the highest recovery of the protein of interest (the amount of clipped bispecific antibody in the purified material was not separately determined for these chromatography runs).

[0140] Thus, these experiments demonstrate that the desired intact bispecific antibody can be efficiently purified by cHA resin chromatography under different pH conditions.

[0141] [Table 4]

[0142] Example 4 Purification of anti-FLT3 / anti-CD3 bispecific antibodies by cHA resin chromatography the purpose: The purpose of this example was to determine whether the bispecific antibody purification method described in Example 1 could be performed efficiently with a different bispecific antibody than that used in Example 1, while also requiring separation of the intact bispecific antibody of interest from various impurities, including a related clipped bispecific antibody of similar mass. In this example, the antibody of interest was a heterodimeric bispecific anti-FLT3 / anti-CD3 antibody.

[0143] material and method: The heterodimeric bispecific anti-FLT3 / anti-CD3 antibody used in this example contained the same anti-CD3 heavy and light chains as in Example 1. The amino acid sequences of the polypeptides in the FLT3 arm of this bispecific antibody are shown in the following SEQ ID NOs: FLT3 heavy chain: SEQ ID NO: 10; FLT3 light chain: SEQ ID NO: 12.

[0144] The clipped version of the bispecific antibody in this example had a clip in the same position of the anti-CD3 heavy chain as described in Example 1.

[0145] The materials and methods for this example were the same as in Example 1, except that the antibody preparation samples contained bispecific anti-FLT3 / anti-CD3 antibodies, as described above. The cHA resin was loaded with the antibody preparation samples to a protein density on the cHA resin of 10 g / L.

[0146] result: Figure 7 shows a graph providing information about the recovery of the desired intact bispecific anti-FLT3 / CD3 antibody from the cHA resin, as well as the relative amount of clipped bispecific antibody in various eluted fractions from the cHA resin. The X-axis, from left to right, indicates the order of elution from the cHA column (i.e., from low salt to high salt; each data point represents a fraction eluted from the column). Along the vertical / Y-axis, three different variables are plotted for each fraction: Variable 1) (diamonds): % POI; Variable 2) (squares): % clip; and Variable 3) (triangles): cumulative clip, each of which was determined as described in Example 1 for Figure 6. Additionally, in the graph of Figure 7, the left Y-axis lists values ​​for % POI, and the right Y-axis lists values ​​for % clip. Figure 7 also includes information showing how specific different fractions in Figure 7 correspond to different points in UV absorbance in the corresponding chromatographic profile (not shown). The points indicated as "Apex," "85%," and "60%" were determined in the same manner as described for FIG.

[0147] Various values ​​from Figure 7 are also provided below in Table 5. As shown in Table 5, the cHA resin purification methods provided herein allow for recovery of greater than 80% of the desired intact bispecific anti-FLT3 / CD3 antibody, for example, with less than 1% clipped bispecific antibody in the purified antibody product.

[0148] Thus, as shown in Figure 7 and Table 5, purification of intact bispecific anti-FLT3 / CD3 from clipped bispecific antibodies can be efficiently achieved via cHA resin.

[0149] [Table 5]

[0150] While the disclosed teachings have been described in connection with various applications, methods, kits, and compositions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the invention as claimed below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the teachings herein have been described in terms of these exemplary embodiments, those skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the teachings herein.

[0151] All references cited herein, including patents, patent applications, articles, textbooks, etc., and references cited therein, are incorporated herein by reference in their entirety to the extent they have not already been incorporated. In the event that one or more of the incorporated literature and similar materials, including but not limited to defined terms, term usage, described technology, etc., differs from or conflicts with this application, this application controls.

[0152] The foregoing description and examples detail certain specific embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the above appears in text, it will be understood that the invention can be practiced in many ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

[0153] Wherever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0154] When aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the invention encompasses not only the entire group recited as a whole, but also each member of the group individually, and all possible subgroups of the main group, as well as the main group in which one or more of the group members are absent. The invention also envisions the explicit exclusion of one or more of any of the group members in the claimed invention.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Any example(s) following the terms "eg" or "for example" are not meant to be exhaustive or limiting. The term "or," when used in the context of a list of multiple alternatives (e.g., "A, B, or C"), shall be interpreted to include any one or more of those alternatives unless the context clearly dictates otherwise.

[0156] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A composition comprising at least two proteins, the two proteins being an intact anti-BCMA / anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, (a) the bispecific antibody comprises an anti-BCMA arm and an anti-CD3 arm, the anti-BCMA arm comprising a heavy chain variable region (VH region) comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region (VL region) comprising the amino acid sequence set forth in SEQ ID NO:7, and the anti-CD3 arm comprising a VH region comprising the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:3; (b) the clipped version of the bispecific antibody comprises the same amino acid sequence as the intact anti-BCMA / anti-CD3 bispecific antibody, except that it comprises a truncation between amino acids 56 and 57 of SEQ ID NO: 1 at the N-terminus of the VH region of the CD3 arm of the clipped version of the bispecific antibody, and the truncated 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody remains attached to the remainder of the clipped version of the bispecific antibody via a disulfide bond; (c) the clipped version of the bispecific antibody has a mass that differs by less than 1% from the mass of the intact anti-BCMA / anti-CD3 bispecific antibody; and the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and a clipped version of the bispecific antibody at 0.1% to 8% by weight of the protein in the composition. composition.

2. 2. The composition of claim 1, wherein the clipped version of the bispecific antibody has a mass 18 Daltons (Da) greater than that of the intact bispecific antibody.

3. 2. The composition of claim 1, wherein the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and a clipped version of the bispecific antibody at 0.1% to 5% by weight of the protein in the composition.

4. 2. The composition of claim 1, wherein the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and a clipped version of the bispecific antibody at 0.1% to 3% by weight of the protein in the composition.

5. A composition comprising at least two proteins, the two proteins being an intact anti-BCMA / anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, (a) the bispecific antibody comprises an anti-BCMA arm and an anti-CD3 arm, the anti-BCMA arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:8, and the anti-CD3 arm comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:4; (b) the clipped version of the bispecific antibody comprises the same amino acid sequence as the intact anti-BCMA / anti-CD3 bispecific antibody, except that it comprises a truncation between amino acids 56 and 57 of SEQ ID NO: 1 at the N-terminus of the VH region of the CD3 arm of the clipped version of the bispecific antibody, and the truncated 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody remains attached to the remainder of the clipped version of the bispecific antibody via a disulfide bond; (c) the clipped version of the bispecific antibody has a mass that differs by less than 1% from the mass of the intact anti-BCMA / anti-CD3 bispecific antibody; and the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and a clipped version of the bispecific antibody at 0.1% to 8% by weight of the protein in the composition. composition.

6. 6. The composition of claim 5, wherein the clipped version of the bispecific antibody has a mass 18 Daltons (Da) greater than that of the intact bispecific antibody.

7. 6. The composition of claim 5, wherein the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and 0.1% to 5% by weight of the protein in the composition of the clipped version of the bispecific antibody.

8. 6. The composition of claim 5, wherein the composition comprises an intact bispecific antibody of interest at least 90% by weight of the protein in the composition, and 0.1% to 3% by weight of the protein in the composition of the clipped version of the bispecific antibody.

9. 1. A method for purifying an antibody preparation comprising at least two proteins, the two proteins being an intact anti-BCMA / anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, (a) the anti-CD3 arm comprises at least one of: i) a heavy chain variable region (VH region) comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region (VL region) comprising the amino acid sequence set forth in SEQ ID NO: 3; or ii) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 4; (b) the anti-BCMA arm comprises at least one of: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO:5 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:7; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:6 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:8; (c) the clipped version of the bispecific antibody comprises the same amino acid sequence as the intact anti-BCMA / anti-CD3 bispecific antibody, except that it comprises a truncation between amino acids 56 and 57 of SEQ ID NO: 1 at the N-terminus of the VH region of the CD3 arm of the clipped version of the bispecific antibody, and the truncated 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody remains attached to the remainder of the clipped version of the bispecific antibody via a disulfide bond; and (d) the clipped version of the bispecific antibody has a mass that differs from the mass of the intact anti-BCMA / anti-CD3 bispecific antibody by less than 1%; The purification method comprises the following steps: A) loading an antibody preparation in a loading buffer onto a hydroxyapatite (HA) resin; and B) eluting the intact bispecific antibody of interest from the HA resin using an elution buffer.

10. 10. The method of claim 9, wherein the clipped version of the bispecific antibody has a mass 18 Daltons (Da) greater than the mass of the intact bispecific antibody.

11. 10. The method of claim 9, wherein the HA resin is a ceramic hydroxyapatite (cHA) resin.

12. 10. The method of claim 9, wherein after loading the antibody preparation onto the HA resin and before eluting the intact bispecific antibody of interest, the HA resin is washed with a wash buffer comprising phosphate ions at a concentration of 10 to 50 mM.

13. 10. The method of claim 9, wherein the elution buffer comprises phosphate ions at a concentration of 40 to 100 mM.

14. 10. The method of claim 9, wherein the elution buffer contains phosphate ions, which are increased from 40 mM to 100 mM during elution.

15. 10. The method of claim 9, wherein the pH of at least one of the load buffer, wash buffer, and elution buffer is between 7.0 and 8.

0.

16. 10. The method of claim 9, wherein the antibody preparation comprises protein previously loaded onto and eluted from at least one of: i) a Protein A resin; and ii) an ion exchange resin.

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