Compositions and Methods

Abstract

The present disclosure relates to methods for purifying binding proteins from undesired components. Such binding proteins may be useful for treating disorders such as cancer.

Description

[Technical Field]

[0001] The present disclosure relates to methods for purifying binding proteins from undesired components. Such binding proteins may be useful for treating disorders such as cancer. [Background technology]

[0002] The economics of large-scale protein purification are important, especially for therapeutic binding proteins, because these molecules account for a large percentage of therapeutic biologics on the market. In addition to their therapeutic value, binding proteins, such as monoclonal antibodies, are also important tools in the diagnostic field. The production of binding proteins for biopharmaceutical applications typically involves the use of cell cultures known to produce undesirable components. While substantial advances have been made in purifying binding proteins, particularly affinity chromatography, such methods may not be particularly suitable for purifying the desired monomer of the binding protein. Therefore, improved methods for purifying binding proteins are needed. Summary of the Invention

[0003] The production of binding proteins using recombinant DNA technology can often result in the accumulation of undesired components in cell culture media. These undesired components include high-molecular-weight aggregates of the binding protein and complexes of the binding protein's heavy chain with free light chains that are not associated with the heavy chain. This problem can be particularly pronounced when Chinese hamster ovary (CHO) cells are used to produce the binding protein of interest, because CHO cells naturally secrete free light chains that are not associated with the heavy chains. The inventors surprisingly identified a method for purifying binding proteins from free light chains that are not associated with the heavy chains by incorporating a basic wash step into the protein purification process. This method allows for a higher yield of unbound binding protein to be recovered, thereby providing a binding protein composition with higher purity. Such compositions may be more therapeutically potent. Thus, in a first aspect, the present disclosure relates to a method for purifying a binding protein from a free light chain that is not associated with a heavy chain, the method comprising: loading a composition comprising the binding protein and a free light chain that is not associated with a heavy chain onto an equilibrated affinity chromatography column at a neutral pH to bind the binding protein in the composition to the affinity chromatography column; washing the affinity chromatography column with a basic wash buffer having a pH at least 2.5-5 above neutral pH to wash the free light chain that is not associated with a heavy chain from the composition; and eluting the binding protein bound to the affinity chromatography column with an elution buffer.

[0004] In one example, the composition is a cell culture medium obtained from CHO cells genetically modified to express the binding protein or a composition derived therefrom.

[0005] In another example, the disclosure encompasses a method for purifying a binding protein that binds to a free light chain that is not associated with a heavy chain from a Chinese hamster ovary (CHO) cell culture containing the binding protein, the method comprising: - binding of binding proteins from CHO cell culture to a Protein A resin at neutral pH; - washing the Protein A resin with a basic wash buffer having a pH at least 2.5-5 above neutral pH to wash free light chains that are not associated with heavy chains from the composition; - eluting the bound proteins bound to the Protein A resin with an elution buffer.

[0006] In one example, the binding protein comprises an antibody. In another example, the binding protein is an antibody. In another example, the antibody is an anti-kappa myeloma antigen (KMA) antibody. In another example, the antibody binds preferentially to KMA over free light chains that are not associated with heavy chains.

[0007] In one example, the basic wash buffer has a pH of 9 to 11. In another example, the basic wash buffer has a pH of 9.5 to 10.5. In another example, the basic wash buffer includes 0.1 M to 0.2 M sodium carbonate. In another example, the basic wash buffer further includes 1 M sodium chloride.

[0008] In one example, washing the affinity chromatography column to remove free light chains not associated with heavy chains includes washing the affinity chromatography column twice with a basic wash buffer. In this example, in the first wash, the basic wash buffer may contain 0.2 M sodium chloride, and in the second wash, the basic wash buffer may contain 0.1 M sodium chloride. In one example, the basic wash buffers have the same pH.

[0009] In another example, washing the affinity chromatography column further comprises washing with an acidic wash buffer. In one example, the acidic wash buffer has a pH of 5.5 to 6.5. In one example, the acidic wash buffer comprises about 35 mM sodium phosphate.

[0010] In one example, the elution buffer is acidic. In one example, the elution buffer has a lower pH than the acidic wash buffer. In one example, the elution buffer has a pH of 2.5 to 3.5. In one example, the elution buffer contains about 10 mM sodium phosphate.

[0011] In one example, the affinity chromatography column is a Protein A chromatography column.

[0012] In one example, the method further comprises a viral inactivation step. In another example, the method further comprises a viral filtration step. In another example, the method further comprises an ultrafiltration step.

[0013] In another example, the composition comprising the binding protein and the free light chain not associated with the heavy chain is cell culture fluid obtained from a cell culture of Chinese hamster ovary (CHO) cells expressing the binding protein. In one example, the cell culture fluid is clarified.

[0014] In another example, the free light chain not associated with a heavy chain is a kappa light chain. In one example, the molecular weight of the free light chain not associated with a heavy chain is about 22.5 to 25 kD. In another example, the free light chain not associated with a heavy chain is a kappa light chain dimer. In this example, the molecular weight of the free light chain not associated with a heavy chain dimer is about 45 to 50 kD. In one example, the molecular weight of the free light chain not associated with a heavy chain or a complex thereof purified from the composition disclosed herein is 20 to 100 kD. In another example, the molecular weight is 22 to 80 kD. In another example, the molecular weight is 22 to 50 kD.

[0015] In one example, it may be desirable to further purify the composition of the present disclosure by removing high molecular weight aggregates. Thus, in one example, the method of the present disclosure may also include a cation exchange chromatography step. In one example, the eluted binding protein is subjected to cation exchange chromatography to remove any potential high molecular weight species.

[0016] In another example, the method further comprises formulating the eluted binding protein into a pharmaceutical or diagnostic composition.

[0017] In one example, the disclosure encompasses pharmaceutical or diagnostic compositions comprising a binding protein purified according to the methods disclosed herein. In one example, the pharmaceutical composition comprises a binding protein comprising a VH region set forth in SEQ ID NO: 1 and a VL region set forth in SEQ ID NO: 3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising the VH region set forth in SEQ ID NO: 1 and the VL region set forth in SEQ ID NO: 3.

[0018] In one example, the eluted binding protein is at least 75% unbound antibody compared to the antibody bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay. In another example, the eluted binding protein is at least 85% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay. In another example, the eluted binding protein is at least 90% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay. In another example, the eluted binding protein is 85%-95% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0019] In another aspect, the disclosure encompasses a composition comprising an anti-KMA binding protein, wherein less than 20% of the binding proteins in the composition are in a complex with a free light chain not associated with a heavy chain. In another example, less than 15%, less than 10%, or less than 6% of the binding proteins in the composition are antibodies in a complex with a free light chain not associated with a heavy chain. In one example, the binding protein comprises a VH region set forth in SEQ ID NO: 1 and a VL region set forth in SEQ ID NO: 3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO: 1 and a VL region set forth in SEQ ID NO: 3. In one example, the binding protein is produced by CHO cells. In one example, the binding protein comprises an antibody. In one example, the binding protein is an antibody.

[0020] Any example herein shall apply mutatis mutandis to any other example unless specifically stated otherwise.

[0021] The present invention is not to be limited in scope by the specific examples described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention as described herein.

[0022] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter is to be deemed to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0023] The invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings.

[0024] Sequence table legend SEQ ID NO: 1 - Kappa Mab variable heavy chain (V H ) amino acid sequence. SEQ ID NO:2 - Kappa Mab epitope amino acid sequence. SEQ ID NO: 3 - Kappa Mab variable light chain (V L ) amino acid sequence. SEQ ID NO:4 - Kappa Mab V H Amino acid sequence of CDR1. SEQ ID NO:5 - Kappa Mab V H Amino acid sequence of CDR2. SEQ ID NO:6 - Kappa Mab V H Amino acid sequence of CDR3. SEQ ID NO:7 - Kappa Mab V L Amino acid sequence of CDR1. SEQ ID NO:8 - Kappa Mab V L Amino acid sequence of CDR2. SEQ ID NO:9 - Kappa Mab V L Amino acid sequence of CDR3. SEQ ID NO: 10—Amino acid sequence of kappa Mab epitope 2 (improved binding). DETAILED DESCRIPTION OF THE INVENTION

[0025] Common Techniques and Selected Definitions Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, antibody production, biochemistry, oncology, and protein purification).

[0026] Unless otherwise indicated, the molecular and statistical techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date) and other sources.

[0027] The phrase "anti-KMA binding protein" is used in the context of this disclosure to refer to a binding protein that binds to or specifically binds to the kappa myeloma antigen. Kappa myeloma antigen (KMA) is a membrane-bound light chain that has selectivity for kappa myeloma cells (Boux, H.A. et al. (1983) J. Exp. Med. 158:1769).

[0028] In one example, an anti-KMA binding protein can bind to KMA-bearing cells. In another example, an anti-KMA binding protein can kill KMA-bearing cells. In one example, an anti-KMA binding protein encompassed by the present disclosure does not bind to intact immunoglobulins. In other words, exemplary anti-KMA binding proteins do not recognize kappa light chains associated with Ig heavy chains, such as intact Ig molecules.

[0029] As used herein, the term "bind" with respect to the interaction of a binding protein and KMA described herein means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the KMA. For example, a binding protein recognizes and binds to a specific antigenic structure rather than an antigen generally. For example, if a binding protein binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction containing labeled "A" and the binding protein will reduce the amount of labeled "A" bound to the binding protein. In one example, a KMA binding protein disclosed herein binds preferentially to KMA (i.e., cell surface antigens) over free kappa light chains (e.g., serum antigens). A binding protein disclosed herein that binds KMA preferentially over free kappa light chains reacts or associates with KMA more frequently, more rapidly, for a longer duration, and / or with higher affinity than with free light chains.

[0030] As used herein, the term "specifically binds" shall be considered to mean that the binding interaction between a binding protein and KMA is dependent on the detection of KMA by the binding protein. Thus, the binding protein specifically binds or recognizes KMA even when present in a mixture of other molecules, cells, or organisms. In one example, the binding protein reacts or associates with KMA more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative antigens or cells. In one example, a binding protein disclosed herein that specifically binds to KMA may also preferentially bind or recognize KMA over free light chains. Reading this definition also indicates that, for example, a binding protein that specifically binds to KMA may or may not specifically bind a second antigen. As such, "specific binding" does not necessarily require exclusive or undetectable binding of another antigen. The term "specifically binds" may be used interchangeably with "selectively bind" herein. Generally, references to binding herein refer to specific binding, with each term being understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to those skilled in the art. For example, a binding protein of the present disclosure is contacted with KMA or a surrogate antigen. The binding of the binding protein to KMA or the surrogate antigen is then determined, and a binding protein that binds to KMA but not to the surrogate antigen as shown above is considered to specifically bind to KMA. Similar methods can be used to identify preferential binding. In this case, the surrogate antigen would be free light chain.

[0031] The term "immunoglobulin" will be understood to include binding proteins of the present disclosure, such as anti-KMA binding proteins, that comprise immunoglobulin domains. Exemplary immunoglobulins are antibodies. Additional proteins encompassed by the term "immunoglobulin" include domain antibodies, camelid antibodies, and antibodies from cartilaginous fish (i.e., immunoglobulin novel antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs are derived from V HIncluding V L They lack the heavy chain immunoglobulins and are often referred to as heavy chain immunoglobulins. Other "immunoglobulins" include T cell receptors.

[0032] The term "binding protein" is used in the context of this disclosure to refer to a human or humanized immunoglobulin molecule that immunoreacts with a specific antigen, and includes both polyclonal and monoclonal antibodies. The term "binding protein" also refers to a fragment having antigen-binding ability (e.g., Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd "Antibody" encompasses antigen-binding forms of antibodies, including Fab', F(ab')2, Fab, Fv, and rIgG, as discussed in "Antibody" by John Wiley & Sons, Inc., Ed., W.H. Freeman & Co., New York (1998). The term is also used to refer to recombinant single-chain Fv fragments (scFv), as well as their bivalent (di-scFv) and trivalent (tri-scFv) forms. The term antibody also includes diabodies, triabodies, and tetrabodies. In one example, the binding proteins of the present disclosure bind to free light chains that are not associated with heavy chains. In another example, the binding proteins bind to free kappa light chains that are not associated with heavy chains.

[0033] The term binding protein as used herein encompasses binding proteins including antibodies, such as bispecific molecules. For example, binding proteins may include the above referenced immunoglobulins, such as antibodies, and the above referenced fragments, such as Fvs.

[0034] An "antigen-binding fragment" of an antibody comprises one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, and single-chain antibody molecules formed from antibody fragments. For example, the term antigen-binding fragment can be used to refer to recombinant single-chain Fv fragments (scFv), as well as their bivalent (di-scFv) and trivalent (tri-scFv) forms. In one example, the binding protein is an antigen-binding fragment. Such fragments can be produced by various methods known in the art.

[0035] The term "complementarity determining region" or "CDR" is used in the context of this disclosure to refer to the portions of the two variable chains (heavy and light chains) of an antibody that recognize and bind to a specific antigen. CDRs are the most variable parts of the variable chains and provide binding proteins with their specificity. Generally, the variable heavy chain (V H ) and variable light chain (V L ) have three CDRs.

[0036] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody defined herein that specifically binds to an antigen and includes, for example, the amino acid sequences of the CDRs, i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three CDRs as well as three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). V H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0037] In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system" or "Kabat").

[0038] Other conventions involving correct or alternate numbering systems for variable domains include IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27:55-77), Chothia (Chothia C, Lesk AM (1987), J Mol Biol 196:901-917, Chothia, et al. (1989), Nature 342:877-883), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309:657-670). For convenience, exemplary binding proteins of the present disclosure may also be labeled according to IMGT.

[0039] The term "antibody heavy chain" is used herein to refer to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0040] Terms such as "host cell," "host cell line," and "host cell culture" are used interchangeably in the context of this disclosure to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. In one example, the host cell is a Chinese hamster ovary (CHO) cell.

[0041] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0042] A "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. A buffer solution resists changes in pH through the action of its acid-base conjugate components. Buffer solutions used with biological reagents generally maintain a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases. Exemplary buffers for use in the purification of biomolecules (e.g., antibodies) include zwitterionic or "Good" buffers; see, for example, Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62. Exemplary buffers include, but are not limited to, TES, MES, PIPES, HEPES, MOPS, MOPSO, TRICINE, and BICINE.

[0043] The term "wash buffer" is used herein to refer to a solution used to carry away impurities, such as free light chains that are not associated with heavy chains, from a given material, e.g., a composition or column or resin, to which a binding protein disclosed herein has bound.

[0044] The term "basic" is used in the context of the present disclosure to refer to a buffer having a basic pH. For example, the term may be used with respect to a wash buffer to refer to a wash buffer having a basic pH. In one example, the term "basic" is used to refer to a wash buffer having a pH of at least 2.5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 3 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 3.5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 4 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 4.5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 4.5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of at least 5.5 above neutral. In another example, the term "basic" is used to refer to a wash buffer having a pH of 2.5 to 5.5 above neutral. In another example, the term basic is used to refer to a wash buffer having a pH 3 to 5 above neutral. In one example, a basic wash buffer has a pH of 9 to 11. In one example, a basic wash buffer has a pH of 9.5 to 10.5. In one example, a basic wash buffer has a pH of at least 9. In one example, a basic wash buffer has a pH of at least 9.5.

[0045] In contrast, the term "acidic" is used in the context of the present disclosure to refer to a buffer having an acidic pH. For example, the term may be used with respect to a wash buffer to refer to a wash buffer having an acidic pH. Acidic buffers disclosed herein have a pH of less than 7. In one example, an acidic wash buffer has a pH of 5 to 6.5. In one example, an acidic wash buffer has a pH of 5.5 to 6.5. In one example, an acidic wash buffer has a pH of 6.5 or less. Other buffers of the present disclosure, such as elution buffers, may be more acidic than the wash buffer. For example, an elution buffer disclosed herein may have a pH of less than 4. In another example, an elution buffer may have a pH of 3.5 or less. In another example, an elution buffer may have a pH of 2.5 to 3.5.

[0046] As used herein, the term "neutral pH" refers to a pH of 7.

[0047] As used herein, the term "affinity chromatography column" refers to a column containing a resin on which affinity chromatography is performed. In one example, affinity chromatography involves subjecting a composition disclosed herein to a column containing a suitable affinity chromatography support. Non-limiting examples of such chromatography supports include, but are not limited to, Protein A resin, Protein G resin, affinity supports containing an antigen against which the binding protein of interest was raised, and affinity supports containing an Fc-binding protein. For example, the affinity chromatography column can contain Protein A chromatography resin, Protein L chromatography resin, or Protein G chromatography resin. In one example, the affinity chromatography column contains Protein A chromatography resin. Commercially available examples of Protein A chromatography resins include MabSelect Xtra and MabSelect SuRe.

[0048] As used herein, the term "viral inactivation step" refers to a process in which viruses remain in the composition but are rendered permanently non-viable. For example, viral inactivation can be performed by adjusting the solution to a low pH.

[0049] As used herein, the term "low pH" shall be understood to mean a pH of 2 to 4, or a pH of 3.4 to 3.6, or a pH of 3.5.

[0050] As used herein, the term "virus filtration step" refers to a process in which viruses are removed from a composition. For example, virus filtration can be performed by passing the composition through a filter, such as a nanofilter (e.g., Planova 20N).

[0051] As used herein, the term "polymeric form" or "high molecular weight form" refers to a binding protein in the form of two or more binding protein monomers. For example, a high molecular weight form of a binding protein (e.g., an anti-KMA binding protein) is a dimer, trimer, or tetramer.

[0052] As used herein, the term "host cell" refers to any cell capable of expressing the recombinant binding proteins disclosed herein, including bacterial, insect, and mammalian cells. For example, the mammalian cell may be a HEK293 cell or a Chinese hamster ovary cell (CHO cell). In one example, the host cell is a CHO cell. For example, the host cell may be a genetically modified host cell that expresses the binding proteins disclosed herein. Thus, in one example, the method of the present disclosure may be used to purify the binding proteins disclosed herein from the broth of a CHO cell culture or a composition derived therefrom.

[0053] As used herein, the term "fermentation" refers to a process in which host cells containing a polynucleotide sequence encoding a binding protein are grown in a cell culture medium to express the binding protein. Optimal fermentation conditions depend on several parameters, including, but not limited to, temperature range, aeration level, feed rate, and medium composition. Fermentation can be carried out under aerobic, anaerobic, or microaerobic conditions. Fermentation can also be carried out in large-scale batch cultures, for example, using one or more bioreactors.

[0054] As used herein, the term "clarified" or "clarification" refers to one or more steps involving the removal of whole cells and / or cell debris using one or more steps including any of the following, alone or in combination: centrifugation, depth filtration, precipitation, flocculation, and / or sedimentation. Clarification generally involves the removal of one or more impurities and is performed before a purification step that involves the capture of binding proteins. For example, clarification can include depth filtration and centrifugation. In one example, a composition of the present disclosure is clarified before being purified according to the methods disclosed herein.

[0055] As used herein, the term "cell culture fluid" refers to cell culture medium containing the binding protein during or after fermentation, but before a purification step involving capture of the binding protein. In one example, the cell culture fluid has been purified or partially purified to provide a preparation containing the binding protein and free light chains that are not associated with heavy chains.

[0056] The terms "purify" or "purifying" or "purification" refer to the complete or partial removal of at least one impurity from a solution containing the binding protein and one or more impurities, thereby improving the level of purity of the binding protein in the solution. Impurities include DNA, RNA, host cell proteins (HCPs), endotoxins, lipids, and one or more additives, which may be present with the binding protein resulting from the methods of the present disclosure, for example, produced by steps performed before or during the purification process. The purified binding protein is preferably essentially pure and desirably essentially homogeneous (i.e., free of contaminating proteins, etc.). In one example, the methods of the present disclosure purify or partially purify free light chains not associated with heavy chains from the compositions disclosed herein. In another example, the methods of the present disclosure purify or partially purify free light chains not associated with heavy chains and high molecular weight aggregates from the compositions disclosed herein. In one example, the high molecular weight aggregates are binding protein complexes, such as dimers. In one example, the purified binding protein is at least 60%, more preferably at least 75%, and more preferably at least 90% free of free light chains not associated with heavy chains. In another example, the purified binding protein is at least 60%, more preferably at least 75%, and more preferably at least 90% free of free light chains not associated with heavy chains and high molecular weight aggregates of the binding protein. In these examples, the free light chains not associated with heavy chains can be kappa light chains. In one example, the free light chains not associated with heavy chains have a molecular weight of 22-25 kD. In one example, the free light chains not associated with heavy chains are kappa light chain dimers. In one example, the free light chains not associated with heavy chains have a molecular weight of 45-50 kD.

[0057] As used herein, the term "pharmaceutical composition" refers to a formulation of a binding protein with compounds generally accepted in the art for delivery of therapeutic proteins to humans. Exemplary compounds include all pharmaceutically acceptable carriers, diluents, or excipients thereof.

[0058] As used herein, the term "diagnostic composition" refers to a formulation of a binding protein disclosed herein with compounds generally accepted in the art for providing the binding protein in a diagnostic form. Such formulations may be used in vitro or in vivo and therefore may be formulated with suitable carriers, diluents, and / or excipients, as appropriate, depending on the intended use.

[0059] As used in this specification and the appended claims, the singular, as well as the singular terms "a," "an," and "the," for example, optionally include plural references unless the content clearly dictates otherwise.

[0060] As used herein, the term "about" refers to + / -10%, more preferably + / -5%, more preferably + / -1% of the specified value, unless stated to the contrary.

[0061] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be understood to provide explicit support for both meanings or either meaning.

[0062] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0063] Binding proteins Binding proteins for production, purification, formulation, or use in the present disclosure include, but are not limited to, the following: In one example, the binding protein is a recombinant binding protein. In one example, the binding protein is produced by CHO cells.

[0064] In one example, the binding protein can include an antibody. For example, the binding protein can be an antibody. For example, the binding protein can be a monoclonal antibody. In one example, the binding protein is a human antibody. In one example, the antibody is humanized. In one example, the antibody is a chimeric antibody.

[0065] In one example, the binding protein is an anti-KMA binding protein. For example, the binding protein can be an anti-KMA antibody. In one example, the anti-KMA binding protein binds KMA preferentially over free light chains that are not associated with heavy chains.

[0066] In one example, the binding protein comprises a VH region and a VL region, wherein the VH region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, and the VL region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. In this example, the binding protein can comprise an antibody. For example, the binding protein can be a bispecific molecule comprising an antibody. In this example, the antibody can comprise the above-referenced CDRs. In another example, the binding protein is an antibody.

[0067] Thus, in one example, the binding protein is an antibody comprising a VH region and a VL region, wherein the VH region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, and the VL region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

[0068] In another example, the binding protein is an antibody comprising a VH region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0069] In another example, the binding protein is an antibody comprising a VH region comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0070] In another example, the binding protein is an antibody comprising a VH region comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0071] In another example, the binding protein is an antibody comprising a VH region comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1 and a VL region comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO:3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0072] In another example, the binding protein is an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3, or binds to the same epitope of kappa myeloma antigen (KMA) as an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0073] In another example, the binding protein is an antibody comprising a VH region set forth in SEQ ID NO:1 and a VL region set forth in SEQ ID NO:3.

[0074] In various examples, the above reference sequence variants having the recited % identity to the recited SEQ ID NOs can also have a VH region and a VL region, wherein the VH region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, and the VL region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9. For example, an anti-KMA binding protein has a VH comprising the CDRs set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO:1, and a VL comprising the CDRs set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO:3.

[0075] In another example, an anti-KMA binding protein has the CDRs set forth in SEQ ID NO: 1 and SEQ ID NO: 3, where the CDRs are assigned using the Kabat numbering system. In another example, an anti-KMA binding protein has the CDRs set forth in SEQ ID NO: 1 and SEQ ID NO: 3, where the CDRs are assigned using the IMGT numbering system. In another example, an anti-KMA binding protein has the CDRs set forth in SEQ ID NO: 1 and SEQ ID NO: 3, where the CDRs are assigned using the EU numbering system of Kabat.

[0076] In one example, the anti-KMA binding protein is a naked antibody. In another example, the anti-KMA binding protein is a full-length, intact, or whole antibody. In one example, the anti-KMA binding protein is monospecific. In one example, the anti-KMA binding protein is bispecific.

[0077] Further, in the above example, the binding protein is capable of binding to a KMA epitope comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:10.

[0078] In another example, an anti-KMA binding protein according to the present disclosure competes with an antibody that binds or specifically binds to an epitope comprising the amino acid sequence set forth in SEQ ID NO: 2. In another example, an anti-KMA binding protein according to the present disclosure competes with an antibody that binds or specifically binds to an epitope consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[0079] Binding proteins can be identified by their ability to compete for binding to KMA or its region or epitope using various methods known in the art.For example, binding to KMA on kappa human myeloma cell lines (κHMCL), such as KMS-11, KMS-26, and JJN3, can be evaluated (Asvadi et al. (2015) British Journal of Hematology, 169, 333-343).In this procedure, anti-KMA binding is conjugated with biotin using established procedures (Hofmann K, et al. (1982) Biochemistry 21:978-84).Then, binding proteins are evaluated by their ability to compete with the binding of biotinylated antibodies to KMA on κHMCL cells.The binding of biotinylated antibodies to κHMCL cells can be evaluated by adding fluorescein-labeled streptavidin, which binds to the biotin on the labeled antibody. Fluorescent staining of the cells is then quantified by flow cytometry, and the competitive effect of the antibodies is expressed as a percentage of the fluorescence level obtained in the absence of competitor.

[0080] In one example, the binding protein includes an immune cell engager. For example, the binding protein can be an immune cell engaging bispecific binding protein. For example, the immune cell engager can cause the binding protein disclosed herein to engage T cells or natural killer (NK) cells. Examples of immune cell engagers include an anti-CD3 binding domain, an anti-CD19 binding domain, and an anti-CD16 binding domain. In one example, the immune cell engager can engage T cells via an anti-CD3 binding domain. In another example, the immune cell engager can engage T cells via an anti-CD4 or anti-CD8 binding domain. Various other examples of immune cell engagers are disclosed in Suurs et al., (2019) Pharmacology and Therapeutics., 201:103-119.

[0081] In the above references, the affinity of the binding proteins disclosed herein for KMAs can be measured using a variety of methods. In one example, the dissociation constant (K D ) or association constant (K A ) or equilibrium constant (K D ) is determined. These constants for a binding protein are measured, in one example, by a radiolabeled or fluorescently labeled KMA binding assay. In this assay, the binding protein is equilibrated with a minimal concentration of labeled KMA in the presence of a titration series of unlabeled KMA. After washing to remove unbound KMA, the amount of label is determined. Similar assays may also be performed using amino acid sequences including SEQ ID NO:2.

[0082] Affinity measurements can be determined by standard antibody reaction methodologies, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin. Biotechnol 11:54, 2000; Englebienne Analyst. 123:1599, 1998), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. In one example, the constant is measured using a surface plasmon resonance assay, for example, BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized LMA. An exemplary SPR method is described in U.S. Patent No. 7,229,619.

[0083] Binding Protein Composition The present inventors have surprisingly identified useful binding protein compositions comprising low levels of binding protein bound to free light chains that are not associated with heavy chains. Such compositions may be particularly advantageous due to increased therapeutic efficacy resulting in more effective treatment, or potentially lower dosing, and therefore increased safety and / or more cost-effective manufacturing. In one example, the present disclosure relates to a composition comprising a referenced binding protein, wherein less than 20% of the binding protein in the composition is in a complex with a free light chain that is not associated with a heavy chain. In another example, the composition comprises a referenced binding protein, wherein less than 15% of the binding protein in the composition is in a complex with a free light chain that is not associated with a heavy chain. In another example, the composition comprises a referenced binding protein, wherein less than 10% of the binding protein in the composition is in a complex with a free light chain that is not associated with a heavy chain. In another example, the composition comprises a referenced binding protein, wherein less than 6% of the binding protein in the composition is in a complex with a free light chain that is not associated with a heavy chain. In one example, the binding protein is an anti-KMA binding protein. In one example, the anti-KMA binding protein comprises a VH and a VL, wherein the VH comprises the CDRs set forth in SEQ ID NOs: 4, 5, and 6, and the VL comprises the CDRs set forth in SEQ ID NOs: 7, 8, and 9. In one example, the anti-KMA binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 3. In one example, the binding protein in the composition is produced by CHO cells. In one example, the binding protein in the composition comprises an antibody. In one example, the binding protein in the composition is an antibody.

[0084] Production of binding proteins In one example, the binding proteins described herein are peptides or polypeptides (e.g., antibodies or antigen-binding fragments thereof). In one example, the binding proteins are recombinant.

[0085] In the case of a recombinant peptide or polypeptide, the nucleic acid encoding it can be cloned into an expression vector, which is then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce immunoglobulin or antibody proteins.

[0086] Suitable molecular cloning techniques are known in the art and are described, for example, in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art. See U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0087] After isolation, the nucleic acid is inserted into an expression construct or expression vector operably linked to a promoter for further cloning (DNA amplification) or for expression in a cell-free system or cell. Thus, another example of the present disclosure provides an expression construct comprising the isolated nucleic acid of the present disclosure and one or more additional nucleotide sequences. Preferably, the expression construct is in the form of, or contains genetic components of, a plasmid, bacteriophage, cosmid, yeast, or bacterial artificial chromosome, as understood in the art. The expression construct may be suitable for maintaining and propagating the isolated nucleic acid in bacteria or other host cells, manipulating it by recombinant DNA technology, and / or expressing the nucleic acid or binding protein of the present disclosure.

[0088] Many vectors for expression in cells are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a binding protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0089] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate-early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the beta-actin promoter, the CMV enhancer / beta-actin promoter, or hybrid regulatory elements containing immunoglobulin or antibody promoters or active fragments thereof. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), or Chinese hamster ovary cells (CHO).

[0090] For example, exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0091] Means for introducing isolated nucleic acids or expression constructs containing them into cells for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, for example, by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and particle bombardment, for example, by using tungsten or gold particles coated with DNA (Agracetus Inc., WI, USA).

[0092] Host cells used to produce binding proteins (e.g., antibodies or antigen-binding fragments) can be cultured in a variety of media, depending on the cell type used. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma), are suitable for culturing mammalian cells. Media for culturing the other cell types discussed herein are known in the art.

[0093] An exemplary protocol for producing a binding protein may include the following steps: Mammalian host cells capable of expressing a recombinant binding protein may be cultured in stirred tank bioreactor systems and fed-batch cultures. In one exemplary fed-batch culture, the mammalian host cells and culture medium are initially fed to a culture vessel, and additional culture nutrients are fed to the culture continuously or in discrete increments during cultivation, with or without periodic removal of the cells and / or binding protein from the culture vessel prior to termination of the fermentation.

[0094] During the growth phase, mammalian host cells are grown under conditions and for a period optimized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO2), and nutrient supplementation, are generally adjusted according to the host cell and will be apparent to those skilled in the art. Generally, pH is adjusted to a level of approximately 6.5 to 7.5. A suitable temperature range for culturing mammalian cells, such as CHO cells, is approximately 30°C to 38°C, with a suitable dO2 of 5 to 90% of air saturation. The cell culture environment during the production phase of fermentation is typically controlled to ensure quality and consistency in batch-to-batch binding protein production. Typically, the binding protein produced by the CHO cells is secreted into the cell culture medium. After fermentation, the cell culture medium containing the binding protein can be clarified.

[0095] Free light chain-binding protein complex The present inventors have experimentally determined that during production of the binding protein, free light chains that are not associated with heavy chains can bind to the binding protein to form a complex. The presence of such complexes in therapeutic formulations is particularly undesirable because it can affect the efficacy of the formulation.

[0096] In one example, the disclosure relates to a method for purifying a binding protein from a free light chain that is not associated with a heavy chain, the method comprising: loading a composition comprising the binding protein and a free light chain that is not associated with a heavy chain onto an equilibrated affinity column at a neutral pH to bind the binding protein in the composition to the affinity chromatography column; washing the affinity chromatography column with a basic wash buffer having a pH at least 2.5-5 above neutral pH to wash the free light chain that is not associated with a heavy chain from the composition; and eluting the binding protein bound to the affinity chromatography column with an elution buffer.

[0097] In one example, "equilibrating," "washing," or "eluting" involves passing at least 1 column volume (CV) of each buffer through the chromatography column. For example, a "washing" step can involve passing at least 1 CV of wash buffer through the chromatography column. In another example, a wash step can involve passing at least 1-25 CV of wash buffer through the chromatography column. In another example, a wash step can involve passing at least 3-20 CV of wash buffer through the chromatography column. In another example, a wash step can involve passing at least 5-15 CV of wash buffer through the chromatography column. In one example, an "eluting" or "eluting" step can involve passing at least 1 CV of elution buffer through the chromatography column. In another example, a elution step can involve passing at least 1-25 CV of elution buffer through the chromatography column. In another example, a elution step can involve passing at least 3-20 CV of elution buffer through the chromatography column. In another example, a elution step can involve passing at least 5-15 CV of elution buffer through the chromatography column. Determining the number of CVs required for each step is considered well within the understanding of one of ordinary skill in the art.

[0098] In one example, the affinity chromatography column is equilibrated with 1× phosphate buffered saline (PBS). For example, the affinity chromatography column can be equilibrated with at least 10 CV of 1×PBS.

[0099] As used herein, the term "wash buffer" refers to a buffer formulated to transfer free light chains not associated with heavy chains from the solid phase of a chromatography column. In one example, the method includes washing with a basic wash buffer having a pH at least 2.5-5 above neutral pH. In one example, the basic wash buffer has a pH of 9.5-10.5. In another example, the basic wash buffer includes sodium carbonate. Exemplary concentrations of sodium carbonate in the wash buffer range from 0.1 M to 0.2 M. In another example, the basic wash buffer also includes sodium chloride. In one example, the basic wash buffer includes 1 M sodium chloride.

[0100] In one example, washing the affinity chromatography column includes two washes with a basic wash buffer. In another example, the affinity chromatography column can be washed three, four, or five times with the basic wash buffer before the bound protein bound to the column is eluted. In one example, the basic wash buffers have the same pH. For example, the wash buffer can have a pH of 10.2. In one example, the column is washed with a basic wash buffer containing 0.2 M sodium carbonate, followed by another basic wash buffer containing 0.1 M sodium carbonate. In this example, approximately 5 to 20 CV of each wash buffer can be passed through the chromatography column. In one example, at least 10 CV is passed through the chromatography column in the first wash, and at least 5 CV is passed through the chromatography column in the second wash.

[0101] In one example, the disclosed method includes washing the affinity chromatography column with an acidic wash buffer. In one example, the disclosed method includes washing the affinity chromatography column with a basic wash buffer followed by washing with an acidic wash buffer. In one example, the acidic wash buffer has a pH of 5.5 to 6.5. In one example, the acidic wash buffer includes sodium phosphate. For example, the acidic wash buffer can include 20 to 50 mM sodium phosphate. In one example, the acidic wash buffer can include approximately 35 mM sodium phosphate.

[0102] In one example, a binding protein bound to an affinity chromatography column can be eluted by washing the affinity chromatography column with an elution buffer. Therefore, as used herein, the term "elution buffer" refers to a buffer formulated to remove binding proteins bound to a chromatography column. The elution buffer acts to dissociate the binding protein. Typical elution substances are well known in the art and may have higher concentrations of salt, free affinity ligands or analogs, or other substances that promote dissociation of the binding protein from a given material. The conductivity and / or pH of the elution buffer are such that the binding protein is eluted from the column. In one example, the elution buffer is acidic. In one example, the elution buffer is more acidic than the wash buffers used in the methods disclosed herein. In one example, the elution buffer has a pH of 2 to 4. In one example, the elution buffer has a pH of 2.5 to 3.5. In one example, the elution buffer has a pH of 3. In one example, the elution buffer contains sodium phosphate. For example, the elution buffer can contain 5 to 15 mM sodium phosphate. In one example, the elution buffer contains 10 mM sodium phosphate.

[0103] In one example, the binding protein eluted from the affinity chromatography column is 75%, or 85%, or 90%, or 95%, or 99% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by size exclusion chromatography HPLC (SEC-HPLC) and / or BioCore assay.

[0104] For example, the binding protein eluted from the affinity chromatography column is at least 75% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0105] In one example, the binding protein eluted from the affinity chromatography column is at least 85% unbound binding protein compared to the binding protein bound to free light chains that are not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0106] In one example, the binding protein eluted from the affinity chromatography column is at least 90% unbound binding protein compared to the binding protein bound to free light chains that are not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0107] In one example, the binding protein eluted from the affinity chromatography column is at least 95% unbound binding protein compared to the binding protein bound to free light chains that are not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0108] In one example, the binding protein eluted from the affinity chromatography column is 85%-95% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0109] In one example, the binding protein eluted from the affinity chromatography column is 90%-95% unbound binding protein compared to the binding protein bound to free light chains not associated with heavy chains, as determined by SEC-HPLC and / or BioCore assay.

[0110] In another example, the disclosure provides a method for purifying a binding protein that binds to a light chain that is not associated with a heavy chain from a Chinese hamster ovary (CHO) cell culture expressing the binding protein, the method comprising binding the binding protein from the CHO cell culture or a composition derived therefrom to an affinity chromatography resin at a neutral pH, washing the resin with a basic wash buffer having a pH at least 2.5-5 above neutral pH, and eluting the binding protein bound to the resin with an elution buffer. In one example, the affinity chromatography column comprises a Protein A resin.

[0111] In one example, the free light chains not associated with heavy chains that are removed using the methods of the present disclosure are kappa light chains. In one example, the molecular weight of the free light chains not associated with heavy chains that are removed using the methods of the present disclosure is 22 to 25 kD. In another example, the free light chains not associated with heavy chains that are removed using the methods of the present disclosure are kappa light chain dimers. In one example, the molecular weight of the free light chains not associated with heavy chains that are removed using the methods of the present disclosure is 45 to 50 kD.

[0112] In one example, the molecular weight of the free light chain or complex thereof not associated with a heavy chain purified from the composition disclosed herein is 20 to 100 kD, in another example, 22 to 80 kD, or in another example, 22 to 50 kD.

[0113] In one example, the affinity chromatography step involves subjecting the composition to a column containing a suitable affinity chromatography support. Non-limiting examples of such chromatography supports include, but are not limited to, Protein A resin, Protein G resin, affinity supports containing an antigen against which the antibody of interest was raised, and affinity supports containing an Fc-binding protein. Protein A resin is useful for binding antibodies (IgG). In one example, the affinity chromatography column comprises Protein A resin. In one example, the affinity chromatography column is a Protein A chromatography column. In another example, the affinity chromatography column is a Protein L chromatography column. In a further example, the affinity chromatography column is a Protein G chromatography column.

[0114] The eluate can be monitored using techniques well known to those skilled in the art. For example, OD 28O The eluted binding protein can be optionally prepared for further processing via one or more of the additional method steps discussed below.

[0115] Starting Composition In one example, the starting composition is a cell culture medium obtained after culturing cells genetically modified to express the binding protein disclosed herein. For example, the starting composition can be a cell culture medium obtained after culturing CHO cells genetically modified to express the binding protein disclosed herein. However, this starting composition may need to be partially purified before being subjected to the method of the present disclosure. For example, the cell culture medium may need to be clarified to remove cellular debris. Therefore, the composition purified according to the method of the present disclosure is not particularly limited, as long as it is derived from a cell culture medium obtained after culturing cells genetically modified to express the binding protein disclosed herein and contains the binding protein and a free light chain that is not associated with a heavy chain. In one example, the starting composition contains the unbound binding protein, the binding protein bound to a free light chain that is not associated with a heavy chain, and the free light chain that is not associated with a heavy chain. In one example, the composition also contains high molecular weight aggregates of the binding protein.

[0116] In one example, the starting composition is derived from cell culture medium obtained after culturing CHO cells that have been genetically modified to express a binding protein disclosed herein, such as an anti-KMA binding protein.

[0117] Additional Method Steps In one example, the disclosed method includes an additional step after the binding protein has been subjected to the above-referenced wash step and eluted from the affinity chromatography column. For example, the eluted binding protein may then be subjected to an additional chromatography step. For example, the eluted binding protein may be subjected to cation exchange chromatography to remove any potential high molecular weight species. For example, cation exchange chromatography may be used to remove high molecular weight aggregates, such as binding protein complexes. Thus, in one example, the disclosed method may further include cation exchange chromatography. Other exemplary additional chromatographic purification steps include, but are not limited to, ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and / or size exclusion chromatography. In one example, the disclosed method further includes a virus inactivation step. In another example, the disclosed method further includes a virus filtration step.

[0118] In one example, the clarified cell culture fluid is purified to remove free light chains not associated with heavy chains according to the present disclosure before being subjected to cation exchange chromatography, ultrafiltration, anion exchange chromatography, phenyl sepharose HP chromatography, viral filtration, and ultrafiltration.

[0119] formulation In one example, the method further comprises formulating the purified binding protein into a pharmaceutical composition.

[0120] The disclosure herein further provides pharmaceutical compositions comprising, for example, a binding protein purified by the methods described herein.

[0121] Suitable pharmaceutical compositions containing the binding protein to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Those skilled in the art will recognize a variety of suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can contain various additives, preservatives, or fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions may optionally contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, and toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The compounds may be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

[0122] The optimal concentration of the active ingredient in the selected medium can be determined empirically according to procedures known to those skilled in the art and will depend on the ultimate pharmaceutical formulation desired.

[0123] Similarly, in one example, a binding protein can be purified according to the present disclosure and formulated into a diagnostic composition that includes one or more of the above-referenced components according to the intended use of the composition (e.g., in vitro vs. in vivo). [Example]

[0124] Example 1: Expression of anti-kappa myeloma antigen (KMA) binding protein The anti-kappa myeloma antigen (anti-KMA) binding protein is expressed by growing Chinese hamster ovary (CHO) cells genetically modified to express the binding protein in fed-batch suspension culture. The production of the genetically modified CHO cells used herein is generally described in WO 2003 / 004056. After suspension culture, the CHO cells are removed from the cell culture fluid (CCF) by depth filtration and 0.2 μm filtration. The resulting CCF is collected and stored at 2°C to 8°C.

[0125] Example 2: Protein A affinity chromatography purification of recombinant anti-kappa myeloma antigen (KMA) binding protein To prepare CCF containing anti-KMA binding proteins for Protein A chromatography purification, the protein concentration of the CCF was adjusted to 3.5 g / L or less and a pH of 6.8 or greater.

[0126] A Protein A affinity chromatography column (MabSelect Xtra Load) was equilibrated with at least 5 column volumes (CV) of 1× PBS (equilibration buffer) to a pH of 7.3–7.5 and a conductivity of 14.0–16.8 mS / cm.

[0127] The protein concentration and pH adjusted CCF was then loaded onto an equilibrated Protein A affinity chromatography column, after which the column was washed with 15 CV or more of a basic wash buffer of 200 mM sodium carbonate, 1 M sodium chloride, pH 10.2. The column was further washed with 5 CV or more of a basic wash buffer of 100 mM sodium carbonate, pH 10.2, followed by 4 CV or more of an acidic wash buffer of 35 mM sodium phosphate, pH 6.2.

[0128] Anti-KMA-binding proteins were eluted from the Protein A affinity chromatography column with an elution buffer of 10 mM sodium phosphate, pH 3.0, and monitored by measuring the absorbance at 280 nm. Single peak fractions were collected, each starting at 15% and ending at 5% above baseline.

[0129] Example 3: Alternative Protein A affinity chromatography purification of recombinant anti-KMA binding protein In an alternative approach, CCF was purified by Protein A affinity chromatography using two washes of basic wash buffer (200 mM sodium carbonate, 1 M sodium chloride, pH 10.2), followed by elution and collection of the anti-kappa myeloma binding protein from the Protein A affinity chromatography column. Results were comparable to those in Example 2.

[0130] Example 4: Low pH Treatment and Adjustment The Protein A affinity chromatography eluate containing the anti-KMA binding protein was further subjected to low-pH viral inactivation, neutralization, and viral filtration. Viral inactivation was performed by adjusting the pH of the Protein A affinity chromatography eluate to 3.40-3.60 with 1N hydrochloric acid and holding the eluate at room temperature for 60-75 minutes to inactivate any potentially contaminating viruses. Following viral inactivation, the eluate was neutralized with 1N sodium hydroxide to a pH of 6.1-6.3, followed by 0.2 μm filtration and storage at 2°C-8°C until further processing.

[0131] The yield of anti-KMA monomer after purification via Examples 2 or 3, and subsequent viral inactivation, neutralization, and filtration was 90% compared to the anti-KMA binding protein bound to free light chains not associated with heavy chains as determined by SEC-HPLC, and activity as determined by BioCore assay.

[0132] Example 4: Cation exchange chromatography purification After low-pH viral inactivation, neutralization, and viral filtration of the Protein A affinity chromatography eluate, the eluate was subjected to cation exchange chromatography to remove any potential high molecular weight species present in the eluate. The cation exchange chromatography column (Fractogel EMD SE HiCap) was equilibrated with 35 mM sodium phosphate at pH 6.2 for over 5 CV. The protein concentration of the eluate was adjusted to 8.5 mg / mL or less at pH 6.1-6.3 before loading onto the cation exchange chromatography column.

[0133] The anti-KMA-binding protein bound to the cation exchange chromatography column was washed with 20 mM sodium phosphate pH 6.2 over 5 CV, and the anti-KMA-binding protein was eluted with 35 mM sodium phosphate and 30 mM sodium chloride pH 6.2, monitored by measuring the absorbance wavelength at 280 nm. Single-peak fractions were collected, rising above baseline and ending when the absorbance decreased to approximately 20%-30% of the maximum peak height.

[0134] The combination of Protein A affinity chromatography and cation exchange chromatography resulted in recovery of 95% pure anti-KMA binding protein monomer compared to anti-KMA binding protein bound to free light chains not associated with heavy chains as determined by SEC-HPLC and activity as determined by the BioCore assay.

[0135] Example 5: Formulation into a pharmaceutical composition The cation exchange chromatography eluate was passed through a Planova 20 N virus removal filter to remove any inadvertent viral contamination and further filtered through a 0.22 μm filter. The filtered preparation was concentrated and diafiltered by tangential flow filtration (TFF) into 20 mM sodium citrate, 100 mM sodium chloride, 1.5% mannitol, 50 μM DTPA pH 6.0, and further filtered through a 0.2 μm filter. Polysorbate 80 (Tween 80) was added to a final concentration of 0.04% as needed to adjust the protein concentration to 10.0 ± 1.0 mg / ml and filtered through a 0.2 μm filter to produce a formulated pharmaceutical composition of the anti-KMA binding protein.

[0136] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0137] All publications discussed above are incorporated herein in their entirety.

[0138] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and should not be construed as an admission that any or all of such matters existed prior to the priority date of each claim of this application and therefore form part of the substrate of the prior art or were common general knowledge in the art relevant to the present invention.

[0139] This application claims priority from Australian Provisional Patent Application No. 2020 / 900948, filed on 27 March 2020, the entire contents of which are incorporated herein by reference.

Claims

1. 1. A method for purifying anti-kappa myeloma antigen (KMA) antibodies that bind to free kappa light chains that are not associated with heavy chains from a Chinese hamster ovary (CHO) cell culture containing anti-KMA antibodies, the method comprising: - binding the anti-KMA antibodies from the CHO cell culture to a Protein A resin at neutral pH; - washing the Protein A resin with a basic wash buffer having a pH at least 3-5 above neutral pH to wash away free kappa light chains that are not associated with heavy chains; - eluting the anti-KMA antibodies bound to the Protein A resin with an elution buffer; the anti-KMA antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a complementarity determining region (CDR) 1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6; The method, wherein the light chain variable region comprises a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:8, and a CDR3 of SEQ ID NO:

9.

2. 2. The method of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

3.

3. The basic wash buffer is one or more of the following: - 0.1M to 0.2M sodium carbonate; - 1M sodium chloride 3. The method of claim 1 or 2, comprising:

4. 4. The method of any one of claims 1 to 3, wherein washing the Protein A resin to remove free kappa light chains not associated with heavy chains comprises washing the Protein A resin twice with a basic wash buffer.

5. 5. The method of claim 4, wherein in the first wash, the basic wash buffer comprises 0.2 M sodium chloride and in the second wash, the basic wash buffer comprises 0.1 M sodium chloride.

6. The method of claim 5 , wherein the basic wash buffers have the same pH.

7. 7. The method of any one of claims 1 to 6, wherein washing the Protein A resin comprises washing with an acidic wash buffer.

8. 8. The method of claim 7, wherein the acidic wash buffer has a pH of 5.5 to 6.

5.

9. 9. The method of claim 7 or 8, wherein the acidic wash buffer comprises 35 mM sodium phosphate.

10. The method of any one of claims 1 to 9, wherein the elution buffer is acidic.

11. The elution buffer may be one or more of the following: - a lower pH than the acidic wash buffer; - pH between 2.5 and 3.5; 10mM sodium phosphate The method according to any one of claims 1 to 10, comprising:

12. One or more of the following: - virus inactivation step; - virus filtration step; - formulating the eluted anti-KMA antibody into a pharmaceutical or diagnostic composition. The method of any one of claims 1 to 11, further comprising:

13. The method of any one of claims 1 to 12, wherein the free kappa light chains not associated with heavy chains are kappa light chain dimers.

14. The method of any one of claims 1 to 13, wherein the molecular weight of the free kappa light chains not associated with heavy chains is between 22 and 100 kDa.

15. The anti-KMA antibody is subjected to cation exchange chromatography to remove high molecular weight species; at least 90% unbound antibody of the total antibody population as determined by SEC-HPLC and / or BiaCore assay; or 85% to 95% unbound antibody of the total antibody population as determined by SEC-HPLC and / or BiaCore assay; The method according to any one of claims 1 to 14.

16. A composition comprising an anti-kappa myeloma antigen (KMA) antibody, wherein less than 10% of the anti-KMA antibody in the composition is in a complex having a free kappa light chain that is not associated with a heavy chain; The anti-KMA antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a complementarity determining region (CDR) 1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6; the light chain variable region comprises a CDR1 of SEQ ID NO: 7, a CDR2 of SEQ ID NO: 8, and a CDR3 of SEQ ID NO: 9; composition.

17. The composition of claim 16, wherein less than 6% of the anti-KMA antibodies in the composition are anti-KMA antibodies in complexes with free kappa light chains that are not associated with heavy chains.

18. 18. The composition of claim 16 or 17, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

3.

19. 18. The composition of claim 16 or 17, wherein the anti-KMA antibody is produced by Chinese hamster ovary (CHO) cells.