Use of cosmotrope for increasing the yield of the affinity chromatography purification step

Abstract

The present disclosure provides a method and kit for purifying an antigen-binding protein comprising a VL domain using a protein L chromatography material comprising a cosmotrope salt in a buffer background.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 353,512, entitled "USE OF KOSMOTROPES TO ENHANCE YIELD OF AN AFFINITY CHROMATOGRAPHY PURIFICATION STEP", filed on June 17, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure generally relates to a method for purifying an antigen - binding protein comprising a VL region, the method comprising binding a polypeptide to a protein L chromatography material, washing the chromatography material with a buffer containing a kosmotrope, and eluting the antigen - binding protein at low pH.

Background Art

[0003] Protein L is an affinity ligand that can be used for the chromatographic purification of antibodies and some antibody fragments such as Fab, scFv, and VHH. All three of them lack the Fc domain and thus are not compatible with protein A. Protein L has an affinity for the VL domain of the kappa light chain, which is the same domain where three complementarity - determining regions (CDRs) are located, and the binding effect can vary in a manner specific to the molecular sequence. Protein L chromatography resin carries the protein L ligand on the surface of beads and is usually operated under conditions similar to those of protein A chromatography (equilibration, loading of highly impure feedstock, washing, and subsequent recovery of the purified protein by elution). There is a need for an improved method for protein L chromatography.

[0004] All references mentioned in this specification, including patent applications and publications, are hereby incorporated by reference in their entirety.

Summary of the Invention

[0005] A method for purifying an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a washing buffer comprising an equilibration buffer and a chaotrope; and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer.

[0006] Also provided herein is a method for improving the protein L purification of an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a washing buffer comprising an equilibration buffer and a chaotrope; and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer.

[0007] In some embodiments, the yield and / or purity of the antigen-binding protein is improved as compared to protein L chromatography where the wash buffer does not contain a chaotrope.

[0008] In some of any such embodiments, the equilibration buffer contains Tris, MES, MOPS, or EDTA. In some of any such embodiments, the equilibration buffer contains Tris and NaCl. In some of any such embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. In some of any such embodiments, NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. In some of any such embodiments, the equilibration buffer has a pH of about 4 to about 10. In some of any such embodiments, the equilibration buffer has a pH of about 6.5 to about 8.5, or about 7.7. In some of any such embodiments, the equilibration buffer has a pH of about 7 to about 8.

[0009] In some of any such embodiments, the wash buffer has a pH of about 4 to about 10. In some of any such embodiments, the wash buffer has a pH of about 4 to about 10, about 4 to about 9.5, about 4 to about 9, about 4 to about 8.5, about 4 to about 8, about 4 to about 7.5, about 5 to about 10, about 5 to about 9.5, about 5 to about 9, about 5 to about 8.5, about 5 to about 8, about 5 to about 7.5, about 6 to about 10, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, about 6 to about 8, or about 6 to about 7.5. In some of any such embodiments, the wash buffer has a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some of any such embodiments, the wash buffer has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0010] In some of any such embodiments, the antigen-binding protein is loaded onto the protein L chromatography material as the host cell culture supernatant in step a).

[0011] In any of some such embodiments, the antigen-binding protein is loaded onto the protein L chromatography material as a purified polypeptide solution in step a).

[0012] In any of some such embodiments, the antigen-binding protein is loaded onto the protein L chromatography material as a mixture from a previous purification step in step a).

[0013] In any of some such embodiments, the antigen-binding protein is prepared in an equilibration buffer before being loaded onto the protein L chromatography material in step a).

[0014] In any of some such embodiments, the antigen-binding protein is prepared in an equilibration buffer and a cosmotrope before being loaded onto the protein L chromatography material in step a).

[0015] In any of some such embodiments, the cosmotrope is a phosphate or a sulfate. In any of some such embodiments, the cosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate.

[0016] In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer or the wash buffer is from about 100 mM to about 800 mM. In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer and the wash buffer is from about 100 mM to about 800 mM. In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer or the wash buffer is from about 120 mM to about 600 mM. In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer or the wash buffer is about 120 mM, about 240 mM, about 360 mM, about 480 mM, or about 600 mM. In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer and / or the wash buffer is from about 100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 100 mM to about 600 mM, from about 100 mM to about 500 mM, from about 200 mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mM to about 600 mM, from about 200 mM to about 500 mM, from about 300 mM to about 800 mM, from about 300 mM to about 700 mM, from about 300 mM to about 600 mM, or from about 300 mM to about 500 mM. In any of some such embodiments, the concentration of the cosmotrope in the equilibration buffer and / or the wash buffer is about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, or 800 mM.

[0017] In any of some such embodiments, the cosmotrope is sodium sulfate. In any of some such embodiments, the cosmotrope is potassium phosphate. In any of some such embodiments, the cosmotrope is ammonium sulfate.

[0018] In some of any such embodiments, the elution buffer has a lower pH than the equilibration buffer. In some of any such embodiments, the elution buffer has a pH of about 2.0 to about 4.0, or about 2.5 to about 3.0. In some of any such embodiments, the elution buffer has a pH of about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3.0, about 3.05, about 3.1, about 3.15, about 3.2, about 3.25, or about 3.3. In some of any such embodiments, the elution buffer has a pH of about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, or about 2.95. In some of any such embodiments, the elution buffer has a higher pH than the equilibration buffer. In some of any such embodiments, the elution buffer has a pH of about 10.0 to about 12.0.

[0019] In some of any such embodiments, the elution buffer contains acetic acid. In some of any such embodiments, the elution buffer contains from about 50 mM acetic acid to about 250 mM acetic acid.

[0020] In some of any such embodiments, the elution buffer contains about 150 mM acetic acid at a pH of about 2.8.

[0021] In some of any such embodiments, the elution buffer contains sodium acetate, citrate, or glycine.

[0022] In some of any such embodiments, the antigen-binding protein does not bind well to Protein L. In some of any such embodiments, the antigen-binding protein binds less well to Protein L compared to a reference antigen-binding protein that is the monoclonal antibody G6-31. In some of any such embodiments, the antigen-binding protein binds more weakly to Protein L than a reference antigen-binding protein that is the monoclonal antibody G6-31.

[0023] In any of some such embodiments, the antigen-binding protein is an antibody or an immunoadhesin or a fragment thereof. In any of some such embodiments, the antigen-binding protein is a monoclonal antibody. In any of some such embodiments, the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody. In any of some such embodiments, the antibody is an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. In any of some such embodiments, the antibody fragment is Fab. In any of some such embodiments, Fab lacks a hydrophobic patch or includes a binding site for binding to Protein L having a weak hydrophobic patch. In any of some such embodiments, the binding site for binding to Protein L lacks a hydrophobic patch. In any of some such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch. In any of some such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. In any of some such embodiments, the binding site for binding to Protein L includes a hydrophilic patch.

[0024] In any of some such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L has a value higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein, which is monoclonal antibody G6-31 for Protein L. In any of some such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L has a value at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein, which is monoclonal antibody G6-31 for Protein L.

[0025] In any of some such embodiments, the antibody does not contain an Fc domain.

[0026] In any of some such embodiments, the antibody is a multispecific antibody. In any of some such embodiments, the multispecific antibody comprises a VL domain and a second VL domain, and the VL domain is a kappa (κ) VL. In any of some such embodiments, the VL domain is a κ2VL. In any of some such embodiments, the VL domain binds poorly to protein L. In any of some such embodiments, the VL domain binds poorly to protein L and the second VL domain does not bind to protein L. In any of some such embodiments, the second VL domain is a lambda (λ) VL. In any of some such embodiments, the VL domain is a λ subtype VL or a κVL. In any of some such embodiments, the VL domain binds poorly to protein L. In any of some such embodiments, the VL domain binds poorly to protein L as compared to the binding of the VL domain of a reference antigen-binding protein that is monoclonal antibody G6-31 to protein L. In any of some such embodiments, the VL domain is a κVL and comprises a modification that weakens the binding of the VL domain to protein L. In any of some such embodiments, the modification comprises one or more amino acid substitutions in the VL domain. In any of some such embodiments, the VL domain is a κ2VL.

[0027] In any of some such embodiments, the antigen-binding protein comprises a binding site for binding to Protein L that lacks a hydrophobic patch or has a weak hydrophobic patch. In some embodiments, the binding site for binding to Protein L lacks a hydrophobic patch. In some embodiments, the binding site for binding to Protein L has a weak hydrophobic patch. In any of some such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. In any of some such embodiments, the binding site for binding to Protein L comprises a hydrophilic patch.

[0028] In any of some such embodiments, the Protein L chromatography material is Pierce™ Protein L chromatography cartridge, Capto™ L chromatography, HiTrap® Protein L chromatography, KanCap™ L chromatography, TOYOPEARL® AF-rProtein L-650F chromatography, or MabSelect™ VL chromatography.

[0029] Furthermore, compositions comprising an antigen-binding protein purified by a method comprising any of the methods described herein are provided herein. In some embodiments, the composition comprises one or more pharmaceutical excipients.

[0030] Furthermore, kits for purifying an antigen-binding protein comprising a VL domain, the kits comprising a Protein L chromatography material and a cosmotrope, are provided herein.

[0031] In some embodiments, the kit further includes a equilibration buffer. In some embodiments, the equilibration buffer includes Tris, MES, MOPS, or EDTA. In any of some such embodiments, the equilibration buffer includes Tris and NaCl. In any of some such embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. In any of some such embodiments, NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM.

[0032] In any of some such embodiments, the equilibration buffer has a pH of about 4 to about 10. In any of some such embodiments, the equilibration buffer has a pH of about 6.5 to about 8.5, or about 7.7. In any of some such embodiments, the equilibration buffer has a pH of about 7 to about 8.

[0033] In any of some such embodiments, the kit further includes a wash buffer. In some embodiments, the wash buffer has a pH of about 4 to about 10. In any of some such embodiments, the wash buffer has a pH of about 4 to about 10, about 4 to about 9.5, about 4 to about 9, about 4 to about 8.5, about 4 to about 8, about 4 to about 7.5, about 5 to about 10, about 5 to about 9.5, about 5 to about 9, about 5 to about 8.5, about 5 to about 8, about 5 to about 7.5, about 6 to about 10, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, about 6 to about 8, or about 6 to about 7.5. In any of some such embodiments, the wash buffer has a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In any of some such embodiments, the wash buffer has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0034] In any of such embodiments, the wash buffer includes a equilibration buffer and a cosmotropic agent. In any of such embodiments, the equilibration buffer further includes a cosmotropic agent.

[0035] In any of some such embodiments, the kosmotrope is a phosphate or a sulfate. In any of some such embodiments, the kosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer or the wash buffer is from about 100 mM to about 800 mM. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer and the wash buffer is from about 100 mM to about 800 mM. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer or the wash buffer is from about 120 mM to about 600 mM. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer or the wash buffer is about 120 mM, about 240 mM, about 360 mM, about 480 mM, or about 600 mM. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer and / or the wash buffer is from about 100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 100 mM to about 600 mM, from about 100 mM to about 500 mM, from about 200 mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mM to about 600 mM, from about 200 mM to about 500 mM, from about 300 mM to about 800 mM, from about 300 mM to about 700 mM, from about 300 mM to about 600 mM, or from about 300 mM to about 500 mM. In any of some such embodiments, the concentration of the kosmotrope in the equilibration buffer and / or the wash buffer is about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, or 800 mM.

[0036] In some of such embodiments, the kosmotrope is sodium sulfate. In some of such embodiments, the kosmotrope is potassium phosphate. In some of such embodiments, the kosmotrope is ammonium sulfate.

[0037] In some of such embodiments, the kit further comprises an elution buffer. In some embodiments, the elution buffer has a lower pH than the equilibration buffer. In some of such embodiments, the elution buffer has a pH of from about 2.0 to about 4.0, or from about 2.5 to about 3.0. In some of such embodiments, the elution buffer has a pH of about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3.0, about 3.05, about 3.1, about 3.15, about 3.2, about 3.25, or about 3.3. In some of such embodiments, the elution buffer has a pH of about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, or about 2.95.

[0038] In some embodiments, the elution buffer has a higher pH than the equilibration buffer. In some of such embodiments, the elution buffer has a pH of from about 10.0 to about 12.0.

[0039] In some of such embodiments, the elution buffer contains acetic acid. In some embodiments, the elution buffer contains from about 50 mM acetic acid to about 250 mM acetic acid. In some of such embodiments, the elution buffer contains about 150 mM acetic acid at a pH of about 2.8. In some of such embodiments, the elution buffer contains sodium acetate, citrate, or glycine.

[0040] In some of such embodiments, the kit is for use in purifying an antibody or an immunoadhesin or a fragment thereof. In some of such embodiments, the antigen-binding protein is a monoclonal antibody. In some of such embodiments, the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody. In some of such embodiments, the antibody is an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. In some of such embodiments, the antibody fragment is Fab.

[0041] In some embodiments, the Fab comprises a binding site for binding to Protein L that lacks a hydrophobic patch or has a weak hydrophobic patch. In some embodiments, the binding site for binding to Protein L lacks a hydrophobic patch. In some embodiments, the binding site for binding to Protein L has a weak hydrophobic patch. In some of such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. In some of such embodiments, the binding site for binding to Protein L comprises a hydrophilic patch.

[0042] In any of some such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L has a value higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein that is monoclonal antibody G6-31 for Protein L. In any of some such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L has a value at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the binding affinity (EC50) and / or dissociation constant of the reference antigen-binding protein that is monoclonal antibody G6-31 for Protein L.

[0043] In any of some such embodiments, the antibody does not contain an Fc domain.

[0044] In any of such embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody comprises a VL domain and a second VL domain, and the VL domain is a kappa (κ) VL. In any of such embodiments, the VL domain is a κ2VL. In any of such embodiments, the VL domain does not bind well to protein L. In any of such embodiments, the VL domain does not bind well to protein L and the second VL domain does not bind to protein L. In any of such embodiments, the second VL domain is a lambda (λ) VL. In any of such embodiments, the antibody comprises a VL domain that is a λ subtype VL or a κVL. In any of such embodiments, the VL domain does not bind well to protein L. In any of such embodiments, the VL domain of the antigen-binding protein binds less well to protein L compared to a reference antigen-binding protein that is the monoclonal antibody G6-31. In any of such embodiments, the VL domain of the antigen-binding protein binds more weakly to protein L than the reference antigen-binding protein that is the monoclonal antibody G6-31.

[0045] In any of some such embodiments, the VL domain is κVL and includes a modification that weakens the binding of the VL domain to Protein L. In some embodiments, the modification includes one or more amino acid substitutions in the VL domain. In any of some such embodiments, the VL domain is κ2VL. In any of some such embodiments, the antigen-binding protein lacks a hydrophobic patch or includes a binding site for binding to Protein L having a weak hydrophobic patch. In some embodiments, the binding site for binding to Protein L lacks a hydrophobic patch. In some embodiments, the binding site for binding to Protein L has a weak hydrophobic patch. In any of some such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. In any of some such embodiments, the binding site for binding to Protein L includes a hydrophilic patch.

Brief Description of Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0055] In some embodiments, the present invention provides a method for purifying an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a chaotrope (e.g., sulfate or phosphate); and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some embodiments, the present invention provides a method for improving the protein L purification of an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a chaotrope (e.g., sulfate or phosphate); and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some embodiments, the yield and / or purity of the antigen-binding protein is improved as compared to protein L chromatography where the wash buffer does not contain a chaotrope.

[0056] Those skilled in the art will also understand that modifications in form and detail of the embodiments described herein can be made without departing from the scope of the present disclosure. Further, while various advantages, aspects, and objects have been described with reference to various embodiments, the scope of the present disclosure should not be limited by reference to such advantages, aspects, and objects.

[0057] Definitions For the purposes of interpreting this specification, the following definitions apply, and whenever appropriate, terms used in the singular include the plural and vice versa. In the event of any conflict between any of the definitions set forth below and any of the documents incorporated herein by reference, the definitions set forth below shall prevail.

[0058] As used herein, "kosmotrope" (sometimes spelled "cosmotrope") is a salt that promotes hydrophobic bonding due to the way it structures water molecules in solution. Two examples of kosmotropic anions used in chromatography are phosphate and sulfate. Another kosmotrope, ammonium sulfate, is very strongly kosmotropic and is frequently used for salting out (precipitating) proteins from solution. In contrast to kosmotropes, chaotropic salts interfere with hydrophobic bonding, and the Hofmeister series is a ranking of various salts along a kosmotrope-versus-chaotrope spectrum according to their properties.

[0059] The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms include naturally modified amino acid polymers, or amino acid polymers modified by any other manipulation or modification such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation to a labeling component. Also included within the scope of this definition are, for example, one or more analogs of amino acids (including, for example, non-natural amino acids), and polypeptides containing other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically include antibodies.

[0060] A "purified" polypeptide (e.g., an antibody or an immunoadhesin) means that the purity of the polypeptide has been increased such that it exists in a more pure form than when the polypeptide is present in its natural environment and / or when it was first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0061] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are more highly conserved than the CDRs and are interposed between adjacent stretches known as framework regions (FRs) that form a scaffold for supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant regions of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a semi-synthetic antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a multispecific antibody such as a bispecific antibody. In some embodiments, the antibody is linked to a fusion protein. In some embodiments, the antibody is linked to an immunostimulatory protein such as interleukin. In some embodiments, the antibody is linked to a protein that facilitates entry across the blood-brain barrier.

[0062] As used herein, the term "antigen-binding fragment" refers to an antibody fragment that includes, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of antibodies that include one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not include a complete antibody structure. An antigen-binding fragment can bind the same antigen to which the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, the antigen-binding fragment can include one or more CDRs from a particular human antibody grafted into framework regions from one or more different human antibodies.

[0063] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, insofar as they exhibit the biological activity of this invention (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0064] As used herein, the term "multispecific antibody" refers to a monoclonal antibody having binding specificity for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, the multispecific antibody has two binding specificities (bispecific antibody). In certain embodiments, the multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0065] The term "semi-synthetic" with respect to an antibody or antibodies means that the antibody or antibodies have one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.

[0066] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are tightly non-covalently associated. The folding of these two domains provides the amino acid residues for antigen binding and generates six hypervariable loops (three loops from each of the heavy and light chains) that confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.

[0067] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment that contains a VH antibody domain and a VL antibody domain connected by a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH domain and the VL domain, which enables the scFv to form a structure desirable for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0068] The term "diabody" refers to a small antibody fragment prepared by constructing an scFv fragment (see preceding paragraph) between the VH and VL domains with a short linker (about 5-10 residues) such that inter-chain pairing, rather than intra-chain pairing of the V domains, is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" scFv fragments where the VH and VL domains of two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example, in European Patent No. 404,097, International Publication No. 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0069] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that contains minimal sequences derived from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable regions (HVRs) are replaced by residues from the hypervariable regions of a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) that have the desired antibody specificity, affinity, and capacity. In some instances, residues in the framework regions (FRs) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody can contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of the non-human immunoglobulin and all or substantially all of the FRs being of human immunoglobulin sequence. Also, a humanized antibody optionally contains at least a portion of the constant region (Fc) of the immunoglobulin, typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0070] As used herein, the term "isolated" typically refers to a molecule that has been separated from at least some of the components in which it is found in nature or is produced. For example, a polypeptide is called "isolated" when it has been separated from at least some of the components of the cell that produced it. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is called "isolated" if it is not part of a larger polynucleotide typically found in nature (such as genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), or, in the case of an RNA polynucleotide, if it has been separated from at least some of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell can be called "isolated".

[0071] "Contaminant" refers to a substance that is different from the desired polypeptide product. Contaminants include, but are not limited to, host cell substances such as CHO host cell protein (CHOP), leached protein A, nucleic acids, variants of the desired polypeptide (base variants or acid variants of the desired polypeptide product), fragments, aggregates, or derivatives (such as high molecular weight species (HMWS) or very high molecular weight species (vHMWS) of the desired polypeptide), other polypeptides, endotoxins, viral contaminants, cell culture medium components, etc. In some examples, contaminants can be host cell proteins (HCP) from, for example, but not limited to, bacterial cells such as Escherichia coli (E. coli) cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells.

[0072] The term "hydrophobic patch" refers to a portion of an antigen-binding protein, such as an antibody or an immunoadhesin or a fragment thereof such as a Fab, that contains one or more hydrophobic amino acid residues that confer overall hydrophobicity. The term "hydrophilic patch" refers to a portion of an antigen-binding protein, such as an antibody or an immunoadhesin, or a fragment thereof such as a Fab, that contains one or more hydrophilic amino acid residues that confer overall hydrophilicity.

[0073] As used herein, the terms "comprising", "having", "containing", and "including", and their grammatical equivalents, are equivalent in meaning and are intended to be open-ended in that any item recited in connection with any one of these words does not mean an exhaustive listing of such items or a limitation to only the recited items. For example, an article "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may contain components A, B, and C as well as one or more other components. Thus, "comprises" and its like forms, and their grammatical equivalents, are intended to and are understood to include disclosures of embodiments "consisting essentially of" or "consisting of".

[0074] Where a range of values is provided, unless the context clearly dictates otherwise, values between the upper and lower limits of that range, each being an intervening value to the tenth of the unit of the lower limit, and any other recited or intervening value within the recited range, are understood to be included within the disclosure subject to any specifically excluded limitations within the recited range. Where the recited range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0075] References in this specification to "about" a value or parameter include (and describe) variations that are directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0076] As used in this specification and the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly dictates otherwise.

[0077] Use of cosmotrope in protein L chromatography In some embodiments, the invention provides a method for purifying an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a cosmotrope (e.g., sulfate or phosphate); and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some embodiments, the invention provides a method for improving the protein L purification of an antigen-binding protein comprising a VL domain, the method comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a cosmotrope (e.g., sulfate or phosphate); and c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some embodiments, the yield and / or purity of the antigen-binding protein is improved as compared to protein L chromatography in which the wash buffer does not contain a cosmotrope.

[0078] In some embodiments, the equilibration buffer can be any suitable equilibration buffer.

[0079] In some embodiments of the present invention, the equilibration buffer contains Tris (i.e., tris(hydroxymethyl)aminomethane or tromethane). In some embodiments of the present invention, the equilibration buffer contains NaCl. In some embodiments of the present invention, the equilibration buffer contains Tris and NaCl.

[0080] In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, Tris is present in the equilibration buffer at any concentration of approximately 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 75 mM to 100 mM, 10 mM to 75 mM, 15 mM to 75 mM, 20 mM to 75 mM, 25 mM to 75 mM, 30 mM to 75 mM, 40 mM to 75 mM, 50 mM to 75 mM, 75 mM to 75 mM, 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 40 mM, 15 mM to 40 mM, 20 mM to 40 mM, 25 mM to 40 mM, 30 mM to 40 mM, 10 mM to 30 mM, 15 mM to 30 mM, 20 mM to 30 mM, 25 mM to 30 mM, 10 mM to 25 mM, 15 mM to 25 mM, 20 mM to 25 mM, 10 mM to 20 mM, 15 mM to 20 mM, or 10 mM to 15 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration greater than about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, or 100 mM.

[0081] In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration of approximately 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 75 mM to 100 mM, 10 mM to 75 mM, 15 mM to 75 mM, 20 mM to 75 mM, 25 mM to 75 mM, 30 mM to 75 mM, 40 mM to 75 mM, 50 mM to 75 mM, 75 mM to 75 mM, 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 40 mM, 15 mM to 40 mM, 20 mM to 40 mM, 25 mM to 40 mM, 30 mM to 40 mM, 10 mM to 30 mM, 15 mM to 30 mM, 20 mM to 30 mM, 25 mM to 30 mM, 10 mM to 25 mM, 15 mM to 25 mM, 20 mM to 25 mM, 10 mM to 20 mM, 15 mM to 20 mM, or 10 mM to 15 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration greater than about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, or 100 mM.

[0082] In some embodiments of the present invention, the equilibration buffer contains MES (2-(N-morpholino)ethanesulfonic acid). In some embodiments, the equilibration buffer contains MES and NaCl. In some embodiments, the equilibration buffer contains MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments, the equilibration buffer contains MOPS and NaCl. In some embodiments, the equilibration buffer contains EDTA (ethylenediaminetetraacetic acid). In some embodiments, the equilibration buffer contains a carbonate buffer.

[0083] In some embodiments, NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, NaCl is present in the equilibration buffer at a concentration of approximately 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 75 mM to 100 mM, 10 mM to 75 mM, 15 mM to 75 mM, 20 mM to 75 mM, 25 mM to 75 mM, 30 mM to 75 mM, 40 mM to 75 mM, 50 mM to 75 mM, 75 mM to 75 mM, 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 40 mM, 15 mM to 40 mM, 20 mM to 40 mM, 25 mM to 40 mM, 30 mM to 40 mM, 10 mM to 30 mM, 15 mM to 30 mM, 20 mM to 30 mM, 25 mM to 30 mM, 10 mM to 25 mM, 15 mM to 25 mM, 20 mM to 25 mM, 10 mM to 20 mM, 15 mM to 20 mM, or 10 mM to 15 mM. In some embodiments, NaCl is present in the equilibration buffer at a concentration greater than about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, or 100 mM.

[0084] In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM and NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM and NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 20 mM to about 30 mM and NaCl is present in the equilibration buffer at a concentration of about 20 mM to about 30 mM. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 25 mM and NaCl is present in the equilibration buffer at a concentration of about 25 mM.

[0085] In some embodiments, the equilibration buffer has a pH of from about 4.0 to about 10.0. In some embodiments, the equilibration buffer has a pH of from about 6.0 to about 9.0. In some embodiments, the equilibration buffer has a pH of from about 7.0 to about 8.0. In some embodiments, the equilibration buffer has a pH of generally any of about 6.0 - 9.0, 6.5 - 8.5, or 7.0 - 8.0. In some embodiments, the equilibration buffer has a pH of any of about 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, or 9.0. In some embodiments, the equilibration buffer has a pH of 7.7 or about 7.7. In some embodiments, Tris is present in the equilibration buffer at a concentration of from about 10 mM to about 100 mM, NaCl is present in the equilibration buffer at a concentration of from about 10 mM to about 100 mM, and the pH of the equilibration buffer is from about 6.0 to about 9.0. In some embodiments, Tris is present in the equilibration buffer at a concentration of from about 10 mM to about 50 mM, NaCl is present in the equilibration buffer at a concentration of from about 10 mM to about 50 mM, and the pH of the equilibration buffer is from about 6.5 to about 8.5. In some embodiments, Tris is present in the equilibration buffer at a concentration of from about 20 mM to about 30 mM, NaCl is present in the equilibration buffer at a concentration of from about 20 mM to about 30 mM, and the pH of the equilibration buffer is from about 7.5 to about 8.0. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 25 mM, NaCl is present in the equilibration buffer at a concentration of about 25 mM, and the pH of the equilibration buffer is about 7.7.

[0086] In some embodiments, the wash buffer has a pH of from about 4 to about 10. In some embodiments, the wash buffer has a pH of from about 4 to about 10, from about 4 to about 9.5, from about 4 to about 9, from about 4 to about 8.5, from about 4 to about 8, from about 4 to about 7.5, from about 5 to about 10, from about 5 to about 9.5, from about 5 to about 9, from about 5 to about 8.5, from about 5 to about 8, from about 5 to about 7.5, from about 6 to about 10, from about 6 to about 9.5, from about 6 to about 9, from about 6 to about 8.5, from about 6 to about 8, or from about 6 to about 7.5. In some embodiments, the wash buffer has a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the wash buffer has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0087] In some embodiments, the antigen-binding protein is loaded onto the Protein L chromatography material as host cell culture supernatant (HCCS) in step a). In some embodiments, the antigen-binding protein is loaded onto the Protein L chromatography material as a purified polypeptide solution in step a). In some embodiments, the antigen-binding protein is loaded onto the Protein L chromatography material as a mixture from a previous purification step in step a). The previous purification step can be any step of a purification process or workflow that occurs prior to the loading onto the Protein L chromatography material in step a). In some embodiments, the antigen-binding protein is prepared in an equilibration buffer prior to being loaded onto the Protein L chromatography material in step a). In some embodiments, the cosmotrope is added to the HCCS containing the antigen-binding protein prior to being loaded onto the Protein L chromatography material in step a). In some embodiments, the antigen-binding protein is prepared in an equilibration buffer and a cosmotrope prior to being loaded onto the Protein L chromatography material in step a). In some embodiments, the cosmotrope is a phosphate or a sulfate. In some embodiments, the cosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to the loading onto the Protein L chromatography material in step a) is any concentration of at least about 100 mM that does not result in precipitation of the antigen-binding protein from the solution. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to the loading onto the Protein L chromatography material in step a) is from about 100 mM to about 1000 mM.In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS before loading onto the Protein L chromatography material in step a) is generally 100 mM to 1000 mM, 120 mM to 1000 mM, 240 mM to 1000 mM, 360 mM to 1000 mM, 480 mM to 1000 mM, 600 mM to 1000 mM, 800 mM to 1000 mM, 100 mM to 800 mM, 120 mM to 800 mM, 240 mM to 800 mM, 360 mM to 800 mM, 480 mM to 800 mM, 100 mM to 600 mM, 120 mM to 600 mM, 240 mM to 600 mM, 360 mM to 600 mM, 480 mM to 600 mM, 100 mM to 480 mM, 120 mM to 480 mM, 240 mM to 480 mM, 360 mM to 480 mM, 100 mM to 360 mM, 120 mM to 360 mM, 240 mM to 360 mM, 100 mM to 240 mM, 120 mM to 240 mM, or 100 mM to 120 mM. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS before loading onto the Protein L chromatography material in step a) is higher than any of about 100 mM, 120 mM, 240 mM, 360 mM, 480 mM, 600 mM, 800 mM, or 1000 mM. In some embodiments, the concentration of the cosmotrope in the equilibration buffer is about 100 mM to about 800 mM. In some embodiments, the concentration of the cosmotrope in the equilibration buffer is about 120 mM to about 600 mM.

[0088] In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer and / or the wash buffer is from about 100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 100 mM to about 600 mM, from about 100 mM to about 500 mM, from about 200 mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mM to about 600 mM, from about 200 mM to about 500 mM, from about 300 mM to about 800 mM, from about 300 mM to about 700 mM, from about 300 mM to about 600 mM, or from about 300 mM to about 500 mM. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer and / or the wash buffer is about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, or 800 mM. In some embodiments, the cosmotrope is sodium sulfate. In some embodiments, the cosmotrope is potassium phosphate. In some embodiments, the cosmotrope is ammonium sulfate.

[0089] In some embodiments, the wash buffer of step b) comprises a cosmotrope. In some embodiments, the cosmotrope in the wash buffer is a phosphate or a sulfate. In some embodiments, the cosmotrope in the wash buffer is potassium phosphate, sodium sulfate, or ammonium sulfate. In some embodiments, the wash buffer of step b) comprises an equilibration buffer and a cosmotrope. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) is at least about 100 mM, any concentration that does not result in precipitation of the antigen-binding protein from the solution. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) is from about 100 mM to about 1000 mM. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) is approximately 100 mM - 1000 mM, 120 mM - 1000 mM, 240 mM - 1000 mM, 360 mM - 1000 mM, 480 mM - 1000 mM, 600 mM - 1000 mM, 800 mM - 1000 mM, 100 mM - 800 mM, 120 mM - 800 mM, 240 mM - 800 mM, 360 mM - 800 mM, 480 mM - 800 mM, 600 mM - 800 mM, 100 mM - 600 mM, 120 mM - 600 mM, 240 mM - 600 mM, 360 mM - 600 mM, 480 mM - 600 mM, 100 mM - 480 mM, 120 mM - 480 mM, 240 mM - 480 mM, 360 mM - 480 mM, 100 mM - 360 mM, 120 mM - 360 mM, 240 mM - 360 mM, 100 mM - 240 mM, 120 mM - 240 mM, or 100 mM - 120 mM. In some embodiments, the concentration of the cosmotrope (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) is higher than any of about 100 mM, 120 mM, 240 mM, 360 mM, 480 mM, 600 mM, 800 mM, or 1000 mM.In some embodiments, the concentration of the cosmotrope in the wash buffer is from about 100 mM to about 800 mM. In some embodiments, the concentration of the cosmotrope in the wash buffer is from about 120 mM to about 600 mM.

[0090] In some embodiments, the concentration of the cosmotrope in the equilibration buffer and the wash buffer is from about 100 mM to about 800 mM. In some embodiments, the concentration of the cosmotrope in the equilibration buffer and the wash buffer is from about 120 mM to about 600 mM.

[0091] In some embodiments, the elution buffer of step c) has a lower pH than the equilibration buffer. In some embodiments, the elution buffer has a pH of from about 2.0 to about 5.0. In some embodiments, the elution buffer has a pH of from about 2.0 to 5.0, 2.5 to 5.0, 3.0 to 5.0, 3.5 to 5.0, 4.0 to 5.0, 2.0 to 4.0, 2.5 to 4.0, 3.0 to 4.0, 3.5 to 4.0, 2.0 to 3.5, 2.5 to 3.5, 3.0 to 3.5, 2.0 to 3.0, 2.5 to 3.0, or 2.0 to 2.5. In some embodiments, the elution buffer has a pH of about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.75, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, or 5.0. In some embodiments, the elution buffer has a pH of about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3.0, about 3.05, about 3.1, about 3.15, about 3.2, about 3.25, or about 3.3. In some embodiments, the elution buffer has a pH of about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, or about 2.95.

[0092] In some embodiments, the elution buffer of step c) has a higher pH than the equilibration buffer. For example, some antigen-binding proteins may not tolerate a low pH elution buffer having a pH of, for example, from about 2.0 to about 5.0. In some embodiments, the elution buffer has a pH of about 10.0 or higher. Thus, in some embodiments, the elution buffer has a pH of any of about 10.0 - 12.0, 10.5 - 12.0, 11.0 - 12.0, 10.0 - 11.5, or 10.0 - 11.0. In some embodiments, the elution buffer has a pH of any of about 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0. In some embodiments, the elution buffer has a pH of about 10.0 - 12.0.

[0093] In some embodiments, the elution buffer contains acetic acid. In some embodiments, the elution buffer contains sodium acetate, citrate, or glycine. In some embodiments, the elution buffer contains acetic acid, sodium acetate, citrate, or glycine at a concentration of about 50 mM to 250 mM, 75 mM to 250 mM, 100 mM to 250 mM, 125 mM to 250 mM, 150 mM to 250 mM, 175 mM to 250 mM, 200 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 200 mM, 125 mM to 200 mM, 150 mM to 200 mM, 175 mM to 200 mM, 50 mM to 175 mM, 75 mM to 175 mM, 100 mM to 175 mM, 125 mM to 175 mM, 150 mM to 175 mM, 50 mM to 150 mM, 75 mM to 150 mM, 100 mM to 150 mM, 125 mM to 150 mM, 50 mM to 125 mM, 75 mM to 125 mM, 100 mM to 125 mM, 50 mM to 100 mM, 75 mM to 100 mM, or 50 mM to 75 mM. In some embodiments, the elution buffer contains acetic acid, sodium acetate, citrate, or glycine at a concentration of about 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 160 mM, 170 mM, 175 mM, 180 mM, 190 mM, 200 mM, 225 mM, or 250 mM.

[0094] In some embodiments, the elution buffer contains from about 50 mM acetic acid to about 250 mM acetic acid. In some embodiments, the elution buffer contains acetic acid at a concentration of any one of from about 50 mM to 250 mM, from 75 mM to 250 mM, from 100 mM to 250 mM, from 125 mM to 250 mM, from 150 mM to 250 mM, from 175 mM to 250 mM, from 200 mM to 250 mM, from 50 mM to 200 mM, from 75 mM to 200 mM, from 100 mM to 200 mM, from 125 mM to 200 mM, from 150 mM to 200 mM, from 175 mM to 200 mM, from 50 mM to 175 mM, from 75 mM to 175 mM, from 100 mM to 175 mM, from 125 mM to 175 mM, from 150 mM to 175 mM, from 50 mM to 150 mM, from 75 mM to 150 mM, from 100 mM to 150 mM, from 125 mM to 150 mM, from 50 mM to 125 mM, from 75 mM to 125 mM, from 100 mM to 125 mM, from 50 mM to 100 mM, from 75 mM to 100 mM, or from 50 mM to 75 mM. In some embodiments, the elution buffer contains acetic acid at a concentration of any one of about 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 160 mM, 170 mM, 175 mM, 180 mM, 190 mM, 200 mM, 225 mM, or 250 mM. In some embodiments, the elution buffer contains about 150 mM acetic acid. In some embodiments, the elution buffer contains about 150 mM acetic acid at a pH of about 2.8.

[0095] In some embodiments, the antigen-binding protein purified by protein L chromatography is an antibody or an immunoadhesin or a fragment thereof. In some embodiments, the VL domain of the antibody or immunoadhesin or fragment thereof is λ subtype VL or κ2VL. In some embodiments, the VL domain does not bind strongly to protein L. In some embodiments, the VL domain is κVL and contains a modification that weakens the binding of the VL domain to protein L. In some embodiments, the modification includes one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is κ2VL.

[0096] In some embodiments, the antigen-binding protein does not bind well to Protein L. For example, in some embodiments, although not bound by theory, the antigen-binding proteins used in the methods described herein do not bind well to Protein L, and while the cosmotrope enhances the binding of the antigen-binding protein to Protein L, impurities such as HMWF and LC-dimers may bind more strongly to Protein L than the antigen-binding protein, and as a result, the yield and quality of the antigen-binding protein may decrease in the absence of the cosmotrope. In some embodiments, the antigen-binding protein that does not bind well to Protein L comprises a VL domain of the κ2 subtype.

[0097] In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, binds less to Protein L compared to a reference antigen-binding protein. In some embodiments, the reference antigen-binding protein is an antigen-binding protein of the same type or a similar type as the antigen-binding protein, such as an antibody. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, binds less to Protein L compared to a reference antigen-binding protein that is the monoclonal antibody G6-31. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, binds more weakly to Protein L than the reference antigen-binding protein. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, binds more weakly to Protein L than the reference antigen-binding protein that is the monoclonal antibody G6-31. The G6-31 (also referred to as "G6.31") monoclonal antibody is an anti-VEGF Fab and is a well-characterized antibody fragment. See, for example, Korsisaari et al., PNAS, 2007, 104(25):10625-10630. In some embodiments, the G6-31 Fab can serve as a reference antigen-binding protein to determine whether another antigen-binding protein exhibits a weaker or poorer binding than the G6-31 Fab, which is thought to exhibit normal binding to Protein L, for example. In some embodiments, an antigen-binding protein that exhibits a weak or poor binding to Protein L may be particularly suitable for the methods described herein that involve the use of cosmotrops to improve the yield and purity of a target antigen-binding protein that can benefit from enhancing the binding of the antigen-binding protein to Protein L.

[0098] In some embodiments, the antigen-binding protein is a monoclonal antibody. In some embodiments, the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody is an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. In some embodiments, the antibody does not contain an Fc domain.

[0099] In some embodiments, the antibody fragment is a Fab. In some embodiments, the Fab lacks a hydrophobic patch or contains a binding site for binding to protein L having a weak hydrophobic patch. In some embodiments, the binding site for binding to protein L lacks a hydrophobic patch. In some embodiments, the binding site for binding to protein L has a weak hydrophobic patch. In some embodiments, the binding site for binding to protein L contains a hydrophilic patch.

[0100] In some embodiments, an antigen-binding protein that does not bind well to protein L lacks a hydrophobic patch or contains a binding site for binding to protein L having a weak hydrophobic patch. In some embodiments, the binding site for binding to protein L has a weaker hydrophobic patch compared to the binding site for binding to protein L of a reference antigen-binding protein that is monoclonal antibody G6-31.

[0101] In some embodiments, an antigen-binding protein that does not bind well to protein L contains a hydrophilic patch.

[0102] In some embodiments, the binding affinity (EC50) and / or dissociation constant of an antigen-binding protein, such as an antibody or immunoadhesin, for Protein L is higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein that is the monoclonal antibody G6-31 for Protein L. In some embodiments, the binding affinity (EC50) and / or dissociation constant of an antigen-binding protein, such as an antibody or immunoadhesin, for Protein L is at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein that is the monoclonal antibody G6-31 for Protein L.

[0103] In some embodiments, an antigen-binding protein, such as a Fab, comprises a VL domain, and the binding site is contained within the VL domain.

[0104] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the multispecific antibody comprises a VL domain and a second VL domain, and the VL domain is a kappa (κ) VL. In some embodiments, the VL domain is a κ2VL. In some embodiments, the VL domain does not bind well to protein L. In some embodiments, the VL domain does not bind well to protein L and the second VL domain does not bind to protein L. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, comprises a VL domain and does not bind well to protein L as compared to a reference antigen-binding protein comprising the VL domain. In some embodiments, the reference antigen-binding protein is an antigen-binding protein of the same type or a similar type as the antigen-binding protein, such as an antibody. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, comprises a VL domain and does not bind well to protein L as compared to a reference antigen-binding protein comprising a VL domain that is the VL domain of monoclonal antibody G6-31. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, comprises a VL domain and binds to protein L more weakly than a reference antigen-binding protein comprising the VL domain. In some embodiments, an antigen-binding protein, such as an antibody or an immunoadhesin, comprises a VL domain and binds to protein L more weakly than a reference antigen-binding protein comprising a VL domain that is the VL domain of monoclonal antibody G6-31. In some embodiments, the VL domain does not bind well to protein L as compared to the binding of the VL domain of a reference antigen-binding protein that is monoclonal antibody G6-31 to protein L.

[0105] In some embodiments, the second VL domain is lambda (λ) VL. In some embodiments, the VL domain is κVL and includes a modification that weakens the binding of the VL domain to Protein L. In some embodiments, the modification includes one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is κ2VL.

[0106] In some embodiments, the VL domain is a λ subtype VL or κ2VL. Protein L chromatography

[0107] Protein L is a cell wall protein of the bacterium Peptostreptococcus magnus (Bjorck et al. (1988) J. Immunol. 140:1194 - 1197) that binds to the variable region of the kappa light chain without interfering with the antigen - binding site of an antibody or antibody fragment (Nilson et al. (1992) J Biol Chem. 267:2234 - 2239). Protein L interacts with FW1 in the V region of the kappa light chain, and its binding is restricted to the VL of the κ1, κ3, and κ4 subtypes, but does not bind or binds weakly to the VL of the κ2 subtype. Commercially available Protein L chromatography materials include, but are not limited to, Pierce™ Protein L chromatography cartridges, Capto™ L chromatography, HiTrap® Protein L chromatography, KanCap™ L chromatography, TOYOPEARL® AF - rProtein L - 650F chromatography, or MabSelect™ VL chromatography.

[0108] Additional steps In some embodiments, the present invention provides additional steps involved in or related to the purification of the antigen-binding proteins described herein. Additional steps involved in or related to purification, and methods for performing such steps, are known. See, for example, Liu et al. mAbs, 2, 2010, which is incorporated herein by reference in its entirety.

[0109] In some embodiments, the purification of the antigen-binding protein further comprises a sample processing step such as a sample preparation step. In some embodiments, the purification of the antigen-binding protein further comprises a clarification step, for example, for clarifying HCCF. In some embodiments, the purification of the antigen-binding protein further comprises a host cell and host cell debris removal step for removing host cells and host cell debris from, for example, a sample and / or composition obtained from a purification platform. In some embodiments, the purification of the antigen-binding protein further comprises a centrifugation step. In some embodiments, the purification of the antigen-binding protein further comprises a sterile filtration step. In some embodiments, the purification of the antigen-binding protein further comprises a tangential flow microfiltration step. In some embodiments, the purification of the antigen-binding protein further comprises an aggregation / precipitation step.

[0110] antigen-binding protein In some embodiments, a method of protein L chromatography using a wash buffer containing cosmotrope is useful for purifying and concentrating an antigen-binding protein comprising a VL domain from an antigen-binding protein preparation. In some embodiments, the antigen-binding protein preparation is derived from a host cell preparation. In some embodiments, the host cell preparation is a host cell culture fluid (HCCF). In some embodiments, the antigen-binding protein preparation comprises a portion of the host cell culture fluid. In some embodiments, the antigen-binding protein preparation is derived from the host cell culture fluid. In some embodiments, the antigen-binding protein preparation comprises host cells. In some embodiments, the antigen-binding protein preparation comprises components of host cells such as host cell debris. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is an Escherichia coli (E. coli) cell.

[0111] Antigen-binding protein Antigen-binding proteins purified by protein L chromatography, including the use of cosmotrope in a wash buffer using the methods described herein, are generally produced using recombinant techniques. Methods for producing recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are specifically incorporated herein by reference. In some embodiments, the protein of interest is produced in CHO cells (see, e.g., WO 94 / 11026). In some embodiments, the polypeptide of interest is produced intracellularly in E. coli cells. See, for example, U.S. Patent Nos. 5,840,523, 5,648,237, and 5,789,199, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. Also see Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of polypeptide fragments in E. coli. When using recombinant techniques, the polypeptide can be produced intracellularly, in the periplasmic space, or secreted directly into the medium.

[0112] The antigen-binding protein can be recovered from the culture medium or host cell lysate. The cells used for the expression of the antigen-binding protein can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents. When the antigen-binding protein is produced intracellularly, as a first step, any particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating polypeptides secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed over about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antigen-binding protein is secreted into the medium, generally, the supernatant from such an expression system is first concentrated using a commercially available polypeptide concentration filter, such as an Amicon® or Millipore Pellicon® ultrafiltration unit. To inhibit proteolysis, a protease inhibitor such as PMSF may be included in any of the foregoing steps, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0113] In some embodiments, the antigen-binding protein is purified or partially purified prior to analysis by the methods of the invention. For example, the antigen-binding protein of the method is in the eluate from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography.

[0114] Examples of antigen-binding proteins purified by protein L chromatography, including the use of cosmotrope in the wash buffer using the methods described herein, include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, and immune complexes.

[0115] (A) Antibody In some embodiments of any of the methods described herein, the antigen-binding protein is an antibody or an immunoadhesin.

[0116] (i) Monoclonal antibody In some embodiments, the antibody is a monoclonal antibody. A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for variants that may arise during production of the monoclonal antibody, and such variants are generally present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being an individual antibody or a mixture of polyclonal antibodies.

[0117] For example, monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0118] In the hybridoma method, a mouse, or other suitable host animal such as a hamster, is immunized as described herein to produce antibodies that specifically bind to the polypeptide used for immunization, or lymphocytes capable of producing such antibodies are induced. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused to myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0119] The hybridoma cells so prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances inhibit the growth of HGPRT-deficient cells.

[0120] In some embodiments, the myeloma cells are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to media such as HAT medium. In particular, in some embodiments, the myeloma cell line is derived from a mouse myeloma line, such as the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and the SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myelomas and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0121] The medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. In some embodiments, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0122] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0123] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as animal ascites tumors.

[0124] Monoclonal antibodies secreted by the subclones are preferably separated from the culture medium, ascites, or serum by conventional immunoglobulin purification procedures such as polypeptide A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0125] DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of a mouse antibody). In some embodiments, the hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed within an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that otherwise does not produce an immunoglobulin polypeptide, to obtain the synthesis of a monoclonal antibody in the recombinant host cell. Review articles regarding the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0126] In further embodiments, an antibody or antibody fragment can be isolated from an antibody phage library made using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe, respectively, the isolation of mouse and human antibodies using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing extremely large phage libraries (Waterhouse et al., Nuc. Acids Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0127] Furthermore, the DNA can be modified, for example, by substituting the coding sequence with the constant domains of the human heavy and light chains instead of the homologous mouse sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

[0128] Typically, such non-immunoglobulin polypeptides are substituted in place of the constant domains of the antibody, or they are substituted in place of the variable domains of one antigen-binding site of the antibody to create a chimeric bivalent antibody that contains one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0129] In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

[0130] (ii) Humanized antibody In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies are described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often, typically, referred to as "grafted" residues, typically obtained from the "grafted" variable domain. Humanization can be essentially carried out by substituting the hypervariable region sequences with the corresponding sequences of a human antibody according to the methods of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Thus, such a "humanized" antibody is a chimeric antibody (U.S. Patent No. 4,816,567) in which substantially less than the entire human variable domain is replaced by the corresponding sequence from a non-human species. In practice, a humanized antibody is typically a human antibody in which some hypervariable region residues, and perhaps some FR residues, are replaced by residues from the analogous sites in a rodent antibody.

[0131] The selection of human variable domains, both heavy and light chains, used in the production of humanized antibodies is extremely important for reducing antigenicity. According to the so-called "best fit" method, the sequences of the variable domains of rodent antibodies are screened against a library of all known human variable-domain sequences. The human sequence closest to the rodent sequence is then identified as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Other methods use specific framework regions derived from the consensus sequences of all human antibodies of a particular subgroup of the light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0132] It is even more important that the antibody be humanized while retaining high affinity for the antigen and other desirable biological properties. To achieve this goal, in some embodiments of the method, the humanized antibody is prepared by an analysis process of the parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the predicted three-dimensional conformation of the selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the putative role of residues in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and donor sequences, resulting in the achievement of desired antibody properties such as improved affinity for the target antigen. Generally, hypervariable region residues are directly and most substantially involved in affecting antigen binding.

[0133] (iii) Human antibodies In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that have the ability to produce a complete repertoire of human antibodies without endogenous immunoglobulin production upon immunization. For example, homozygous deletion of the antibody heavy chain joining region (J H ) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Pat. Nos. 5,591,669; 5,589,369; and 5,545,807.

[0134] Alternatively, the phage display technique (McCafferty et al., Nature 348:552-553(1990)) can be used to produce human antibodies and antibody fragments in vitro from an immunoglobulin variable (V) domain gene repertoire from non-immunized donors. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat polypeptide genes of filamentous bacteriophages such as M13 or fd and presented as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain a copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also leads to the selection of the gene encoding the antibody with those properties. Thus, phage mimics some of the properties of B cells. Phage display can be performed in various formats, for an overview of which, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes can be constructed from non-immunized human donors, and antibodies against a variety of antigens (including autoantigens) of the diverse array can be isolated essentially according to the techniques described in Marks et al., J. Mol. Biol. 222:581-597(1991), or Griffith et al., EMBO J. 12:725-734(1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0135] Also, human antibodies can be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

[0136] (iv) Antibody fragments In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is a Fab, Fab’-SH, Fv, scFv, or (Fab’)2 fragment. In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody is an antibody fragment that includes an Fc receptor binding domain. Various techniques have been developed for producing antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of a complete antibody (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above.

[0137] In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antibody fragment is an antigen-binding fragment that includes an Fc receptor binding domain. In some embodiments, the antibody fragment is an antigen-binding fragment that includes an Fcγ receptor binding domain.

[0138] (v) Bispecific antibody In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes. Alternatively, the bispecific antibody binding arms may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), in order to focus on the cell defense mechanism against cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies).

[0139] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where these two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random pairing of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed by an affinity chromatography step, is quite cumbersome and the yield of the product is low. Similar procedures are disclosed in WO 93 / 08829, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0140] According to different methods, antibody variable domains (antibody-antigen binding sites) having a desired binding specificity are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides excellent flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where the unequal ratios of the three polypeptide chains used in construction provide an optimal yield. However, if expression of at least two polypeptide chains at equal ratios results in a high yield or if their ratios are not particularly meaningful, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector.

[0141] In some embodiments of this approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of the immunoglobulin light chain only in half of the bispecific molecule provides an easy separation method. This approach is disclosed in WO 94 / 04690. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0142] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises at least a portion of the C H 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory "hole" of the same or similar size as the large side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

[0143] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the heteroconjugate antibodies can be conjugated to avidin and the other to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and also for the treatment of HIV infection (International Publication No. 91 / 00360, International Publication No. 92 / 200373, and European Patent No. 0308936). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with several cross-linking techniques.

[0144] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are cleaved by proteolysis to yield F(ab’)2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab’ fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to Fab’-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab’-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0145] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides derived from the Fos and Jun proteins were linked by gene fusion to the Fab’ portions of two different antibodies. This antibody homodimer was reduced at the hinge region to form monomers, which were then re-oxidized to form antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described in Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for generating bispecific antibody fragments. This fragment contains a heavy chain variable domain (V L ) connected to a light chain variable domain (V H ) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the V H domain and V L domain of one fragment are complementary to the V L domain and VH It is paired with a domain, thereby forming two antigen-binding sites. Another method for producing a bispecific antibody fragment by using a single-chain Fv (sFv) dimer has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0146] Antibodies having a valence greater than 2 are also conceivable. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0147] (v) Multivalent antibodies In some embodiments, the antibody is a multivalent antibody. Multivalent antibodies can be internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies provided herein can be multivalent antibodies (e.g., tetravalent antibodies) other than those of the IgM class that have three or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. Multivalent antibodies can include a dimerization domain and three or more antigen-binding sites. Preferred dimerization domains include (or consist of) the Fc region or the hinge region. In this scenario, the antibody will include the Fc region and three or more antigen-binding sites on the amino-terminal side of the Fc region. Preferred multivalent antibodies herein include (or consist of) from three to about eight, preferably four, antigen-binding sites. Multivalent antibodies include at least one polypeptide chain (and preferably two polypeptide chains), and the polypeptide chain includes two or more variable domains. For example, the polypeptide chain can include VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 represent an amino acid or a polypeptide, and n is 0 or 1. For example, the polypeptide chain can include VH-CH1-flexible linker-VH-CH1-Fc region chain, or VH-CH1-VH-CH1-Fc region chain. Multivalent antibodies herein preferably further include at least two (preferably four) light chain variable domain polypeptides. Multivalent antibodies herein can include, for example, from about two to about eight light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein include the light chain variable domain and optionally further include the CL domain.

[0148] In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, antibodies that include heavy chain variable domains (V H ) and light chain variable domains (V L ) (V H V Lunits having polyepitope specificity), two or more V L domains and V H domains (each V H V L unit binds to a different epitope), antibodies having two or more single variable domains (each single variable domain binds to a different epitope), full-length antibodies, antibody fragments, such as Fab, Fv, dsFv, scFv, diabody, bispecific diabody, triabody, trifunctional antibody, covalently or non-covalently linked antibody fragments. In some embodiments, the multispecific antibody comprises a VH domain and a second VH domain that each bind to a different epitope. In some embodiments, the antibody has polyepitope specificity and has the ability to specifically bind to two or more different epitopes, for example, on the same target or different targets. In some embodiments, the antibody is monospecific, for example, an antibody that binds to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0149] (vi) Other antibody modifications For example, in order to enhance the antigen - dependent cell - mediated cytotoxicity (ADCC) and / or complement - dependent cytotoxicity (CDC) of an antibody, it may be desirable to modify the antibodies provided herein with respect to effector function. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or in addition, cysteine residues can be introduced into the Fc region to allow for the formation of inter - chain disulfide bonds in this region. The homodimeric antibodies thus produced can have improved internalization ability and / or increased complement - mediated cell killing and antibody - dependent cell - mediated cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191 - 1195 (1992) and Shopes, B.J., Immunol. 148:2918 - 2922 (1992). Further, homodimeric antibodies with enhanced antitumor activity can be prepared using the heterobifunctional cross - linking agents described in Wolff et al., Cancer Research 53:2560 - 2565 (1993). Alternatively, antibodies can be created that have a dual Fc region and thereby can have enhanced complement - mediated lysis and ADCC capabilities. See Stevenson et al., Anti - Cancer Drug Design 3:219 - 230 (1989).

[0150] To extend the serum half - life of an antibody, changes can be made to the amino acids of the antibody as described in U.S. Patent Application Publication No. 2006 / 0067930, which is hereby incorporated by reference in its entirety.

[0151] (B) Polypeptide Variants and Modifications Modifications of the amino acid sequences of polypeptides (e.g., antigen - binding proteins) containing the antibodies described herein can be used in the methods for purifying the polypeptides (e.g., antigen - binding proteins) described herein.

[0152] (i) Variant Polypeptides "Polypeptide variant" means a polypeptide (e.g., an antigen-binding protein) as defined herein that has at least about 80% amino acid sequence identity with the full-length native sequence of a polypeptide, a polypeptide sequence lacking a signal peptide, or the extracellular domain of a polypeptide with or without a signal peptide, preferably an active polypeptide. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the full-length native amino acid sequence. Usually, a TAT polypeptide variant has at least about 80% amino acid sequence identity, or any of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity with the full-length native sequence polypeptide sequence, a polypeptide sequence lacking a signal peptide, or the extracellular domain of a polypeptide with or without a signal peptide. Optionally, the variant polypeptide has one or fewer conservative amino acid substitutions compared to the native polypeptide sequence, or any of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or fewer conservative amino acid substitutions compared to the native polypeptide sequence.

[0153] Variant polypeptides may be truncated at the N-terminus or C-terminus or may lack internal residues, for example, compared to the full-length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with deletions, insertions, and truncations can be prepared by any of several conventional techniques. The desired variant polypeptide may be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding the desired variant polypeptide by polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the nucleic acid fragment are used in the 5' and 3' primers of the PCR. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptides disclosed herein.

[0154] Amino acid insertions include amino and / or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies fused to an antibody or cytotoxic polypeptide having an N-terminal methionyl residue. Other insertion variants of antibody molecules include N-terminal or C-terminal fusions of antibodies to enzymes or polypeptides that increase the serum half-life of the antibody.

[0155] For example, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide (e.g., an antigen-binding protein). Amino acid sequence variants of a polypeptide are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the polypeptide. Any combination of deletions, insertions, and substitutions is made so long as the final construct has the desired characteristics. Furthermore, amino acid changes may also modify post-translational processes of the polypeptide (e.g., an antibody), such as changes in the number or position of glycosylation sites.

[0156] A guide in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity is found by comparing the sequence of the polypeptide with the sequences of known polypeptide molecules of the same species and minimizing the number of amino acid sequence changes made in regions of high homology.

[0157] A particularly useful method for identifying specific residues or regions of a polypeptide (e.g., an antigen-binding protein) that are preferred positions for mutagenesis-induced generation is what is termed "alanine scanning mutagenesis" as described by Cunningham and Wells, Science 244:1081-1085 (1989). Here, the target residue or group of residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu), and replaced by a neutral or negatively charged amino acid (most preferably, alanine or polyalanine), which affects the interaction between the antigen and the amino acid. Those amino acid positions that show functional sensitivity to the substitution are then refined by introducing additional variants or other variants at the site of substitution, or for the site of substitution. Thus, the site at which the amino acid sequence variant is introduced is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0158] Other types of variants are amino acid substitution variants. These variants have at least one amino acid residue within the antibody molecule that has been replaced by a different residue. The most interesting sites in substitution mutagenesis include the hypervariable regions, although FR modifications are also conceivable. Conservative substitutions are shown in Table 1 below under the heading "Exemplary Substitutions". If such a substitution results in a change in biological activity, it is represented as a "substitution" in Table 1, or more substantial changes can be introduced as further described below in relation to amino acid classes, and the product can be screened.

Table 1

[0159] Substantial modification of the biological properties of a polypeptide is achieved by selecting substitutions that differ significantly in their effect on the maintenance of (a) the structure of the polypeptide backbone in the substituted region as, for example, a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, Biochemistry second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp (D), Glu (E) (4) Basic: Lys (K), Arg (R), His (H)

[0160] Alternatively, naturally occurring residues can be grouped into classes based on common side chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will involve the exchange of one member of one of these classes for another.

[0161] Any cysteine residues not involved in maintaining the proper conformation of the antibody may also generally be substituted with serine in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the polypeptide to improve its stability (particularly if the antibody is an antibody fragment such as an Fv fragment).

[0162] Particularly preferred types of substituted variants involve substitution of one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody). Generally, the resulting variants selected for further development have improved biological properties relative to the parent antibody from which they are generated. A convenient way to generate such substituted variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutagenized to generate all possible amino substitutions at each site. The antibody variants thus generated are presented in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it can be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the target. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants is subjected to the screening described herein, and antibodies having excellent properties in one or more relevant assays can be selected for further development.

[0163] Another type of amino acid variant of a polypeptide (e.g., an antigen-binding protein) modifies the original glycosylation pattern of the antibody. The polypeptide can include non-amino acid moieties. For example, the polypeptide can be glycosylated. Such glycosylation can occur naturally during expression of the polypeptide in a host cell or host organism, or can be a planned modification resulting from human intervention. Modification means deletion of one or more carbohydrate moieties found in the polypeptide and / or addition of one or more glycosylation sites not present in the polypeptide.

[0164] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0165] The addition of glycosylation sites to a polypeptide is conveniently achieved by modifying the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). This modification may be done by the addition or substitution of one or more serine or threonine residues to the sequence of the original antibody (in the case of O-linked glycosylation sites).

[0166] Removal of carbohydrate moieties present on a polypeptide can be achieved chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on a polypeptide can be achieved by the use of various endo- and exoglycosidases.

[0167] Other modifications include deamidation of glutaminyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0168] (ii) Chimeric polypeptide The polypeptides described herein (e.g., antigen-binding proteins) can be modified in ways that result in the formation of chimeric molecules that include a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule includes a fusion of a polypeptide and a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of the polypeptide. The presence of a polypeptide in such an epitope-tagged form can be detected using an antibody to the tag polypeptide. The provision of an epitope tag also enables the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0169] In alternative embodiments, the chimeric molecule can include a fusion of a polypeptide and an immunoglobulin or a specific region of an immunoglobulin. A chimeric molecule in a bivalent form is referred to as an "immunoadhesin."

[0170] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide and the effector functions of an immunoglobulin constant domain. Structurally, an immunoadhesin includes a fusion of an amino acid sequence having a desired binding specificity other than the antigen recognition and binding site of an antibody (i.e., "heterologous") and an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 subtype, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0171] The Ig fusion preferably involves substitution of a polypeptide in a soluble form (membrane-spanning domain deleted or inactivated) instead of at least one variable region within the Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3 of the IgG1 molecule, or the hinge, CH1, CH2, and CH3 regions.

[0172] (iii) polypeptide complex The antigen-binding proteins used in the methods described herein can be conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radiolabeled complex).

[0173] Chemotherapeutic agents useful in the production of such complexes can be used. Further, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene. A variety of radionuclides are available for the production of radiolabeled polypeptides. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186Examples of Re include. The complex of a polypeptide and a cytotoxic agent is prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, as described in Vitetta et al., Science 238:1098 (1987), lysine immunotoxins can be prepared. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the complexation of radioactive nucleotides to polypeptides.

[0174] Complexes of antigen-binding proteins with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothecenes, and CC1065, and derivatives of these toxins having toxin activity are also contemplated herein.

[0175] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from Maytenus serrata, a shrub from East Africa. Subsequently, certain microorganisms have also been found to produce maytansinoids such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also contemplated. There are many linking groups known in the art for making polypeptide-maytansinoid conjugates, such as those disclosed in, for example, U.S. Patent No. 5,208,020. Linking groups include disulfide groups, thioether groups, acid-labile groups, photocleavable groups, peptidase-labile groups, or esterase-labile groups as disclosed in the above-mentioned patents, with disulfide and thioether groups being preferred.

[0176] Depending on the type of bond, the linker can bind to the maytansinoid molecule at various positions. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0177] Another conjugate of interest comprises an antigen-binding protein conjugated to one or more calicheamicin molecules. The antibiotic calicheamicin family has the ability to cause double-strand DNA breakage at subpicomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, for example, U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG, and θ1 Iis included, but is not limited thereto. Another anti-tumor drug that can be conjugated to the antibody is QFA, an anti-folate agent. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents through polypeptide (e.g., antibody)-mediated internalization greatly enhances their cytotoxic effects.

[0178] Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozocin, vincristine, and 5-fluorouracil, a family of agents collectively known as the LL-E33288 complex, and esperamicin.

[0179] In some embodiments, the antigen-binding protein may be a complex between a polypeptide and a compound having nuclease activity (e.g., ribonuclease, or a DNA endonuclease, such as deoxyribonuclease, DNase).

[0180] In yet another embodiment, the antigen-binding protein may be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, in which case the polypeptide-receptor complex is administered to the patient, and then the unbound complex is removed from the bloodstream using a scavenger agent, and thereafter a "ligand" (such as avidin) conjugated to a cytotoxic agent (e.g., a radioactive nucleotide) is administered.

[0181] In some embodiments, the antigen-binding protein may be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) to an active anti-cancer agent. Enzyme components of the immunocomplex include any enzyme that can act on the prodrug in such a way as to convert the prodrug to a more active cytotoxic form.

[0182] Enzymes that are useful include, but are not limited to, alkaline phosphatase useful for converting a phosphate-containing prodrug to a free drug; arylsulfatase useful for converting a sulfate-containing prodrug to a free drug; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anticancer drug, 5-fluorouracil; proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsin (e.g., cathepsin B and L) useful for converting a peptide-containing prodrug to a free drug; D-alanyl carboxypeptidase useful for converting a prodrug containing a D-amino acid substituent; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting a glycosylated prodrug to a free drug; β-lactamase useful for converting a drug derivatized with a β-lactam to a free drug; and penicillin amidase such as penicillin V amidase or penicillin G amidase useful for converting a drug derivatized with a phenoxyacetyl group or a phenylacetyl group, respectively, at the amine nitrogen to a free drug. Alternatively, an antibody having enzyme activity, also known in the art as an "abzyme," can be used to convert a prodrug to a free active drug.

[0183] (iv) Others Another type of covalent modification of an antigen-binding protein involves conjugating the antigen-binding protein to one of various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. Also, the antigen-binding protein may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively) prepared by, for example, coacervation techniques or interfacial polymerization, or may be within a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or within a microemulsion. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Ed., (1990).

[0184] The antigen-binding protein for use in the methods described herein may include modifications that weaken the binding of the VL domain to protein L. In some embodiments, the modification includes one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is κ2VL. In some embodiments, the modification includes an S12P substitution in the VL domain.

[0185] Obtaining the polypeptide for use in the method The antigen-binding proteins used in the purification methods described herein can be obtained using methods well known in the art, including recombinant methods. The following sections provide guidance regarding these methods.

[0186] (A) Polynucleotide As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA.

[0187] Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues that are thought to carry the polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides can be readily obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0188] For example, a polynucleotide can encode an entire immunoglobulin molecular chain, such as a light or heavy chain. A complete heavy chain includes not only the heavy chain variable region (V H ), but typically three constant domains, namely C H 1, C H 2, and C H 3, as well as the heavy chain constant region (C H ) including the "hinge" region. In some situations, the presence of the constant region is desirable.

[0189] Other antigen-binding proteins that can be encoded by a polynucleotide include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fv, scFv, Fab', and F(ab')2, and "minibodies". Minibodies typically include (C H 1 and C K or C LIt is a bivalent antibody fragment from which the domain has been excised. Minibodies are smaller than conventional antibodies, so they should achieve better tissue penetration in clinical / diagnostic applications, but are bivalent and should retain a higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context dictates otherwise, the term "antibody" as used herein includes not only all antibody molecules, but also antigen-binding antibody fragments of the types described above. Preferably, each framework region present in the encoded polypeptide contains at least one amino acid substitution relative to the corresponding human receptor framework. Thus, for example, the framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the receptor framework region.

[0190] Pharmaceutical composition In some embodiments, the disclosure provides a pharmaceutical composition comprising an antigen-binding protein obtained from the purification process described herein (e.g., Protein L chromatography using a cosmotrope as described herein). In some embodiments, the pharmaceutical composition is a purified composition. In some embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0191] The pharmaceutical composition can be prepared for storage in the form of a lyophilized formulation or an aqueous solution by mixing an antigen-binding protein having the desired purity with a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), optionally.

[0192] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations used. In many cases, the physiologically acceptable carrier is an aqueous pH buffer solution.

[0193] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0194] Kit In some aspects of the present invention, a kit for use in the method for purifying an antigen-binding protein described herein. In some embodiments, the kit comprises one or more of a protein L chromatography material, an equilibration buffer, a wash buffer, an elution buffer, and a cosmotrope. In some embodiments, the kit further provides instructions for its use. A container may hold the formulation, and a label on the container or a label associated with the container may indicate instructions for use. The manufactured product may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, culture supplies, reagents for detecting reporter molecules, and an accompanying document with instructions for use. The kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, culture supplies, reagents for detecting reporter molecules, and an accompanying document with instructions for use.

[0195] Exemplary embodiments The following exemplary embodiments are provided herein. 1. A method for purifying an antigen-binding protein comprising a VL domain, comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a cosmotrope; c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer and comprising. 2. A method for improving the protein L purification of an antigen-binding protein comprising a VL domain, comprising: a) binding the antigen-binding protein to a protein L chromatography material; b) washing the protein L chromatography material with a wash buffer comprising an equilibration buffer and a cosmotrope; c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer A method comprising 3. The method according to embodiment 2, wherein the yield and / or purity of the antigen-binding protein is improved as compared to protein L chromatography in which the washing buffer does not contain a cosmotrope. 4. The method according to any one of embodiments 1 to 3, wherein the equilibration buffer contains Tris, MES, MOPS, or EDTA. 5. The method according to any one of embodiments 1 to 4, wherein the equilibration buffer contains Tris and NaCl. 6. The method according to embodiment 4 or embodiment 5, wherein the Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. 7. The NaCl is 6. The method according to embodiment 5 or embodiment 6, wherein the NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. 8. The method according to any one of embodiments 1 to 7, wherein the equilibration buffer has a pH of about 4 to about 10. 9. The method according to any one of embodiments 1 to 8, wherein the equilibration buffer has a pH of about 6.5 to about 8.5, or about 7.7. 10. The method according to any one of embodiments 1 to 9, wherein the equilibration buffer has a pH of about 7 to about 8. 11. The method according to any one of embodiments 1 to 10, wherein the washing buffer has a pH of about 4 to about 10. 12. The method according to any one of embodiments 1 to 11, wherein the washing buffer has a pH of about 4 to about 10, about 4 to about 9.5, about 4 to about 9, about 4 to about 8.5, about 4 to about 8, about 4 to about 7.5, about 5 to about 10, about 5 to about 9.5, about 5 to about 9, about 5 to about 8.5, about 5 to about 8, about 5 to about 7.5, about 6 to about 10, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, about 6 to about 8, or about 6 to about 7.5. 13. The method according to any one of embodiments 1 to 12, wherein the washing buffer has a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. 14. The washing buffer has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, and is the method according to any one of Embodiments 1 to 12. 15. The antigen-binding protein is loaded onto the protein L chromatography material as a host cell culture supernatant in step a), and is the method according to any one of Embodiments 1 to 14. 16. The antigen-binding protein is loaded onto the protein L chromatography material as a purified polypeptide solution in step a), and is the method according to any one of Embodiments 1 to 14. 17. The antigen-binding protein is loaded onto the protein L chromatography material as a mixture from a previous purification step in step a), and is the method according to any one of Embodiments 1 to 14. 18. The antigen-binding protein is prepared in an equilibration buffer before being loaded onto the protein L chromatography material in step a), and is the method according to any one of Embodiments 1 to 14. 19. The antigen-binding protein is prepared in an equilibration buffer and a cosmotrope before being loaded onto the protein L chromatography material in step a), and is the method according to any one of Embodiments 1 to 14 and 18. 20. The cosmotrope is a phosphate or sulfate, and is the method according to any one of Embodiments 1 to 19. 21. The cosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate, and is the method according to any one of Embodiments 1 to 20. 22. The concentration of the cosmotrope in the equilibration buffer or the washing buffer is about 100 mM to about 800 mM, and is the method according to any one of Embodiments 1 to 21. 23. The concentration of the cosmotrope in the equilibration buffer and the washing buffer is about 100 mM to about 800 mM, and is the method according to any one of Embodiments 1 to 21. 24. The method according to any one of embodiments 1 to 23, wherein the concentration of the cosmotrope in the equilibration buffer or the washing buffer is from about 120 mM to about 600 mM. 25. The method according to any one of embodiments 1 to 24, wherein the concentration of the cosmotrope in the equilibration buffer or the washing buffer is about 120 mM, about 240 mM, about 360 mM, about 480 mM, or about 600 mM. 26. The method according to any one of embodiments 1 to 21, wherein the concentration of the cosmotrope in the equilibration buffer and / or the washing buffer is from about 100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 100 mM to about 600 mM, from about 100 mM to about 500 mM, from about 200 mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mM to about 600 mM, from about 200 mM to about 500 mM, from about 300 mM to about 800 mM, from about 300 mM to about 700 mM, from about 300 mM to about 600 mM, or from about 300 mM to about 500 mM. 27. The method according to any one of embodiments 1 to 21, wherein the concentration of the cosmotrope in the equilibration buffer and / or the washing buffer is about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, or 800 mM. 28. The method according to any one of embodiments 1 to 27, wherein the cosmotrope is sodium sulfate. 29. The method according to any one of embodiments 1 to 27, wherein the cosmotrope is potassium phosphate. 30. The method according to any one of embodiments 1 to 27, wherein the cosmotrope is ammonium sulfate. 31. The method according to any one of embodiments 1 to 30, wherein the elution buffer has a lower pH than the equilibration buffer. 32. The elution buffer has a pH of from about 2.0 to about 4.0, or from about 2.5 to about 3.0, according to the method of any one of Embodiments 1 to 31. 33. The elution buffer has a pH of about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3.0, about 3.05, about 3.1, about 3.15, about 3.2, about 3.25, or about 3.3, according to the method of any one of Embodiments 1 to 32. 34. The elution buffer has a pH of about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, or about 2.95, according to the method of any one of Embodiments 1 to 32. 35. The elution buffer has a higher pH than the equilibration buffer, according to the method of any one of Embodiments 1 to 30. 36. The elution buffer has a pH of from about 10.0 to about 12.0, according to the method of Embodiment 35. 37. The elution buffer contains acetic acid, according to the method of any one of Embodiments 1 to 36. 38. The elution buffer contains from about 50 mM acetic acid to about 250 mM acetic acid, according to the method of Embodiment 37. 39. The elution buffer contains about 150 mM acetic acid at a pH of about 2.8, according to the method of Embodiment 37 or Embodiment 38. 40. The elution buffer contains sodium acetate, citrate, or glycine, according to the method of any one of Embodiments 1 to 36. 41. The antigen-binding protein does not bind well to Protein L, according to the method of any one of Embodiments 1 to 40. 42. The antigen-binding protein does not bind well to Protein L as compared to a reference antigen-binding protein that is monoclonal antibody G6-31, according to the method of any one of Embodiments 1 to 41. 43. The antigen-binding protein binds to Protein L more weakly than a reference antigen-binding protein that is monoclonal antibody G6-31, according to the method of any one of Embodiments 1 to 41. 44. The method according to any one of Embodiments 1 to 43, wherein the antigen-binding protein is an antibody, an immunoadhesin, or a fragment thereof. 45. The method according to any one of Embodiments 1 to 44, wherein the antigen-binding protein is a monoclonal antibody. 46. The method according to any one of Embodiments 1 to 45, wherein the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody. 47. The method according to any one of Embodiments 44 to 46, wherein the antibody is an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. 48. The method according to Embodiment 47, wherein the antibody fragment is Fab. 49. The method according to Embodiment 48, wherein the Fab lacks a hydrophobic patch or includes a binding site for binding to protein L having a weak hydrophobic patch. 50. The method according to Embodiment 49, wherein the binding site for binding to protein L lacks a hydrophobic patch. 51. The method according to Embodiment 49, wherein the binding site for binding to protein L has a weak hydrophobic patch. 52. The method according to any one of Embodiments 49 to 51, wherein the binding site for binding to protein L has a weak hydrophobic patch as compared with the binding site for binding to protein L of a reference antigen-binding protein which is monoclonal antibody G6-31. 53. The method according to any one of Embodiments 48 to 52, wherein the binding site for binding to protein L includes a hydrophilic patch. 54. The method according to any one of Embodiments 1 to 53, wherein the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for protein L has a value higher than the value of the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein which is monoclonal antibody G6-31 for protein L. 55. The binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L is at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the binding affinity (EC50) and / or dissociation constant of the reference antigen-binding protein, which is the monoclonal antibody G6-31 for Protein L, according to the method of embodiment 54. 56. The method according to any one of embodiments 44 to 55, wherein the antibody does not contain an Fc domain. 57. The method according to any one of embodiments 44 to 46, wherein the antibody is a multispecific antibody. 58. The method according to embodiment 57, wherein the multispecific antibody comprises the VL domain and a second VL domain, and the VL domain is a kappa (κ) VL. 59. The method according to embodiment 58, wherein the VL domain is κ2VL. 60. The method according to any one of embodiments 57 to 59, wherein the VL domain does not bind Protein L very well. 61. The method according to any one of embodiments 58 to 60, wherein the VL domain does not bind Protein L very well and the second VL domain does not bind Protein L. 62. The method according to any one of embodiments 58 to 61, wherein the second VL domain is a lambda (λ) VL. 63. The method according to any one of embodiments 1 to 56, wherein the VL domain is a λ subtype VL or a κVL. 64. The method according to embodiment 63, wherein the VL domain does not bind Protein L very well. 65. The method according to any one of embodiments 60 to 64, wherein the VL domain does not bind Protein L very well as compared to the binding of the VL domain of the reference antigen-binding protein, which is the monoclonal antibody G6-31, to Protein L. 66. The method according to any one of embodiments 63 to 65, wherein the VL domain is κVL and comprises a modification that weakens the binding of the VL domain to Protein L. 67. The method according to embodiment 66, wherein the modification comprises one or more amino acid substitutions in the VL domain. 68. The method according to any one of embodiments 63 to 67, wherein the VL domain is κ2VL. 69. The method according to any one of embodiments 1 to 48 and 56 to 68, wherein the antigen-binding protein lacks a hydrophobic patch or comprises a binding site for binding to Protein L having a weak hydrophobic patch. 70. The method according to embodiment 69, wherein the binding site for binding to Protein L lacks a hydrophobic patch. 71. The method according to embodiment 69, wherein the binding site for binding to Protein L has a weak hydrophobic patch. 72. The method according to any one of embodiments 69 to 71, wherein the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. 73. The method according to any one of embodiments 69 to 71, wherein the binding site for binding to Protein L comprises a hydrophilic patch. 74. The method according to any one of embodiments 1 to 73, wherein the Protein L chromatography material is Pierce™ Protein L chromatography cartridge, Capto™ L chromatography, HiTrap® Protein L chromatography, KanCap™ L chromatography, TOYOPEARL® AF-rProtein L-650F chromatography, or MabSelect™ VL chromatography. 75. A composition comprising an antigen-binding protein purified by a method comprising the method according to any one of embodiments 1 to 74. 76. The composition according to embodiment 75, wherein the composition comprises one or more pharmaceutical excipients. A kit for purifying an antigen-binding protein containing a VL domain, the kit comprising a protein L chromatography material and a cosmotrope. 78. The kit according to embodiment 77, further comprising an equilibration buffer. 79. The kit according to embodiment 78, wherein the equilibration buffer contains Tris, MES, MOPS, or EDTA. 80. The kit according to embodiment 78 or 79, wherein the equilibration buffer contains Tris and NaCl. 81. The kit according to embodiment 79 or 80, wherein the Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. 82. The kit according to embodiment 80 or 81, wherein the NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM. 83. The kit according to any one of embodiments 78 to 82, wherein the equilibration buffer has a pH of about 4 to about 10. 84. The kit according to any one of embodiments 78 to 83, wherein the equilibration buffer has a pH of about 6.5 to about 8.5, or about 7.7. 85. The kit according to any one of embodiments 78 to 84, wherein the equilibration buffer has a pH of about 7 to about 8. 86. The kit according to any one of embodiments 77 to 85, further comprising a washing buffer. 87. The kit according to embodiment 86, wherein the washing buffer has a pH of about 4 to about 10. 88. The kit according to embodiment 86 or 87, wherein the washing buffer has a pH of about 4 to about 10, about 4 to about 9.5, about 4 to about 9, about 4 to about 8.5, about 4 to about 8, about 4 to about 7.5, about 5 to about 10, about 5 to about 9.5, about 5 to about 9, about 5 to about 8.5, about 5 to about 8, about 5 to about 7.5, about 6 to about 10, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, about 6 to about 8, or about 6 to about 7.5. 89. The washing buffer is the kit according to any one of embodiments 86 to 88, having a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. 90. The washing buffer is the kit according to any one of embodiments 86 to 88, having a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. 91. The washing buffer is the kit according to any one of embodiments 86 to 90, comprising the equilibration buffer and the cosmotrope. 92. The equilibration buffer further comprises the cosmotrope, and is the kit according to any one of embodiments 78 to 91. 93. The cosmotrope is phosphate or sulfate, and is the kit according to any one of embodiments 77 to 92. 94. The cosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate, and is the kit according to any one of embodiments 77 to 93. 95. The concentration of the cosmotrope in the equilibration buffer or the washing buffer is about 100 mM to about 800 mM, and is the kit according to any one of embodiments 78 to 94. 96. The concentration of the cosmotrope in the equilibration buffer and the washing buffer is about 100 mM to about 800 mM, and is the kit according to any one of embodiments 78 to 94. 97. The concentration of the cosmotrope in the equilibration buffer or the washing buffer is about 120 mM to about 600 mM, and is the kit according to any one of embodiments 78 to 96. 98. The concentration of the cosmotrope in the equilibration buffer or the washing buffer is about 120 mM, about 240 mM, about 360 mM, about 480 mM, or about 600 mM, and is the kit according to any one of embodiments 78 to 97. 99. The concentration of the cosmotrope in the equilibration buffer and / or the wash buffer is from about 100 mM to about 800 mM, from about 100 mM to about 700 mM, from about 100 mM to about 600 mM, from about 100 mM to about 500 mM, from about 200 mM to about 800 mM, from about 200 mM to about 700 mM, from about 200 mM to about 600 mM, from about 200 mM to about 500 mM, from about 300 mM to about 800 mM, from about 300 mM to about 700 mM, from about 300 mM to about 600 mM, or from about 300 mM to about 500 mM. The kit according to any one of embodiments 77 to 94. 100. The concentration of the cosmotrope in the equilibration buffer and / or the wash buffer is about 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, 525 mM, 550 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, or 800 mM. The kit according to any one of embodiments 77 to 94. 101. The cosmotrope is sodium sulfate. The kit according to any one of embodiments 77 to 100. 102. The cosmotrope is potassium phosphate. The kit according to any one of embodiments 77 to 100. 103. The cosmotrope is ammonium sulfate. The kit according to any one of embodiments 77 to 100. 104. The kit according to any one of embodiments 77 to 103, further comprising an elution buffer. 105. The elution buffer has a lower pH than the equilibration buffer. The kit according to embodiment 104. 106. The elution buffer has a pH of from about 2.0 to about 4.0, or from about 2.5 to about 3.0. The kit according to embodiment 104 or embodiment 105. 107. The elution buffer has a pH of about 2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, about 2.95, about 3.0, about 3.05, about 3.1, about 3.15, about 3.2, about 3.25, or about 3.3, and is the kit according to any one of Embodiments 104 to 106. 108. The elution buffer has a pH of about 2.7, about 2.75, about 2.8, about 2.85, about 2.9, or about 2.95, and is the kit according to any one of Embodiments 104 to 106. 109. The elution buffer has a higher pH than the equilibration buffer, and is the kit according to Embodiment 104. 110. The elution buffer has a pH of about 10.0 to about 12.0, and is the kit according to Embodiment 104 or Embodiment 109. 111. The elution buffer contains acetic acid, and is the kit according to any one of Embodiments 104 to 110. 112. The elution buffer contains about 50 mM acetic acid to about 250 mM acetic acid, and is the kit according to Embodiment 111. 113. The elution buffer contains about 150 mM acetic acid at a pH of about 2.8, and is the kit according to Embodiment 111 or Embodiment 112. 114. The elution buffer contains sodium acetate, citrate, or glycine, and is the kit according to any one of Embodiments 104 to 110. 115. The kit according to any one of Embodiments 77 to 114 for use in the purification of an antibody or an immunoadhesin or a fragment thereof. 116. The antigen-binding protein is a monoclonal antibody, and is the kit according to any one of Embodiments 77 to 115. 117. The antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody, and is the kit according to any one of Embodiments 77 to 116. 118. The antibody is an antibody fragment selected from Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments, and is the kit according to any one of Embodiments 115 to 117. 119. The kit according to embodiment 118, wherein the antibody fragment is Fab. 120. The kit according to embodiment 119, wherein the Fab comprises a binding site for binding to Protein L that lacks a hydrophobic patch or has a weak hydrophobic patch. 121. The kit according to embodiment 120, wherein the binding site for binding to Protein L lacks a hydrophobic patch. 122. The kit according to embodiment 120, wherein the binding site for binding to Protein L has a weak hydrophobic patch. 123. The kit according to embodiment 120 or 122, wherein the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. 124. The kit according to any one of embodiments 120 to 123, wherein the binding site for binding to Protein L comprises a hydrophilic patch. 125. The kit according to any one of embodiments 77 to 124, wherein the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L has a value higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein that is monoclonal antibody G6-31 for Protein L. 126. The kit according to embodiment 125, wherein the binding affinity (EC50) and / or dissociation constant of the antigen-binding protein for Protein L is at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the binding affinity (EC50) and / or dissociation constant of a reference antigen-binding protein that is monoclonal antibody G6-31 for Protein L. 127. The kit according to any one of embodiments 115 to 126, wherein the antibody does not contain an Fc domain. 128. The kit according to any one of embodiments 115 to 127, wherein the antibody is a multispecific antibody. 129. The kit according to embodiment 128, wherein the multispecific antibody comprises a VL domain and a second VL domain, and the VL domain is a kappa (κ) VL. 130. The kit according to embodiment 129, wherein the VL domain is a κ2VL. 131. The kit according to embodiment 129 or 130, wherein the VL domain does not bind well to protein L. 132. The kit according to any one of embodiments 129 to 131, wherein the VL domain does not bind well to protein L, and the second VL domain does not bind to protein L. 133. The kit according to any one of embodiments 129 to 132, wherein the second VL domain is a lambda (λ) VL. 134. The kit according to any one of embodiments 115 to 127, wherein the antibody comprises a VL domain that is a λ subtype VL or a κVL. 135. The kit according to embodiment 134, wherein the VL domain does not bind well to protein L. 136. The kit according to any one of embodiments 129 to 135, wherein the VL domain of the antigen-binding protein does not bind well to protein L as compared to a reference antigen-binding protein that is a monoclonal antibody G6-31. 137. The kit according to any one of embodiments 129 to 136, wherein the VL domain of the antigen-binding protein binds to protein L less strongly than a reference antigen-binding protein that is a monoclonal antibody G6-31. 138. The kit according to any one of embodiments 134 to 137, wherein the VL domain is a κVL and comprises a modification that weakens the binding of the VL domain to protein L. 139. The kit according to embodiment 138, wherein the modification comprises one or more amino acid substitutions in the VL domain. 140. The kit according to any one of embodiments 134 to 139, wherein the VL domain is a κ2VL. 141. The kit according to any one of Embodiments 77 to 140, wherein the antigen-binding protein lacks a hydrophobic patch or comprises a binding site for binding to Protein L having a weak hydrophobic patch. 142. The kit according to Embodiment 141, wherein the binding site for binding to Protein L lacks a hydrophobic patch. 143. The kit according to Embodiment 141, wherein the binding site for binding to Protein L has a weak hydrophobic patch. 144. The kit according to Embodiment 141 or Embodiment 143, wherein the binding site for binding to Protein L has a weak hydrophobic patch as compared to the binding site for binding to Protein L of a reference antigen-binding protein which is monoclonal antibody G6-31. 145. The kit according to any one of Embodiments 141 to 144, wherein the binding site for binding to Protein L comprises a hydrophilic patch.

[0196] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the disclosure of this application. The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described therein.

Examples

[0197] Example 1. Protein L Chromatography of Antibody Fragments Using Cosmotrope When developing a purification process for a pipeline molecule called Fab 1 (Fab antibody fragment) herein, Protein L was used because this Fab molecule lacks the Fc region which is the binding site required for the use of Protein A.

[0198] In the development of Protein L chromatography runs (Figure 1), high UV absorbance was observed during the wash steps after loading and before elution. Further, the UV readings decreased during the step containing potassium phosphate (a non-corrosive buffer salt used to remove ionic impurities such as host cell DNA), but after this wash step ended, the UV baseline increased again. This result suggests protein dropout from the column after the loading step, but the inventors of the present invention discovered that this escape of protein is temporarily interrupted and suppressed by the presence of the chaotrope phosphate.

[0199] To investigate the use of chaotropes to enhance Protein L chromatography, several tests were conducted to purify Fab 1 by Protein L chromatography using wash buffers supplemented with various concentrations of potassium phosphate (a chaotrope), sodium sulfate (a stronger chaotrope), and sodium chloride (a neutral salt with little or no chaotropic properties). A host cell culture fluid (HCCF) containing the Fab 1 antibody fragment was loaded onto a 7 ml Capto L column at a density of 3.74 g / L and a flow rate of 2.23 ml / min. The column was washed with an equilibration buffer (25 mM Tris, 25 mM NaCl, pH 7.7) containing a chaotrope (sodium sulfate or potassium phosphate) or NaCl at concentrations of 600 mM, 480 mM, 360 mM, 240 mM, or 120 mM. An equilibration buffer without a chaotrope or additional NaCl was used as a negative control. The antibody was then eluted from the column using 100 mM acetic acid, pH 2.9. Protein concentration, conductivity, and pH, measured by A280, were monitored throughout the chromatography run. The chromatography run was analyzed for yield and quality using SEC-HPLC (TSK Gel 2000SW column). As shown below, the process using chaotropic salts improved both yield and purity.

[0200] Test 1: Sodium sulfate (strong chaotrope) Six purification runs in which a equilibration buffer was used with five different concentrations of sodium sulfate to evaluate antibody loss from the column. A host cell culture fluid (HCCF) containing the Fab 1 antibody fragment was loaded onto a 7 ml Capto L column at a density of 3.74 g / L and a flow rate of 2.23 ml / min. The column was washed with an equilibration buffer (25 mM Tris, 25 mM NaCl, pH 7.7) containing sodium sulfate (600 mM, 480 mM, 360 mM, 240 mM, 120 mM) or without sulfate (0 mM; as negative control). The antibody was then eluted from the column using 100 mM acetic acid, pH 2.9. Protein concentration, conductivity, and pH, measured by A280, were monitored throughout the chromatography run.

[0201] Examination of the chromatogram (Figure 2) revealed a correlation that the higher the sulfate concentration, the less protein eluted from the column during the wash step and the more protein was recovered during the elution step.

[0202] The pools recovered from the elution step were collected and analyzed by concentration and size exclusion HPLC (Figure 3). As shown in Table 2, the yield and purity were improved when the chaotrope was incorporated into the wash buffer. Without being bound by theory, this result suggests that the chaotrope strengthens the binding of the major species to Protein L. Impurities such as HMWF and LC dimers have multiple binding sites and may bind more strongly to Protein L than the major species, thereby potentially reducing the yield and quality of the major species.

Table 2

[0203] Test 2: Potassium phosphate (chaotrope) In this test, different salts with common cosmotropic properties were evaluated. Six purification runs were performed where the equilibration buffer used five different concentrations of potassium sulfate (600 mM, 480 mM, 360 mM, 240 mM, 120 mM) or no sulfate (0 mM; as a negative control). Other chromatography parameters were the same as those for the runs with sodium sulfate. As shown in Figure 4 and Table 3, similar results were obtained although the effects were not significant in previous studies. In the chromatogram (Figure 4), the UV absorbance signal in the post - load wash was increasingly suppressed by increasing the concentration of potassium phosphate. Although not to the same extent as for equivalent concentrations of sodium sulfate, increasing the concentration of potassium phosphate (Figure 5, Table 3) also improved the yield and purity similarly.

Table 3

[0204] Test 3: Sodium chloride (neutral salt, control) The addition of sodium chloride to the wash buffer had minimal impact on the salvage performance of this step. Comparing the chromatograms (Figure 6), the UV absorbance after washing remained high even with high concentrations of sodium chloride, which was accompanied by only a much more modest improvement in yield and purity over the course of the test (Figure 7, Table 4) compared to equivalent concentrations of cosmotropic salts.

Table 4

[0205] Example 2. Protein L chromatography of bispecific antibodies using cosmotropes Poor binding of the antibody to Protein L was observed in another molecule, namely a bispecific antibody having one light chain against target antigen A (referred to as "anti-A light chain") and one light chain against target antigen B (referred to as "anti-B light chain") called bispecific antibody 1 herein. The anti-B light chain was designed not to bind to Protein L by engineering S12P within the VL region, leaving only the anti-A light chain to function as the "anchor" to Protein L. The anti-A LC has a VK2 kappa backbone, which is a poor binder of Protein L.

[0206] To investigate the use of kosmotropes to enhance Protein L chromatography of bispecific antibody 1, chromatography runs were performed in the absence or presence of kosmotropes. Host cell culture fluid (HCCF) containing bispecific antibody 1 fragment was filtered using 0.22 μm, diluted to 0.345 g / L with equilibration buffer (25 mM Tris, 25 mM NaCl, pH 7.7), and loaded onto a 1 ml HiTrap Protein L column at a flow rate of 0.4 ml / min. The column was washed with equilibration buffer without kosmotropes or equilibration buffer with 600 mM sodium sulfate. Then, the antibody was eluted from the column using 170 mM acetic acid, pH 2.75. Protein concentration, conductivity, and pH measured by A280 were monitored throughout the chromatography run.

[0207] The results of the chromatography run when the wash buffer did not contain a kosmotrope are shown in Figure 8, and the results of the chromatography run when the wash buffer contained a kosmotrope (600 mM sodium sulfate) are shown in Figure 9. In the sulfate-containing experiment, protein breakthrough and wash-off decreased, resulting in greater protein retention and recovery of a larger elution peak when sulfate was included compared to when it was not. Please compare Figure 9 with Figure 8.

[0208] The process yield of the non-sulfate run was calculated to be 10.11%, while in the experiment using sulfate, it was calculated to be 31.49%, which is three times the yield.

Claims

1. A method for purifying antigen-binding proteins containing a VL domain, a) The step of binding the antigen-binding protein to the protein L chromatography material, b) The step of washing the protein L chromatography material with a washing buffer containing an equilibration buffer and a cosmotrope, c) The step of eluting the antigen-binding protein from the protein L chromatography material with elution buffer. Methods that include...

2. A method for improving the purification of protein L of antigen-binding proteins containing a VL domain, a) The step of binding the antigen-binding protein to the protein L chromatography material, b) The step of washing the protein L chromatography material with a washing buffer containing an equilibration buffer and a cosmotrope, c) The step of eluting the antigen-binding protein from the protein L chromatography material with elution buffer. Methods that include...

3. The method according to claim 2, wherein the yield and / or purity of the antigen-binding protein is improved compared to protein L chromatography in which the washing buffer does not contain cosmotrope.

4. The method according to claim 1 or 2, wherein the equilibration buffer comprises Tris, MES, MOPS, or EDTA.

5. The method according to claim 1 or 2, wherein the equilibration buffer comprises Tris and NaCl.

6. The equilibration buffer has a pH of about 7 to about 8, The Tris is present in the equilibration buffer at a concentration of approximately 10 mM to approximately 50 mM, and / or The NaCl is present in the equilibration buffer at a concentration of approximately 10 mM to approximately 50 mM. The method according to claim 5.

7. The method according to claim 1 or 2, wherein the equilibration buffer and / or washing buffer has a pH of about 4 to about 10.

8. The antigen-binding protein in step a) Host cell culture supernatant; Purified polypeptide solution; or, Mixture from the previous purification process; The protein L chromatography material is then loaded as follows: The method according to claim 1 or 2.

9. The method according to claim 1 or 2, wherein the antigen-binding protein is prepared in equilibration buffer and cosmotrope before being loaded onto the protein L chromatography material in step a).

10. The method according to claim 1 or 2, wherein the cosmotrope is a phosphate or sulfate.

11. The method according to claim 1 or 2, wherein the cosmotrope is potassium phosphate, sodium sulfate, or ammonium sulfate.

12. The method according to claim 1 or 2, wherein the concentration of the cosmotrope in the equilibration buffer and / or the washing buffer is about 100 mM to about 800 mM.

13. The method according to claim 1 or 2, wherein the concentration of the cosmotrope in the equilibration buffer and / or the washing buffer is about 300 mM to about 700 mM.

14. The method according to claim 1 or 2, wherein the cosmotrope is sodium sulfate or potassium phosphate.

15. The method according to claim 1 or 2, wherein the elution buffer has a pH of about 2.0 to about 4.

0.

16. The method according to claim 1 or 2, wherein the elution buffer comprises acetic acid, sodium acetate, citrate, or glycine.

17. The method according to claim 1 or 2, wherein the antigen-binding protein binds to protein L more weakly than the reference antigen-binding protein, which is the monoclonal antibody G6-31.

18. The method according to claim 1 or 2, wherein the antigen-binding protein is an antibody, an immunoadhesin, or a fragment thereof.

19. The method according to claim 1 or 2, wherein the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody.

20. The aforementioned antibody: The antibody fragment is selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments, and / or Does not include the Fc domain, The method according to claim 18.

21. The method according to claim 20, wherein the antibody fragment is Fab.

22. The method according to claim 1 or 2, wherein the antigen-binding protein is a multispecific antibody.

23. The method according to claim 1 or 2, wherein the VL domain is κ2VL.

24. The antigen-binding protein includes a binding site for binding to protein L, and the binding site for binding to protein L is: Lacking a hydrophobic patch, or having a weak hydrophobic patch compared to the binding site for protein L of the reference antigen-binding protein, which is the monoclonal antibody G6-31, and / or Including hydrophilic patches, The method according to claim 1 or 2.

25. The method according to claim 1 or 2, wherein the protein L chromatography material is Pierce® Protein L Chromatography Cartridge, Capto® L Chromatography, HiTrap® Protein L Chromatography, KanCap® L Chromatography, TOYOPEARL® AF-rProtein L-650F Chromatography, or MabSelect® VL Chromatography.

26. A composition comprising an antigen-binding protein purified by a method according to claim 1 or 2.

27. A kit for purifying antigen-binding proteins containing a VL domain, comprising protein L chromatography material and a cosmotrope.

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