Use of lyophilic agent to increase yield of affinity chromatography purification step

Abstract

The present disclosure provides methods and kits for purifying antigen binding proteins comprising a VL domain using a protein L chromatography material comprising a lyophilic agent salt in a buffer background.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 353,512, filed on June 17, 2022, entitled “USE OF KOSMOTROPES TO ENHANCEYIELD OF AN AFFINITY CHROMATOGRAPHY PURIFICATION STEP,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to methods for purifying an antigen binding protein comprising a VL region, the method comprising binding the polypeptide to a Protein L chromatography material, washing the chromatography material with a buffer comprising a kosmotropic agent, and eluting the antigen binding protein at low pH. Background Art

[0004] Protein L is a kind of affinity ligand, and it can be used for the chromatographic purification of antibodies and some antibody fragments, and these antibody fragments are such as Fab, scFv and VHH.All three lack Fc domain, and therefore are incompatible with protein A.Protein L has affinity to the VL domain of kappa light chain, and this domain is the identical domain that three complementary determining regions (CDRs) are located, and this may change binding efficiency in the mode specific to molecular sequence.Protein L chromatography resin carries protein L ligand on the surface of bead, and is usually operated under conditions similar to protein A chromatography (balance, load highly impure raw material, washing, then by elution to recover purified protein).What is needed is a kind of method through improved protein L chromatography.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the Invention

[0006] Provided herein is 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, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and c) eluting the antigen binding protein from the protein L chromatography material with an elution buffer.

[0007] Also provided herein is a method for improving 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, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and c) eluting the antigen binding protein from the Protein L chromatography material with an elution buffer.

[0008] In some embodiments, the yield and / or purity of the antigen binding protein is increased compared to Protein L chromatography wherein the wash buffer does not comprise a kosmotropic agent.

[0009] In some embodiments of any such embodiments, the equilibrium buffer comprises Tris, MES, MOPS or EDTA. In some embodiments of any such embodiments, the equilibrium buffer comprises Tris and NaCl. In some embodiments of any such embodiments, Tris is present in the equilibrium buffer at a concentration of about 10mM to about 50mM or about 25mM. In some embodiments of any such embodiments, NaCl is present in the equilibrium buffer at a concentration of about 10mM to about 50mM or about 25mM. In some embodiments of any such embodiments, the equilibrium buffer has a pH of about 4 to about 10. In some embodiments of any such embodiments, the equilibrium buffer has a pH of about 6.5 to about 8.5 or about 7.7. In some embodiments of any such embodiments, the equilibrium buffer has a pH of about 7 to about 8.

[0010] In some embodiments of any such embodiments, the wash buffer has a pH of about 4 to about 10. In some embodiments 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 embodiments 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.

[0011] In some of any such embodiments, the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a host cell culture supernatant.

[0012] In some of any such embodiments, the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a purified polypeptide solution.

[0013] In some of any such embodiments, the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a mixture from a previous purification step.

[0014] In some of any such embodiments, the antigen binding protein is formulated in an equilibration buffer prior to loading onto the Protein L chromatography material in step a).

[0015] In some of any such embodiments, the antigen binding protein is formulated in an equilibration buffer and a kosmotropic agent prior to loading onto the Protein L chromatography material in step a).

[0016] In some embodiments of any such embodiments, the kosmotropic agent is a phosphate or sulfate. In some embodiments of any such embodiments, the kosmotropic agent is potassium phosphate, sodium sulfate, or ammonium sulfate.

[0017] In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM. In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM. In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM. In some embodiments of any such embodiments, the concentration of the lyophile 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 some of any such embodiments, the concentration of the kosmotropic agent in the equilibration buffer and / or wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM. In some embodiments of any such embodiments, the concentration of the kosmotropic agent in the equilibration buffer and / or 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.

[0018] In some embodiments of any such embodiments, the kosmotropic agent is sodium sulfate. In some embodiments of any such embodiments, the kosmotropic agent is potassium phosphate. In some embodiments of any such embodiments, the kosmotropic agent is ammonium sulfate.

[0019] In some embodiments of any such embodiments, the elution buffer has a lower pH than the equilibration buffer. In some embodiments 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 embodiments 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 embodiments 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 embodiments 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.

[0020] In some of any such embodiments, the elution buffer comprises acetic acid. In some of any such embodiments, the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid.

[0021] In some embodiments of any such embodiments, the elution buffer comprises about 150 mM acetic acid at a pH of about 2.8.

[0022] In some of any such embodiments, the elution buffer comprises sodium acetate, citrate, or glycine.

[0023] In some embodiments of any such embodiments, the antigen binding protein binds poorly to Protein L. In some embodiments of any such embodiments, the antigen binding protein binds poorly to Protein L as compared to a reference antigen binding protein that is monoclonal antibody G6-31. In some embodiments of any such embodiments, the antigen binding protein binds weaker to Protein L as compared to a reference antigen binding protein that is monoclonal antibody G6-31.

[0024] In some embodiments of any such embodiments, the antigen-binding protein is an antibody or immunoadhesin or a fragment thereof. In some embodiments of any such embodiments, the antigen-binding protein is a monoclonal antibody. In some embodiments of any such embodiments, the antigen-binding protein is a human antibody, a chimeric antibody, or a humanized antibody. In some embodiments of any such embodiments, the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments of any such embodiments, the antibody fragment is Fab. In some embodiments of any such 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 of any such embodiments, the binding site for binding to Protein L lacks a hydrophobic patch. In some embodiments of any such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch. In some embodiments of any such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch compared to the binding site for binding to Protein L of a reference antigen-binding protein that is the monoclonal antibody G6-31. In some of any such embodiments, the binding site for binding to Protein L comprises a hydrophilic patch.

[0025] In some embodiments of any such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for Protein L has a value that is higher than the value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L. In some embodiments of any such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for Protein L has a value that 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 value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L.

[0026] In some embodiments of any such embodiments, the antibody does not contain an Fc domain.

[0027] In some embodiments of any such embodiments, the antibody is a multispecific antibody. In some embodiments of any such embodiments, the multispecific antibody comprises a VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL. In some embodiments of any such embodiments, the VL domain is a κ2 VL. In some embodiments of any such embodiments, the VL domain binds poorly to Protein L. In some embodiments of any such embodiments, the VL domain binds poorly to Protein L, and the second VL domain does not bind to Protein L. In some embodiments of any such embodiments, the second VL domain is a lambda (λ) VL. In some embodiments of any such embodiments, the VL domain is a λ subtype VL or a κ VL. In some embodiments of any such embodiments, the VL domain binds poorly to Protein L. In some embodiments of any 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, monoclonal antibody G6-31, to Protein L. In some embodiments of any such embodiments, the VL domain is a kappa VL and comprises a modification that weakens binding of the VL domain to protein L. In some embodiments of any such embodiments, the modification comprises one or more amino acid substitutions in the VL domain. In some embodiments of any such embodiments, the VL domain is a kappa 2 VL.

[0028] In some embodiments of any 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 some embodiments of any such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch compared to the binding site for binding to Protein L of a reference antigen binding protein that is monoclonal antibody G6-31. In some embodiments of any such embodiments, the binding site for binding to Protein L comprises a hydrophilic patch.

[0029] In some embodiments of any such embodiments, the Protein L chromatography material is Pierce TM Protein L chromatography box, Capto TM L chromatography, Protein L chromatography, KanCap TM L chromatography, AF-rProtein L-650F chromatography or MabSelect TM VL chromatography.

[0030] Also provided herein is a composition comprising an antigen binding protein purified by a method comprising any of the methods described herein. In some embodiments, the composition comprises one or more pharmaceutical excipients.

[0031] Also provided herein is a kit for purifying an antigen binding protein comprising a VL domain, wherein the kit comprises a protein L chromatography material and a kosmotropic agent.

[0032] In some embodiments, the kit further comprises an equilibrium buffer. In some embodiments, the equilibrium buffer comprises Tris, MES, MOPS or EDTA. In some embodiments of any such embodiments, the equilibrium buffer comprises Tris and NaCl. In some embodiments of any such embodiments, Tris is present in the equilibrium buffer at a concentration of about 10mM to about 50mM or about 25mM. In some embodiments of any such embodiments, NaCl is present in the equilibrium buffer at a concentration of about 10mM to about 50mM or about 25mM.

[0033] In some embodiments of any such embodiments, the equilibration buffer has a pH of about 4 to about 10. In some embodiments of any such embodiments, the equilibration buffer has a pH of about 6.5 to about 8.5, or about 7.7. In some embodiments of any such embodiments, the equilibration buffer has a pH of about 7 to about 8.

[0034] In some embodiments of any such embodiments, the kit further comprises a wash buffer. In some embodiments, the wash buffer has a pH of about 4 to about 10. In some embodiments 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 embodiments 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.

[0035] In some of any such embodiments, the wash buffer comprises an equilibration buffer and a kosmotropic agent. In some of any such embodiments, the equilibration buffer further comprises a kosmotropic agent.

[0036] In some embodiments of any such embodiments, the lyophile is a phosphate or sulfate. In some embodiments of any such embodiments, the lyophile is potassium phosphate, sodium sulfate or ammonium sulfate. In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM. In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM. In some embodiments of any such embodiments, the concentration of the lyophile in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM. In some embodiments of any such embodiments, the concentration of the lyophile 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 some of any such embodiments, the concentration of the kosmotropic agent in the equilibration buffer and / or wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM. In some embodiments of any such embodiments, the concentration of the kosmotropic agent in the equilibration buffer and / or 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.

[0037] In some embodiments of any such embodiments, the kosmotropic agent is sodium sulfate. In some embodiments of any such embodiments, the kosmotropic agent is potassium phosphate. In some embodiments of any such embodiments, the kosmotropic agent is ammonium sulfate.

[0038] In some embodiments of any such embodiments, the kit further comprises an elution buffer. In some embodiments, the elution buffer has a lower pH than the equilibrium buffer. In some embodiments 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 embodiments 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 embodiments 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.

[0039] In some embodiments, the elution buffer has a higher pH than the equilibration buffer. In some embodiments of any such embodiments, the elution buffer has a pH of about 10.0 to about 12.0.

[0040] In some embodiments of any such embodiments, the elution buffer comprises acetic acid. In some embodiments, the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid. In some embodiments of any such embodiments, the elution buffer comprises about 150 mM acetic acid at a pH of about 2.8. In some embodiments of any such embodiments, the elution buffer comprises sodium acetate, citrate, or glycine.

[0041] In some embodiments of any such embodiments, the kit is used to purify antibodies or immunoadhesins or fragments thereof. In some embodiments of any such embodiments, the antigen binding protein is a monoclonal antibody. In some embodiments of any such embodiments, the antigen binding protein is a human antibody, a chimeric antibody, or a humanized antibody. In some embodiments of any such embodiments, the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments of any such embodiments, the antibody fragment is Fab.

[0042] 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 embodiments of any such embodiments, the binding site for binding to Protein L has a weak hydrophobic patch compared to the binding site for binding to Protein L of a reference antigen-binding protein that is monoclonal antibody G6-31. In some embodiments of any such embodiments, the binding site for binding to Protein L comprises a hydrophilic patch.

[0043] In some embodiments of any such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for Protein L has a value that is higher than the value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L. In some embodiments of any such embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for Protein L has a value that 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 value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L.

[0044] In some embodiments of any such embodiments, the antibody does not contain an Fc domain.

[0045] In some embodiments of any such embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody comprises a VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL. In some embodiments of any such embodiments, the VL domain is a κ2 VL. In some embodiments of any such embodiments, the VL domain binds poorly to protein L. In some embodiments of any such embodiments, the VL domain binds poorly to protein L, and the second VL domain does not bind to protein L. In some embodiments of any such embodiments, the second VL domain is a lambda (λ) VL. In some embodiments of any such embodiments, the antibody comprises a VL domain that is a lambda subtype VL or a kappa VL. In some embodiments of any such embodiments, the VL domain binds poorly to protein L. In some embodiments of any such embodiments, the VL domain of the antigen-binding protein binds poorly to protein L compared to a reference antigen-binding protein that is monoclonal antibody G6-31. In some of any such embodiments, the VL domain of the antigen binding protein binds weaker to Protein L than a reference antigen binding protein that is monoclonal antibody G6-31.

[0046] In some embodiments of any such embodiments, the VL domain is a kappa VL and comprises modifications that weaken the binding of the VL domain to protein L. In some embodiments, the modifications comprise one or more amino acid substitutions in the VL domain. In some embodiments of any such embodiments, the VL domain is a kappa 2 VL. In some embodiments of any 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 some embodiments of any such embodiments, the binding site for binding to protein L has a weak hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein that is monoclonal antibody G6-31. In some embodiments of any such embodiments, the binding site for binding to protein L comprises a hydrophilic patch. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Shown is a chromatogram from the purification of Fab 1 (Fab antibody fragment) on Protein L resin. The UV absorbance is suppressed during the phosphate-containing wash phase with high conductivity but recovers after this phase.

[0048] Figure 2Shown are the UV absorbance signal overlaps across six chromatographic runs using wash buffers containing different amounts of sodium sulfate (0mM, 120mM, 240mM, 360mM, 480mM, and 600mM). The protein leaving the column is detected by UV traces (intensity expressed in milli-absorbance units "mAU" on the Y-axis). In each run, the column is equilibrated with a volume = 0 mL (on the X-axis) before injection. Due to the high load of host impurities in the raw material (harvested cell culture fluid), a high UV signal is generated during the loading phase. The loading phase ends at marker "1.A.1" and the wash phase begins, which is performed by marker 1.A.2. The elution phase recovers the purified protein at marker 1.A.3 on the X-axis at a volume of approximately 120 mL.

[0049] Figure 3 Shown in Figure 2 SEC-HPLC analysis of Fab 1 after the chromatographic run shown in. In addition to the Fab, a high molecular weight fragment (HMWF) and a light chain (LC) dimer were also detected.

[0050] Figure 4 Shown is an overlay of UV absorbance signals across six chromatographic runs using wash buffers containing varying amounts of potassium phosphate (0 mM, 120 mM, 240 mM, 360 mM, 480 mM, and 600 mM).

[0051] Figure 5 Shown in Figure 4 SEC-HPLC analysis of Fab 1 after the chromatographic run shown in. In addition to the Fab, a high molecular weight fragment (HMWF) and a light chain (LC) dimer were also detected.

[0052] Figure 6 Shown is an overlay of UV absorbance signals across six chromatographic runs using wash buffers containing different amounts of sodium chloride (0 mM, 120 mM, 240 mM, 360 mM, 480 mM, and 600 mM).

[0053] Figure 7 Shown in Figure 6 SEC-HPLC analysis of Fab 1 after the chromatographic run shown in. In addition to the Fab, a high molecular weight fragment (HMWF) and a light chain (LC) dimer were also detected.

[0054] Figure 8Shown are chromatograms of protein L chromatography using a bispecific antibody with no sulfate in the wash buffer (i.e., a control experiment). The chromatograms show the UV absorbance signal as well as signals for pH and conductivity. The rise in UV early indicates that the protein breaks through during the load phase and is washed away during the wash phase. A modest elution peak is restored around the 34 minute mark.

[0055] Figure 9 Shown is a chromatogram from a protein L chromatography run using a bispecific antibody with 600 mM sulfate present in both the loading material and the wash buffer. The run chromatogram contains UV absorbance signals as well as signals for pH and conductivity. An increase in UV early indicates protein breakthrough during the loading phase and washes away during the wash phase. A modest elution peak is recovered around the 34 minute mark. DETAILED DESCRIPTION

[0056] In some aspects, 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, wherein the wash buffer comprises an equilibration buffer and a lyophile (e.g., sulfate or phosphate), c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some aspects, the present invention provides a method for improving 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, wherein the wash buffer comprises an equilibration buffer and a lyophile (e.g., sulfate or phosphate), 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 increased compared to protein L chromatography in which the wash buffer does not comprise a lyophile.

[0057] Those skilled in the art will also appreciate that, without departing from the scope of the present disclosure, changes may be made to the form and details of the embodiments described herein. In addition, although various advantages, aspects, and objects have been described with reference to various embodiments, the scope of the present disclosure should not be limited by these advantages, aspects, and objects.

[0058] definition

[0059] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition will control.

[0060] As used herein, a "kosmotrope," less frequently spelled "cosmotrope," is a salt that promotes hydrophobic binding due to the way it structures water molecules in solution. Two examples of kosmotropic anions used in chromatography are sulfate and phosphate. Another kosmotrope, ammonium sulfate, has a strong affinity for lyophiles and is often used to salt out (precipitate) proteins from solution. The opposite of kosmotropes are chaotropic salts, which disrupt hydrophobic binding; the Hofmeister sequence is an ordering of different salts along the kosmotropic and chaotropic spectra based on their properties.

[0061] The terms "polypeptide" or "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically include antibodies.

[0062] By "purified" polypeptide (e.g., antibody or immunoadhesin) is meant a polypeptide that has been increased in purity such that it exists in a purer form than it exists in its own natural environment and / or when initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0063] 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 in the two chains typically contain three highly variable loops, called complementarity determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2, and LC-CDR3, and 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 by 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 inserted between flanking extensions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. 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. Several major antibody classes are 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 semisynthetic 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 antibodies are linked to immunostimulatory proteins such as interleukins. In some embodiments, the antibodies are linked to proteins that facilitate entry across the blood-brain barrier.

[0064] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen bound by a parent antibody or parent antibody fragment (e.g., a parent scFv). In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted to framework regions from one or more different human antibodies.

[0065] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain 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 these antibodies, as long as they exhibit the biological activity of the present invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0066] As used herein, the term "multispecific antibody" refers to a monoclonal antibody that has binding specificity for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some aspects, a multispecific antibody has two binding specificities (bispecific antibodies). In some aspects, a multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0067] 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 (ie, synthetic) sequences.

[0068] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. The folding of these two domains produces six hypervariable loops (3 loops each from the heavy chain and light chain), which contribute amino acid residues to achieve antigen binding and give the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site.

[0069] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired antigen-binding structure. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0070] The term "diabody" refers to small antibody fragments made by constructing scFv fragments (see preceding paragraph), wherein there is typically a short linker (such as about 5 to about 10 residues) between the VH and VL domains, thereby achieving interchain pairing of the V domains rather than intrachain pairing, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are more fully described in, for example: EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0071] The non-human (e.g., rodent) antibody of the "humanized" form is a chimeric antibody comprising a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the hypervariable region (HVR) of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, which has the desired antibody specificity, affinity, and function. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may include residues not found in the receptor antibody or the donor antibody. These modifications are intended to further improve antibody performance. In general, the humanized antibody will include substantially all of at least one variable domain, and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody also optionally includes at least a portion of an immunoglobulin constant region (Fc), which is typically 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).

[0072] As used herein, the term "isolated" refers to a molecule that has been separated from at least some components that are typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some components of the cell that produces it. In the case where the polypeptide is secreted by the cell after expression, physical separation of the supernatant containing the polypeptide from the cell that produces it is considered to be "isolated" polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide that is typically found in nature (such as, for example, genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or when it is separated from at least some components of the cell that produces it, for example, in the case of an RNA polynucleotide. Therefore, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated."

[0073] "Contaminants" refer to materials that are different from the desired polypeptide product. Contaminants include, but are not limited to, host cell material, such as CHO host cell protein (CHOP); leached protein A; nucleic acids; desired polypeptide variants (e.g., basic or acidic variants of the desired polypeptide product), fragments, aggregates, or derivatives (e.g., high molecular weight species (HMWS) or very high molecular weight species (vHMWS) of the desired polypeptide); another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc. In some examples, contaminants can be host cell proteins (HCPs) from, for example, but not limited to, bacterial cells (such as E. coli cells), insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells.

[0074] The term "hydrophobic patch" refers to a portion of an antigen binding protein (e.g., an antibody or immunoadhesin or a fragment thereof, such as a Fab) that contains one or more hydrophobic amino acid residues, which renders it generally hydrophobic. The term "hydrophilic patch" refers to a portion of an antigen binding protein (e.g., an antibody or immunoadhesin or a fragment thereof, such as a Fab) that contains one or more hydrophilic amino acid residues, which renders it generally hydrophilic.

[0075] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents are intended to be equivalent in meaning and to be open-ended, i.e., one or more items following any of these words are not meant to be an exhaustive list of the one or more items, or to be limited to only the listed one or more items. For example, an article that "comprising" components (assemblies) A, B, and C can consist of (i.e., contain only) components (assemblies) A, B, and C, or can contain not only components (assemblies) A, B, and C, but also one or more other components (assemblies). Thus, it is intended and understood that "comprising" and its similar forms and its grammatical equivalents include disclosure of embodiments that "consist essentially of" or "consist of."

[0076] Where a range of values ​​is provided, it is understood that each intervening value (based on the tenth of the unit of the lower limit unless the context clearly dictates otherwise) between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the disclosure, subject to any specifically excluded limit in that stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0077] Reference herein to "about" a value or parameter includes (and describes) variations involving that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0078] As used herein, including the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0079] Use of lyophiles in protein L chromatography

[0080] In some aspects, 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, wherein the wash buffer comprises an equilibration buffer and a lyophile (e.g., sulfate or phosphate), c) eluting the antigen-binding protein from the protein L chromatography material with an elution buffer. In some aspects, the present invention provides a method for improving 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, wherein the wash buffer comprises an equilibration buffer and a lyophile (e.g., sulfate or phosphate), 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 increased compared to protein L chromatography in which the wash buffer does not comprise a lyophile.

[0081] In some embodiments, the equilibration buffer can be any suitable equilibration buffer.

[0082] In some embodiments of the invention, the equilibration buffer comprises Tris (i.e., tris(hydroxymethyl)aminomethane or tromethamine). In some embodiments of the invention, the equilibration buffer comprises NaCl. In some embodiments of the invention, the equilibration buffer comprises Tris and NaCl.

[0083] In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, the concentration of Tris present in the equilibration buffer is between about 10 mM and 100 mM, 15 mM and 100 mM, 20 mM and 100 mM, 25 mM and 100 mM, 30 mM and 100 mM, 40 mM and 100 mM, 50 mM and 100 mM, 75 mM and 100 mM, 10 mM and 75 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, 10 mM and 50 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, Any between 5mM and 50mM, 20mM and 50mM, 25mM and 50mM, 30mM and 50mM, 40mM and 50mM, 10mM and 40mM, 15mM and 40mM, 20mM and 40mM, 25mM and 40mM, 30mM and 40mM, 10mM and 30mM, 15mM and 30mM, 20mM and 30mM, 25mM and 30mM, 10mM and 25mM, 15mM and 25mM, 20mM and 25mM, 10mM and 20mM, 15mM and 20mM, or 10mM and 15mM. 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.

[0084] In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, the concentration of Tris present in the equilibration buffer is between about 10 mM and 100 mM, 15 mM and 100 mM, 20 mM and 100 mM, 25 mM and 100 mM, 30 mM and 100 mM, 40 mM and 100 mM, 50 mM and 100 mM, 75 mM and 100 mM, 10 mM and 75 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, 10 mM and 50 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, Any between 5mM and 50mM, 20mM and 50mM, 25mM and 50mM, 30mM and 50mM, 40mM and 50mM, 10mM and 40mM, 15mM and 40mM, 20mM and 40mM, 25mM and 40mM, 30mM and 40mM, 10mM and 30mM, 15mM and 30mM, 20mM and 30mM, 25mM and 30mM, 10mM and 25mM, 15mM and 25mM, 20mM and 25mM, 10mM and 20mM, 15mM and 20mM, or 10mM and 15mM. 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.

[0085] In some embodiments of the present invention, the equilibration buffer comprises MES (2-(N-morpholino)ethanesulfonic acid). In some embodiments, the equilibration buffer comprises MES and NaCl. In some embodiments, the equilibration buffer comprises MOPS (3-(N-morpholino)propanesulfonic acid). In some embodiments, the equilibration buffer comprises MOPS and NaCl. In some embodiments, the equilibration buffer comprises EDTA (ethylenediaminetetraacetic acid). In some embodiments, the equilibration buffer comprises a carbonate buffer.

[0086] In some embodiments, NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 100 mM. In some embodiments, the concentration of NaCl present in the equilibration buffer is between about 10 mM and 100 mM, 15 mM and 100 mM, 20 mM and 100 mM, 25 mM and 100 mM, 30 mM and 100 mM, 40 mM and 100 mM, 50 mM and 100 mM, 75 mM and 100 mM, 10 mM and 75 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, 10 mM and 50 mM, 15 mM and 75 mM, 20 mM and 75 mM, 25 mM and 75 mM, 30 mM and 75 mM, 40 mM and 75 mM, 50 mM and 75 mM, 75 mM and 75 mM, Any between 5mM and 50mM, 20mM and 50mM, 25mM and 50mM, 30mM and 50mM, 40mM and 50mM, 10mM and 40mM, 15mM and 40mM, 20mM and 40mM, 25mM and 40mM, 30mM and 40mM, 10mM and 30mM, 15mM and 30mM, 20mM and 30mM, 25mM and 30mM, 10mM and 25mM, 15mM and 25mM, 20mM and 25mM, 10mM and 20mM, 15mM and 20mM, or 10mM and 15mM. 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.

[0087] In certain embodiments, Tris is present in the equilibrium buffer at a concentration of about 10mM to about 100mM, and NaCl is present in the equilibrium buffer at a concentration of about 10mM to about 100mM. In certain embodiments, Tris is present in the equilibrium buffer at a concentration of about 10mM to about 50mM, and NaCl is present in the equilibrium buffer at a concentration of about 10mM to about 50mM. In certain embodiments, Tris is present in the equilibrium buffer at a concentration of about 20mM to about 30mM, and NaCl is present in the equilibrium buffer at a concentration of about 20mM to about 30mM. In certain embodiments, Tris is present in the equilibrium buffer at a concentration of about 25mM, and NaCl is present in the equilibrium buffer at a concentration of about 25mM.

[0088] In some embodiments, the equilibration buffer has a pH of about 4.0 to about 10.0. In some embodiments, the equilibration buffer has a pH of about 6.0 to about 9.0. In some embodiments, the equilibration buffer has a pH of about 7.0 to about 8.0. In some embodiments, the equilibration buffer has a pH between about 6.0 and 9.0, 6.5 and 8.5, or 7.0 and 8.0. In some embodiments, the equilibration buffer has a pH 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 about 10mM to about 100mM, NaCl is present in the equilibration buffer at a concentration of about 10mM to about 100mM, and the pH of the equilibration buffer is about 6.0 to about 9.0. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, and the pH of the equilibration buffer is about 6.5 to about 8.5. In some embodiments, Tris is present in the equilibration buffer at a concentration of about 20 mM to about 30 mM, NaCl is present in the equilibration buffer at a concentration of about 20 mM to about 30 mM, and the pH of the equilibration buffer is 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.

[0089] In some embodiments, the wash buffer has a pH of about 4 to about 10. In some 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 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.

[0090] In some embodiments, the antigen-binding protein is loaded onto the protein L chromatographic material in step a) with a host cell culture supernatant (HCCS). In some embodiments, the antigen-binding protein is loaded onto the protein L chromatographic material in step a) with a purified polypeptide solution. In some embodiments, the antigen-binding protein is loaded onto the protein L chromatographic material in step a) with a mixture from a previous purification step. The previous purification step can be any step in the purification process or workflow that occurs before being loaded onto the protein L chromatographic material in step a). In some embodiments, the antigen-binding protein is formulated in an equilibration buffer before being loaded onto the protein L chromatographic material in step a). In some embodiments, a previous lyophile is added to the HCCS comprising the antigen-binding protein. In some embodiments, the antigen-binding protein is formulated in an equilibration buffer and a lyophile before being loaded onto the protein L chromatographic material in step a). In some embodiments, the lyophile is a phosphate or sulfate. In some embodiments, the lyophile is potassium phosphate, sodium sulfate, or ammonium sulfate. In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to loading onto the Protein L chromatography material in step a) is any concentration of at least about 100 mM that does not cause the antigen binding protein to precipitate out of solution. In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to 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 kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to loading onto the protein L chromatography material in step a) is between about 100 mM and 1000 mM, 120 mM and 1000 mM, 240 mM and 1000 mM, 360 mM and 1000 mM, 480 mM and 1000 mM, 600 mM and 1000 mM, 800 mM and 1000 mM, 100 mM and 800 mM, 120 mM and 800 mM, 240 mM and 800 mM, 360 mM and 800 mM, 480 mM and 1000 mM, or between any of: 100mM and 240mM, 120mM and 240mM, 360mM and 600mM, 480mM and 600mM, 100mM and 480mM, 120mM and 480mM, 240mM and 480mM, 360mM and 480mM, 100mM and 360mM, 120mM and 360mM, 240mM and 360mM, 100mM and 240mM, 120mM and 240mM, or 100mM and 120mM.In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer or HCCS prior to loading onto the protein L chromatography material in step a) exceeds 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 kosmotropic agent in the equilibration buffer is about 100 mM to about 800 mM. In some embodiments, the concentration of the kosmotropic agent in the equilibration buffer is about 120 mM to about 600 mM.

[0091] In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer and / or wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM. In some embodiments, the concentration of the lyophile (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the equilibration buffer and / or 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 lyophile is sodium sulfate. In some embodiments, the lyophile is potassium phosphate. In some embodiments, the lyophile is ammonium sulfate.

[0092] In certain embodiments, the washing buffer of step b) comprises a lyophile. In certain embodiments, the lyophile in the washing buffer is phosphate or sulfate. In certain embodiments, the lyophile in the washing buffer is potassium phosphate, sodium sulfate or ammonium sulfate. In certain embodiments, the washing buffer of step b) comprises an equilibrium buffer and a lyophile. In certain embodiments, the concentration of the lyophile (e.g., potassium phosphate, sodium sulfate or ammonium sulfate) in the washing buffer of step b) is any concentration of at least about 100mM, which does not cause the antigen-binding proteins to precipitate from the solution. In certain embodiments, the concentration of the lyophile (e.g., potassium phosphate, sodium sulfate or ammonium sulfate) in the washing buffer of step b) is about 100mM to about 1000mM. In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) is between about 100 mM and 1000 mM, 120 mM and 1000 mM, 240 mM and 1000 mM, 360 mM and 1000 mM, 480 mM and 1000 mM, 600 mM and 1000 mM, 800 mM and 1000 mM, 100 mM and 800 mM, 120 mM and 800 mM, 240 mM and 800 mM, 360 mM and 1000 mM, 480 mM and 1000 mM, 600 mM and 1000 mM, 800 mM and 1000 mM, 100 mM and 800 mM, Between any of 0mM and 800mM, 100mM and 600mM, 120mM and 600mM, 240mM and 600mM, 360mM and 600mM, 480mM and 600mM, 100mM and 480mM, 120mM and 480mM, 240mM and 480mM, 360mM and 480mM, 100mM and 360mM, 120mM and 360mM, 240mM and 360mM, 100mM and 240mM, 120mM and 240mM, or 100mM and 120mM. In some embodiments, the concentration of the kosmotropic agent (e.g., potassium phosphate, sodium sulfate, or ammonium sulfate) in the wash buffer of step b) exceeds 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 kosmotropic agent in the wash buffer is about 100 mM to about 800 mM. In some embodiments, the concentration of the kosmotropic agent in the wash buffer is about 120 mM to about 600 mM.

[0093] In some embodiments, the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM. In some embodiments, the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 120 mM to about 600 mM.

[0094] 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 about 2.0 to about 5.0. In some embodiments, the elution buffer has a pH between about 2.0 and 5.0, 2.5 and 5.0, 3.0 and 5.0, 3.5 and 5.0, 4.0 and 5.0, 2.0 and 4.0, 2.5 and 4.0, 3.0 and 4.0, 3.5 and 4.0, 2.0 and 3.5, 2.5 and 3.5, 3.0 and 3.5, 2.0 and 3.0, 2.5 and 3.0, or 2.0 and 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.

[0095] 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 the low pH of an elution buffer having a pH between, for example, about 2.0 and about 5.0. In some embodiments, the elution buffer has a pH of about 10.0 or higher. Therefore, in some embodiments, the elution buffer has a pH between about 10.0 and 12.0, 10.5 and 12.0, 11.0 and 12.0, 10.0 and 11.5, or 10.0 and 11.0. In some embodiments, the elution buffer has a pH of about any one of 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 between about 10.0 and 12.0.

[0096] In some embodiments, the elution buffer comprises acetic acid. In some embodiments, the elution buffer comprises sodium acetate, citrate, or glycine. In some embodiments, the elution buffer comprises 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, or between any of: 1 M, 175 mM and 200 mM, 50 mM and 175 mM, 75 mM and 175 mM, 100 mM and 175 mM, 125 mM and 175 mM, 150 mM and 175 mM, 50 mM and 150 mM, 75 mM and 150 mM, 100 mM and 150 mM, 125 mM and 150 mM, 50 mM and 125 mM, 75 mM and 125 mM, 100 mM and 125 mM, 50 mM and 100 mM, 75 mM and 100 mM, or 50 mM and 75 mM. In some embodiments, the elution buffer comprises acetic acid, sodium acetate, citrate, or glycine at a concentration of about any of 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.

[0097] In some embodiments, the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid. In some embodiments, the elution buffer comprises acetic acid at a concentration between about 50 mM and 250 mM, 75 mM and 250 mM, 100 mM and 250 mM, 125 mM and 250 mM, 150 mM and 250 mM, 175 mM and 250 mM, 200 mM and 250 mM, 50 mM and 200 mM, 75 mM and 200 mM, 100 mM and 200 mM, 125 mM and 200 mM, 150 mM and 200 mM, 175 mM and 200 mM , 50mM and 175mM, 75mM and 175mM, 100mM and 175mM, 125mM and 175mM, 150mM and 175mM, 50mM and 150mM, 75mM and 150mM, 100mM and 150mM, 125mM and 150mM, 50mM and 125mM, 75mM and 125mM, 100mM and 125mM, 50mM and 100mM, 75mM and 100mM, or any of 50mM and 75mM. In some embodiments, the elution buffer comprises acetic acid at a concentration of about any of 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 comprises about 150 mM acetic acid. In some embodiments, the elution buffer comprises about 150 mM acetic acid at a pH of about 2.8.

[0098] In some embodiments, the antigen binding protein purified by protein L chromatography is an antibody or immunoadhesin or fragment thereof. In some embodiments, the VL domain of the antibody or immunoadhesin or fragment thereof is a lambda subtype VL or a kappa 2 VL. In some embodiments, the VL domain binds poorly to protein L. In some embodiments, the VL domain is a kappa VL and comprises modifications that weaken binding of the VL domain to protein L. In some embodiments, the modifications comprise one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is a kappa 2 VL.

[0099] In some embodiments, the antigen binding protein binds poorly to Protein L. For example, in some embodiments, and without being bound by theory, the antigen binding protein used in the methods described herein binds poorly to Protein L, and while the kosmotropic agent enhances the binding of the antigen binding protein to Protein L, impurities (such as HMWF and LC-dimer) may bind more tightly to Protein L than to the antigen binding protein, which may result in poorer yield and quality of the antigen binding protein in the absence of the kosmotropic agent. In some embodiments, the antigen binding protein that binds poorly to Protein L comprises a VL domain of the κ2 subtype.

[0100] In some embodiments, the antigen binding protein (e.g., antibody or immunoadhesin) binds poorly to Protein L compared to a reference antigen binding protein. In some embodiments, the reference antigen binding protein is of the same or similar type of antigen binding protein as the antigen binding protein, e.g., an antibody. In some embodiments, the antigen binding protein (e.g., antibody or immunoadhesin) binds poorly to Protein L compared to a reference antigen binding protein, which is monoclonal antibody G6-31. In some embodiments, the antigen binding protein (e.g., antibody or immunoadhesin) binds weaker to Protein L than the reference antigen binding protein. In some embodiments, the antigen binding protein (e.g., antibody or immunoadhesin) binds weaker to Protein L than the reference antigen binding protein, which is monoclonal antibody G6-31. The G6-31 (also referred to as "G6.31") monoclonal antibody is an antibody fragment that is an anti-VEGF Fab and has been widely published. See, e.g., Korsisaari et al., PNAS, 2007, 104(25):10625-10630. In some embodiments, G6-31 Fab can serve as a reference antigen-binding protein for determining whether another antigen-binding protein exhibits weaker or poorer binding to Protein L, for example, compared to a G6-31 Fab that is considered to exhibit normal binding to Protein L. In some embodiments, antigen-binding proteins that exhibit weaker or poorer binding to Protein L may be particularly suitable for use in the methods described herein involving the use of kosmotropes to improve the yield and purity of an antigen-binding protein of interest, which may benefit from enhanced binding of the antigen-binding protein to Protein L.

[0101] 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.

[0102] In some embodiments, the antibody fragment is a Fab. 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 embodiments, the binding site for binding to protein L comprises a hydrophilic patch.

[0103] In some embodiments, the antigen-binding protein that binds poorly to protein L 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 has a weaker hydrophobic patch than the binding site for binding to protein L of a reference antigen-binding protein that is monoclonal antibody G6-31.

[0104] In some embodiments, the antigen binding protein that binds poorly to Protein L comprises a hydrophilic patch.

[0105] In some embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein (e.g., antibody or immunoadhesin) for Protein L has a value that is higher than the value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L. In some embodiments, the binding affinity (EC50) and / or dissociation constant of the antigen binding protein (e.g., antibody or immunoadhesin) for Protein L has a value that 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 value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L.

[0106] In some embodiments, the antigen binding protein, e.g., Fab, comprises a VL domain, and the binding site is contained within the VL domain.

[0107] 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 wherein the VL domain is a kappa (κ) VL. In some embodiments, the VL domain is a κ2 VL. In some embodiments, the VL domain binds poorly to protein L. In some embodiments, the VL domain binds poorly to protein L, and the second VL domain does not bind to protein L. In some embodiments, an antigen-binding protein (e.g., an antibody or immunoadhesin) comprises a VL domain and binds poorly to protein L compared to a reference antigen-binding protein comprising a VL domain. In some embodiments, the reference antigen-binding protein belongs to an antigen-binding protein of the same or similar type as the antigen-binding protein, for example, an antibody. In some embodiments, an antigen-binding protein (e.g., an antibody or immunoadhesin) comprises a VL domain and binds poorly to protein L compared to a reference antigen-binding protein comprising a VL domain, which is the monoclonal antibody G6-31. In some embodiments, the antigen binding protein (e.g., an antibody or immunoadhesin) comprises a VL domain and binds weaker to Protein L than a reference antigen binding protein comprising a VL domain. In some embodiments, the antigen binding protein (e.g., an antibody or immunoadhesin) comprises a VL domain and binds weaker to Protein L than a reference antigen binding protein comprising a VL domain, which is monoclonal antibody G6-31. In some embodiments, the VL domain binds poorly to Protein L as compared to the binding of the VL domain of a reference antigen binding protein, which is monoclonal antibody G6-31.

[0108] In some embodiments, the second VL domain is a lambda (λ) VL. In some embodiments, the VL domain is a kappa VL and comprises a modification that weakens the binding of the VL domain to protein L. In some embodiments, the modification comprises one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is a kappa 2 VL.

[0109] In some embodiments, the VL domain is a lambda subtype VL or a kappa 2 VL.

[0110] Protein L chromatography

[0111] Protein L is a cell wall protein of the bacterium Peptostreptococcus magna ( et al. (1988) J. Immunol. 140: 1194-1197), this cell wall protein binds to the variable region of the kappa light chain without interfering with the antigen binding site of the 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 limited to VLs of the kappa 1, kappa 3, and kappa 4 subtypes, but does not bind to VLs of the kappa 2 subtype or binds weakly to VLs of the kappa 2 subtype. Commercially available protein L chromatography materials include, but are not limited to, Pierce TM Protein L chromatography box, Capto TM L chromatography, Protein L chromatography, KanCap TM L chromatography, AF-rProtein L-650F chromatography or MabSelect TM VL chromatography.

[0112] Additional steps

[0113] In some aspects, the present invention provides additional steps involved in or associated with the purification of the antigen-binding proteins as described herein. Additional steps involved in or associated with purification, and methods for performing these steps, are known. See, for example, Liu et al., mAbs, 2, 2010, which is hereby incorporated by reference in its entirety.

[0114] In certain embodiments, the purification of antigen-binding proteins further comprises a sample treatment step, such as a sample preparation step. In certain embodiments, the purification of antigen-binding proteins further comprises a clarification step, such as clarification HCCF. In certain embodiments, the purification of antigen-binding proteins further comprises a host cell and host cell debris removal step, such as removing host cells and host cell debris from a sample and / or composition obtained from a purification platform. In certain embodiments, the purification of antigen-binding proteins further comprises a centrifugation step. In certain embodiments, the purification of antigen-binding proteins further comprises a sterile filtration step. In certain embodiments, the purification of antigen-binding proteins further comprises a tangential flow microfiltration step. In certain embodiments, the purification of antigen-binding proteins further comprises a flocculation / precipitation step.

[0115] Antigen binding proteins

[0116] In some aspects, protein L chromatography using a wash buffer solution comprising a kosmotropic agent can be used to purify and concentrate antigen-binding proteins comprising VL domains from antigen-binding protein preparations. In some embodiments, antigen-binding protein preparations are derived from host cell preparations. In some embodiments, host cell preparations are host cell culture fluid (HCCF). In some embodiments, antigen-binding protein preparations comprise a portion of host cell culture fluid. In some embodiments, antigen-binding protein preparations are derived from host cell culture fluid. In some embodiments, antigen-binding protein preparations comprise host cells. In some embodiments, antigen-binding protein preparations comprise components of host cells, such as host cell fragments. In some embodiments, host cells are bacterial cells. In some embodiments, host cells are insect cells. In some embodiments, host cells are mammalian cells. In some embodiments, host cells are Chinese hamster ovary (CHO) cells. In some embodiments, host cells are Escherichia coli cells.

[0117] Antigen binding proteins

[0118] Antigen-binding proteins to be purified by protein L chromatography (including using a lyophile in a washing buffer using methods described herein) are typically produced using recombinant technology. Methods for producing recombinant proteins are described in, for example, U.S. Patent Nos. 5,534,615 and 4,816,567, which are specifically incorporated herein by reference. In certain embodiments, the protein of interest is produced in CHO cells (see, for example, WO 94 / 11026). In certain embodiments, the polypeptide of interest is produced in Escherichia coli cells. See, for example, U.S. Patent No. 5,648,237; U.S. Patent No. 5,789,199, and U.S. Patent No. 5,840,523, which describe translation initiation regions (TIR) ​​and signal sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describe the expression of polypeptide fragments in Escherichia coli. When using recombinant techniques, the polypeptide can be produced intracellularly in the periplasmic space or directly secreted into the culture medium.

[0119] Antigen binding proteins can be recovered from culture medium or from host cell lysates. Cells used to express antigen binding proteins can be destroyed by various physical or chemical means (such as freeze-thaw cycles, sonication, mechanical disruption or cell lysis agents). If the antigen binding protein is produced intracellularly, as a first step, the microparticle debris of the host cells or lysed fragments is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a method for separating polypeptides secreted into the periplasmic space of E. coli. In short, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. In the case where the antigen binding protein is secreted into the culture medium, a commercially available polypeptide concentration filter, such as or Millipore Ultrafiltration units concentrate supernatants from such expression systems.Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[0120] In some embodiments, the antigen binding protein has been purified or partially purified prior to analysis by the methods of the invention, for example, in the eluent from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography.

[0121] Examples of antigen binding proteins to be purified by Protein L chromatography (including but not limited to the use of a kosmotropic agent in a wash buffer using the methods described herein) include but are not limited to immunoglobulins, immunoadhesins, antibodies, and immunoconjugates.

[0122] (A) Antibodies

[0123] In some embodiments of any of the methods described herein, the antigen binding protein is an antibody or an immunoadhesin.

[0124] (i) Monoclonal antibodies

[0125] In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that arise during the production of the monoclonal antibodies, which variants are typically present in small amounts. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete or polyclonal antibodies.

[0126] For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0127] In the hybridoma method, mice or other suitable host animals (such as hamsters) are immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with tumor 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)).

[0128] The hybridoma cells thus 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, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of cells lacking HGPRT.

[0129] In certain embodiments, myeloma cells are those that are effectively fused, support selected antibody-producing cells to stably produce antibodies at high levels, and are sensitive to culture media such as HAT culture media. Wherein, in certain embodiments, myeloma cell lines are mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors that can be obtained from Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells that can be obtained from American Type Culture Collection, Rockville, Maryland USA. Human myeloma 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 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0130] Culture medium in which hybridoma cells are grown is analyzed for production of monoclonal antibodies directed against the antigen.In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0131] The binding affinity of a monoclonal antibody can be determined by, for example, Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0132] After hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0133] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0134] The DNA encoding the monoclonal antibody is easy to separate and sequence using conventional procedures (for example, by using oligonucleotide probes that can bind specifically to the genes encoding the heavy and light chains of murine antibodies). In some embodiments, these hybridoma cells serve as the source of such DNA. Once separated, the DNA can be placed in an expression vector, which is then transfected into host cells such as Escherichia coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin polypeptides, to synthesize monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding antibodies include Skerra et al., Curr. Opinion in Immunol. 5: 256-262 (1993) and Plückthun, Immunol. Revs., 130: 151-188 (1992).

[0135] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the technology 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 the use of phage libraries to separate murine antibodies and human antibodies, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies (Marks et al., Bio / Technology 10: 779-783 (1992)) by chain shuffling and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21: 2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0136] The DNA also can be modified, for example, by substituting coding sequences for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently linking to the immunoglobulin coding sequence the coding sequence for all or part of a non-immunoglobulin polypeptide.

[0137] Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site specific for an antigen and a second antigen-binding site specific for a different antigen.

[0138] 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.

[0139] (ii) Humanized antibodies

[0140] In certain embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In certain embodiments, the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization can be carried out substantially according to the method of Winter and his colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Therefore, such a "humanized" antibody is a chimeric antibody (U.S. Patent No. 4,816,567), in which substantially less than a complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0141] The selection of light chain and heavy chain for the human variable domains of the preparation of humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domains of rodent antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to rodents is then used as the human framework region (FR) of humanized antibodies (Sims et al., J.Immunol.151:2296 (1993); Chothia et al., J.Mol.Biol., 196:901 (1987)). Another method uses the specific framework region of the consensus sequence of all human antibodies from a specific subgroup of light chain or heavy chain variable region. The same framework can be used for several different humanized antibodies (Carter et al., Proc.Natl.AcadSci.USA 89:4285 (1992); Presta et al., J.Immunol.151:2623 (1993)).

[0142] It is further important that the antibody is humanized while retaining high affinity to antigen and other favorable biological properties. To achieve this purpose, in some embodiments of the method, humanized antibodies are prepared by analyzing the process of the parental sequence and various conceptual humanized products using the three-dimensional model of the parental and humanized sequences. The three-dimensional immunoglobulin model is conventionally obtainable and is familiar to those skilled in the art. A computer program is provided, which illustrates and shows the possible three-dimensional conformational structure of a selected candidate immunoglobulin sequence. Inspection of these displays allows analysis of the possible effects of residues in the function of the candidate immunoglobulin sequence, that is, analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, it is possible to select and combine FR residues from receptor and imported sequence, thereby obtaining required antibody characteristics, such as an increase in affinity for the target antigen. Usually, hypervariable region residues directly and to the greatest extent participate in influencing antigen binding.

[0143] (iii) Human antibodies

[0144] In some embodiments, the antibodies are human antibodies. As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that are able to produce a complete library of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, the antibody heavy chain joining region (J-J) has been described in chimeric and germline mutant mice. H Homozygous deletion of the ) gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in these germline mutant mice will result in the production of human antibodies after 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. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

[0145] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from a complete set of immunoglobulin variable (V) domain genes from unimmunized donors. According to this technology, the antibody V domain genes are cloned in frame into the major or minor coat polypeptide genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particles contain single-stranded DNA copies of the phage genome, the selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that exhibit these properties. Therefore, phage mimics some characteristics of B cells. Phage display can be carried out in various forms; its relevant review is seen, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene fragments from several sources can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes from the spleens of immunized mice. Libraries of V genes from unimmunized human donors can be constructed essentially following the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993), and antibodies against a diverse array of antigens, including self-antigens, can be isolated. See also, U.S. Patent Nos. 5,565,332 and 5,573,905.

[0146] Human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0147] (iv) Antibody fragments

[0148] In certain embodiments, the antibody is an antibody fragment. In certain embodiments, the antibody fragment is Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment. In certain embodiments, the antibody fragment is Fab. In certain embodiments, the antibody is an antibody fragment comprising an Fc receptor binding domain. Various technologies for producing antibody fragments have been developed. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, 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 by recombinant host cells. For example, antibody fragments can be isolated from the above-mentioned antibody phage library.

[0149] 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 comprising an Fc receptor binding domain. In some embodiments, the antibody fragment is an antigen-binding fragment comprising an Fcγ receptor binding domain.

[0150] (v) Bispecific antibodies

[0151] In certain embodiments, the antibody is a bispecific antibody. Bispecific antibodies are antibodies with binding specificity to at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes. Alternatively, the bispecific antibody binding arm can be combined with the arm of the triggering molecule on the leukocyte, such as T cell receptor molecules (e.g., CD2 or CD3) or IgG Fc receptors (FcγR) such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), thereby focusing the cellular defense mechanism on the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0152] Methods for preparing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random diversity of immunoglobulin heavy and light chains, these hybridomas (quadrama) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecules usually completed by affinity chromatography steps is quite cumbersome, and the product yield is low. Similar processes are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0153] According to different methods, the antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused with the immunoglobulin constant domain sequence. In certain embodiments, the fusion uses the immunoglobulin heavy chain constant domain, comprising at least a portion of the hinge, CH2 and CH3 regions. In certain embodiments, the first heavy chain constant region (CH1) comprising the light chain binding required site is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and (if necessary) immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. When the unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in regulating the mutual ratios of the three polypeptide fragments in an embodiment. However, when at least two polypeptide chains are expressed in equal ratios, resulting in high yields or when the ratio has no particular significance, it is possible to insert the coding sequence of two or all three polypeptide chains in one expression vector.

[0154] In some embodiments of the method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with 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. It has been found that this asymmetric structure promotes the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, because the presence of immunoglobulin light chains in only one half of the bispecific molecule provides a simple separation method. The method is disclosed in WO 94 / 04690. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0155] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises the C-terminal regions of the antibody constant domains.H At least a portion of the 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). Compensatory "cavities" with the same or similar size as the large side chains are created on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism that can increase the yield of heterodimers rather than other unwanted end products (such as homodimers).

[0156] Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Patent number 4,676,980), and for treating HIV infection (WO 91 / 00360, WO 92 / 200373 and EP 0308936). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are described in U.S. Patent number 4,676,980 and many cross-linking techniques.

[0157] The technology of producing bispecific antibodies from antibody fragments has also been described in the literature. For example, chemical connection can be used to prepare bispecific antibodies. Brennan et al., Science, 229:81 (1985) described a procedure in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfides. The generated Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted into a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as reagents for selective enzyme immobilization.

[0158] Various techniques for preparing and isolating bispecific antibody fragments directly 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). By gene fusion, leucine zipper peptides from Fos protein and Jun protein are connected to the Fab' portion of two different antibodies. Antibody homodimers are reduced to form monomers at the hinge region and then oxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragment comprises a light chain variable domain (V) connected to the light chain variable domain (V) by a linker. H ) of the heavy chain variable domain (V L ), the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to mate with the complementary V L and V H The domains pair to form two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

[0159] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0160] (v) Multivalent Antibodies

[0161] In certain embodiments, the antibody is a multivalent antibody. By expressing cells of the antigen bound by the antibody, the multivalent antibody can be internalized (and / or catabolized) faster than a bivalent antibody. The antibody provided herein can be a multivalent antibody (which is not an IgM antibody) (e.g., a tetravalent antibody) with three or more antigen binding sites, which can be easily produced by recombinantly expressing nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody can include a dimerization domain and three or more antigen binding sites. The preferred dimerization domain includes an Fc region or a hinge region (or is composed of it). In this case, the antibody will include an Fc region and three or more antigen binding sites located at the amino-terminal end of the Fc region. The preferred multivalent antibody herein includes three to about eight, but preferably four antigen binding sites (or is composed of it). The multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and, optionally, further comprise a CL domain.

[0162] In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V H ) and light chain variable domain (V L ) of the antibody, wherein V H V L Units have multiple epitope specificity, with two or more V L and V H Domain antibody, each V H V LUnits bind to different epitopes, antibodies having two or more single variable domains, each single variable domain binding to a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. In some embodiments, multispecific antibodies comprise a VH domain and a second VH domain, each of which binds to a different epitope. In some embodiments, the antibody has polyepitopic specificity; for example, the ability to specifically bind to two or more different epitopes on the same or different targets. In some embodiments, the antibody is monospecific; for example, an antibody that binds 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.

[0163] (vi) Other antibody modifications

[0164] It may be desirable to modify the antibodies provided herein in terms of effector function, for example to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues can be introduced in the Fc region, thereby allowing the formation of interchain disulfide bonds in the region. The resulting homodimeric antibodies may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, BJ, Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents, as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, which can result in enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0165] To increase the serum half-life of an antibody, amino acid changes may be made in the antibody as described in US 2006 / 0067930, which is incorporated herein by reference in its entirety.

[0166] (B) Polypeptide Variants and Modifications

[0167] Amino acid sequence modifications of the polypeptides (eg, antigen binding proteins), including antibodies, described herein can be used in methods for purifying the polypeptides (eg, antigen binding proteins) described herein.

[0168] (i) Variant polypeptides

[0169] " Polypeptide variant " refers to a polypeptide (for example, antigen binding proteins) as defined herein, full-length native sequence, lack of a signal peptide polypeptide sequence, polypeptide (with or without a signal peptide) extracellular domain with at least about 80% amino acid sequence identity, preferably an active polypeptide. Such polypeptide variants include, for example, polypeptides, wherein one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the full-length natural amino acid sequence. Typically, TAT polypeptide variants and full-length native sequence polypeptide sequences, lack of a signal peptide polypeptide sequence, polypeptide (with or without a signal peptide) extracellular domain will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity. Optionally, compared to the native polypeptide sequence, the variant polypeptide will have no more than one conservative amino acid substitution, or compared to the native polypeptide sequence, including no more than about 2, 3, 4, 5, 6, 7, 8, 9 or 10 any one conservative amino acid substitution.

[0170] For example, compared to the full-length native polypeptide, the variant polypeptide may be truncated at the N-terminus or C-terminus, or may lack internal residues. Some variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncation, deletion and insertion can be prepared by any of many conventional techniques. The desired variant polypeptide can be chemically synthesized. Another suitable technique includes separating and amplifying nucleic acid fragments encoding the desired variant polypeptides by polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the nucleic acid fragments are used at the 5' and 3' primers in PCR. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptide disclosed herein.

[0171] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include fusions to the N-terminus or C-terminus of an antibody to an enzyme or polypeptide that increases the serum half-life of the antibody.

[0172] 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 antibody nucleic acids or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the polypeptide amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct has the desired properties. Amino acid changes may also alter post-translational processes of the polypeptide (e.g., an antibody), such as changing the number or position of glycosylation sites.

[0173] Guidance in determining which amino acid residues may be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the polypeptide sequence to the sequences of homologous, known polypeptide molecules and minimizing the number of amino acid sequence changes in regions of high homology.

[0174] The method that can be used to identify certain residues or regions of polypeptides (such as antigen-binding proteins) (which are preferred positions for mutagenesis) is called "alanine scanning mutagenesis", as described in Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or a group of target residues (for example, charged residues, such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of amino acids with antigens. Then, by introducing further or other variants at or for the substitution site, those amino acid positions that show functional sensitivity to the substitution are refined. Therefore, although the site for introducing amino acid sequence variation is predetermined, it is not necessary to predetermine the nature of the mutation itself. For example, in order to analyze the performance of the mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the desired activity of the expressed antibody variants is screened.

[0175] The variant of another type is amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The most interesting site of substitution mutagenesis includes the hypervariable region, but FR changes are also considered. Conservative substitutions are shown under the "exemplary substitutions" heading in Table 1 below. If this type of substitution causes a change in biological activity, then a more substantial change, named "substitution" in Table 1 or as further described below with reference to the amino acid class, can be introduced and the product screened.

[0176] Table 1.

[0177]

[0178]

[0179] Substantial modification of the biological properties of a polypeptide is accomplished by selecting substitutions that significantly differ in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as 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 their side chain properties (Allehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)):

[0180] (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

[0181] (2) Non-charged polarity: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)

[0182] (3) Acidic: Asp (D), Glu (E)

[0183] (4) Basic: Lys(K), Arg(R), His(H)

[0184] Alternatively, naturally occurring residues can be divided into the following groups based on common side-chain properties:

[0185] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0186] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0187] (3) Acidic: Asp, Glu;

[0188] (4) Basic: His, Lys, Arg;

[0189] (5) Residues that affect chain orientation: Gly, Pro;

[0190] (6) Aromatic: Trp, Tyr, Phe.

[0191] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0192] Any cysteine ​​residues not involved in maintaining the proper conformation of the antibody may also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to a polypeptide to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0193] A particularly preferred type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Typically, the variants selected for further development will have improved biological properties relative to the parent antibody from which they are produced. A convenient way to produce such substitution variants involves affinity maturation using phage phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of the M13 packaged within each particle from filamentous phage particles in a monovalent manner. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened, as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the target. According to the technology set forth herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0194] Another type of amino acid variant of a polypeptide (e.g., an antigen binding protein) alters the original glycosylation pattern of the antibody. The polypeptide may contain non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be an intentional modification caused by human intervention. The alteration refers to the deletion of one or more carbohydrate moieties found in the polypeptide and / or the addition of one or more glycosylation sites not present in the polypeptide.

[0195] Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the 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 enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. 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, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0196] Glycosylation sites are conveniently added to the polypeptide by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such alterations can also be accomplished by adding or substituting one or more serine or threonine residues into the sequence of the original antibody (for O-linked glycosylation sites).

[0197] Removal of carbohydrate moieties present on a polypeptide can be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that are targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.

[0198] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group 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.

[0199] (ii) Chimeric polypeptides

[0200] The polypeptides described herein (e.g., antigen binding proteins) can be modified in a manner to form a chimeric molecule comprising a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises a fusion of a polypeptide and a tag polypeptide, wherein the tag polypeptide provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically placed at the amino or carboxyl terminus of the polypeptide. Antibodies to the tag polypeptide can be used to detect the presence of such epitope-tagged polypeptides. In addition, the provision of the epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0201] In alternative embodiments, the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or specific regions of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

[0202] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector functions of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with a desired binding specificity that is different from the antigen recognition and binding site binding specificity (i.e., "heterologous") of an antibody. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that comprises at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be derived from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0203] Ig fusions preferably include substituting a soluble (transmembrane domain deleted or inactivated) form of a polypeptide at the position of at least one variable region in an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge regions CH2 and CH3, or the hinge regions CH1, CH2, and CH3, of an IgG1 molecule.

[0204] (iii) Polypeptide Conjugates

[0205] Antigen binding proteins for use in the methods described herein can be conjugated to a cytotoxic agent (such as a chemotherapeutic agent), a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0206] Chemotherapeutic agents that can be used to produce such conjugates can be used. In addition, 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, α-sarcin, Aleurites fordii protein, dianthus protein, pokeweed antiviral proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcumin, crotonin, saponaria officinalis inhibitor, gelatin, mitocin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides can be used to produce radioconjugated polypeptides. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186Re. Conjugates of polypeptides and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of iminoesters (such as dimethyl adipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to polypeptides.

[0207] Also contemplated herein are conjugates of antigen binding proteins with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothene, and CC1065, as well as derivatives of these toxins that possess toxin activity.

[0208] Maytansinoids are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine I was first isolated from the East African shrub Maytenus serrata. Subsequently, it was found that certain microorganisms also produced maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also considered. Many linking groups for preparing polypeptide-maytansinoid conjugates are known in the art, including those disclosed in, for example, U.S. Patent number 5,208,020. Linking groups include disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups or esterase-labile groups, and as disclosed in the above-mentioned patents, disulfide and thioether groups are preferred.

[0209] Linking group can be connected with maytansinol molecule at different positions, and this depends on the type of connection.For example, conventional coupling technology can be used to form ester bond by reacting with hydroxyl group.Reaction can be carried out at the C-3 position with hydroxyl, the C-14 position modified by hydroxyl, the C-15 position modified by hydroxyl and the C-20 position with hydroxyl.In a preferred embodiment, connection is formed at the C-3 position of maytansinol or maytansinol analog.

[0210] Another conjugate of interest comprises an antigen binding protein conjugated to one or more calicheamicin molecules. The calicheamicin antibiotic family is capable of producing double-stranded DNA breaks at sub-picomolar 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, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ1 I Another anti-tumor drug that can be conjugated to an antibody is QFA, an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents via polypeptide (e.g., antibody)-mediated internalization greatly enhances their cytotoxic effects.

[0211] 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), as well as esperamicin.

[0212] In some embodiments, the antigen binding protein can be a conjugate between a polypeptide and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0213] In yet another example, the antigen binding protein can be conjugated to a "receptor" (such as streptavidin) for tumor pretargeting, wherein the polypeptide receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionucleotide).

[0214] In some embodiments, the antigen binding protein can be conjugated to a prodrug activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme that can act on a prodrug in this manner to convert it into its more active cytotoxic form.

[0215] Useful enzymes include, but are not limited to, alkaline phosphatase, which can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatase, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminase, which can be used to convert the non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as serrapeptides, thermolysins, subtilisins, carboxypeptidases, and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs; D-alanyl carboxypeptidases, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drugs; β-lactamase, which can be used to convert drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derivatized at their amine nitrogens to have phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs into free active drugs.

[0216] (iv) Others

[0217] Another type of covalent modification of antigen binding proteins comprises linking the antigen binding protein to one of a variety of non-protein polymers, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylene or a copolymer of polyethylene glycol and polypropylene glycol. Antigen binding proteins can also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, AR, ed., (1990).

[0218] The antigen binding proteins used in the methods described herein can comprise modifications that weaken the binding of the VL domain to protein L. In some embodiments, the modifications comprise one or more amino acid substitutions in the VL domain. In some embodiments, the VL domain is a κ2 VL. In some embodiments, the modifications comprise an S12P substitution in the VL domain.

[0219] Obtaining polypeptides for use in these methods

[0220] 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 section provides guidance on these methods.

[0221] (A) Polynucleotide

[0222]

[0014] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA.

[0223] Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues believed to have polypeptide mRNA and express the polypeptide at detectable levels. Thus, polynucleotides encoding polypeptides can be conveniently obtained from cDNA libraries prepared from human tissues. Polypeptide encoding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0224] For example, a polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only the heavy chain variable region (V H ), also including the heavy chain constant region (C H ), the heavy chain constant region generally includes three constant domains: C H 1. C H 2 and C H 3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0225] Other antigen-binding proteins that can be encoded by polynucleotides include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and F(ab')2, and "minibodies". Minibodies are (usually) C-terminal fragments of H 1 and C K or C L In some embodiments, the present invention relates to a bivalent antibody fragment of a polypeptide comprising a small antibody domain. Because small antibodies are smaller than ordinary antibodies, they should achieve better tissue penetration in clinical / diagnostic use, but as bivalent bodies, they should maintain a higher binding affinity than monovalent antibody fragments (such as dAb). Therefore, unless the context otherwise provides, the term "antibody" as used herein not only includes whole antibody molecules, but also includes the antigen-binding antibody fragment of the above type. Each framework region preferably present in the encoded polypeptide will include at least one amino acid replacement relative to the corresponding human receptor framework. Therefore, for example, relative to the receptor framework region, the framework region includes three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid replacements in total.

[0226] Pharmaceutical composition

[0227] In some aspects, the present disclosure provides pharmaceutical compositions comprising an antigen binding protein obtained from a purification process described herein (e.g., protein L chromatography using a kosmotropic agent as described herein). In some embodiments, the pharmaceutical composition is a purified composition. In some embodiments, the pharmaceutical composition is a sterile pharmaceutical composition.

[0228] Pharmaceutical compositions can be prepared by mixing antigen binding proteins having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized preparations or aqueous solutions for storage.

[0229] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are typically pH-buffered aqueous solutions.

[0230] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl 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; m-cresol); low molecular weight (less than about 10 residues) (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0231] Reagent test kit

[0232] In some aspects of the invention, a kit for use in a method for purifying an antigen-binding protein as 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 lyophile. In some embodiments, the kit further provides instructions for its use. The container holds the formulation, and a label on or associated with the container may indicate instructions for use. The article may further include other materials desired from a commercial and user perspective, including other buffers, diluents, culture vessels, reagents for detecting reporter molecules, and a package insert with instructions for use. The kit may further include other materials desired from a commercial and user perspective, including other buffers, diluents, culture vessels, reagents for detecting reporter molecules, and a package insert with instructions for use.

[0233] Exemplary embodiments

[0234] The following exemplary embodiments are provided herein:

[0235] 1. A method for purifying an antigen-binding protein comprising a VL domain, the method comprising:

[0236] a) binding the antigen binding protein to the Protein L chromatography material,

[0237] b) washing the protein L chromatography material with a wash buffer, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and

[0238] c) Eluting the antigen binding protein from the Protein L chromatography material with elution buffer.

[0239] 2. A method for improving the purification of Protein L of an antigen binding protein comprising a VL domain, the method comprising:

[0240] a) binding the antigen binding protein to the Protein L chromatography material,

[0241] b) washing the protein L chromatography material with a wash buffer, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and

[0242] c) Eluting the antigen binding protein from the Protein L chromatography material with elution buffer.

[0243] 3. The method of embodiment 2, wherein the yield and / or purity of the antigen binding protein is increased compared to Protein L chromatography wherein the wash buffer does not contain a kosmotropic agent.

[0244] 4. The method of any one of embodiments 1 to 3, wherein the equilibration buffer comprises Tris, MES, MOPS, or EDTA.

[0245] 5. The method of any one of embodiments 1 to 4, wherein the equilibration buffer comprises Tris and NaCl.

[0246] 6. The method of embodiment 4 or claim 5, wherein Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM.

[0247] 7. The method of embodiment 5 or embodiment 6, wherein NaCl

[0248] is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM.

[0249] 8. The method of any one of embodiments 1 to 7, wherein the equilibration buffer has a pH of about 4 to about 10.

[0250] 9. The method of 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.

[0251] 10. The method of any one of embodiments 1 to 9, wherein the equilibration buffer has a pH of about 7 to about 8.

[0252] 11. The method of any one of embodiments 1 to 10, wherein the wash buffer has a pH of about 4 to about 10.

[0253] 12. The method of any one of embodiments 1-11, wherein 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.

[0254] 13. The method of any one of embodiments 1 to 12, wherein 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.

[0255] 14. The method of any one of embodiments 1 to 12, wherein 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.

[0256] 15. The method of any one of embodiments 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a host cell culture supernatant.

[0257] 16. The method of any one of embodiments 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a purified polypeptide solution.

[0258] 17. The method of any one of embodiments 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a mixture from a previous purification step.

[0259] 18. The method of any one of embodiments 1 to 14, wherein the antigen binding protein is formulated in an equilibration buffer prior to loading onto the Protein L chromatography material in step a).

[0260] 19. The method of any one of embodiments 1 to 14 and 18, wherein the antigen binding protein is formulated in an equilibration buffer and a kosmotropic agent before being loaded onto the Protein L chromatography material in step a).

[0261] 20. The method of any one of embodiments 1 to 19, wherein the kosmotropic agent is a phosphate or sulfate salt.

[0262] 21. The method of any one of embodiments 1 to 20, wherein the kosmotropic agent is potassium phosphate, sodium sulfate, or ammonium sulfate.

[0263] 22. The method of any one of embodiments 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM.

[0264] 23. The method of any one of embodiments 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM.

[0265] 24. The method of any one of embodiments 1 to 23, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM.

[0266] 25. The method of any one of embodiments 1 to 24, wherein the concentration of the kosmotropic agent 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.

[0267] 26. The method of any one of embodiments 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM.

[0268] 27. The method of any one of embodiments 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or 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.

[0269] 28. The method of any one of embodiments 1 to 27, wherein the kosmotropic agent is sodium sulfate.

[0270] 29. The method of any one of embodiments 1 to 27, wherein the kosmotropic agent is potassium phosphate.

[0271] 30. The method of any one of embodiments 1 to 27, wherein the kosmotropic agent is ammonium sulfate.

[0272] 31. The method of any one of embodiments 1 to 30, wherein the elution buffer has a lower pH than the equilibration buffer.

[0273] 32. The method of any one of embodiments 1 to 31, wherein the elution buffer has a pH of about 2.0 to about 4.0, or about 2.5 to about 3.0.

[0274] 33. The method of any one of embodiments 1 to 32, wherein 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.

[0275] 34. The method of any one of embodiments 1 to 32, wherein 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.

[0276] 35. The method of any one of embodiments 1 to 30, wherein the elution buffer has a higher pH than the equilibration buffer.

[0277] 36. The method of embodiment 35, wherein the elution buffer has a pH of about 10.0 to about 12.0.

[0278] 37. The method of any one of embodiments 1 to 36, wherein the elution buffer comprises acetic acid.

[0279] 38. The method of embodiment 37, wherein the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid.

[0280] 39. The method of embodiment 37 or embodiment 38, wherein the elution buffer comprises about 150 mM acetic acid at a pH of about 2.8.

[0281] 40. The method of any one of embodiments 1 to 36, wherein the elution buffer comprises sodium acetate, citrate, or glycine.

[0282] 41. The method of any one of embodiments 1 to 40, wherein the antigen binding protein binds poorly to Protein L.

[0283] 42. The method of any one of embodiments 1 to 41, wherein the antigen binding protein binds poorly to Protein L compared to a reference antigen binding protein that is monoclonal antibody G6-31.

[0284] 43. The method of any one of embodiments 1 to 41, wherein the antigen binding protein binds to Protein L less strongly than a reference antigen binding protein that is monoclonal antibody G6-31.

[0285] 44. The method of any one of embodiments 1 to 43, wherein the antigen binding protein is an antibody or immunoadhesin or a fragment thereof.

[0286] 45. The method of any one of embodiments 1 to 44, wherein the antigen binding protein is a monoclonal antibody.

[0287] 46. ​​The method of any one of embodiments 1 to 45, wherein the antigen binding protein is a human antibody, a chimeric antibody, or a humanized antibody.

[0288] 47. The method of any one of embodiments 44 to 46, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0289] 48. The method of embodiment 47, wherein the antibody fragment is Fab.

[0290] 49. The method of embodiment 48, wherein the Fab comprises a binding site for binding to protein L that lacks a hydrophobic patch or has a weak hydrophobic patch.

[0291] 50. The method of embodiment 49, wherein the binding site for binding to protein L lacks a hydrophobic patch.

[0292] 51. The method of embodiment 49, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

[0293] 52. The method of any one of embodiments 49 to 51, wherein the binding site for binding to protein L has a less hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein that is monoclonal antibody G6-31.

[0294] 53. The method of any one of embodiments 48 to 52, wherein the binding site for binding to Protein L comprises a hydrophilic patch.

[0295] 54. The method of 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 that is greater than the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for Protein L.

[0296] 55. The method of embodiment 54, wherein the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for protein L has a value that 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 value of the binding affinity (EC50) and / or dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for protein L.

[0297] 56. The method of any one of embodiments 44 to 55, wherein the antibody does not contain an Fc domain.

[0298] 57. The method of any one of embodiments 44 to 46, wherein the antibody is a multispecific antibody.

[0299] 58. The method of embodiment 57, wherein the multispecific antibody comprises a VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL.

[0300] 59. The method of embodiment 58, wherein the VL domain is κ2 VL.

[0301] 60. The method of any one of embodiments 57 to 59, wherein the VL domain binds poorly to protein L.

[0302] 61. The method of any one of embodiments 58 to 60, wherein the VL domain binds poorly to protein L and the second VL domain does not bind to protein L.

[0303] 62. The method of any one of embodiments 58 to 61, wherein the second VL domain is a lambda (λ) VL.

[0304] 63. The method of any one of embodiments 1 to 56, wherein the VL domain is a lambda subtype VL or a kappa VL.

[0305] 64. The method of embodiment 63, wherein the VL domain binds poorly to protein L.

[0306] 65. The method of any one of embodiments 60 to 64, wherein the VL domain binds poorly to Protein L compared to the binding of the VL domain of a reference antigen binding protein that is monoclonal antibody G6-31 to Protein L.

[0307] 66. The method of any one of embodiments 63 to 65, wherein the VL domain is a kappa VL and comprises a modification that weakens binding of the VL domain to protein L.

[0308] 67. The method of embodiment 66, wherein the modification comprises one or more amino acid substitutions in the VL domain.

[0309] 68. The method of any one of embodiments 63 to 67, wherein the VL domain is a κ2 VL.

[0310] 69. The method of any one of embodiments 1 to 48 and 56 to 68, wherein the antigen binding protein comprises a binding site for binding to protein L that lacks a hydrophobic patch or has a weakly hydrophobic patch.

[0311] 70. The method of embodiment 69, wherein the binding site for binding to protein L lacks a hydrophobic patch.

[0312] 71. The method of embodiment 69, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

[0313] 72. The method of any one of embodiments 69 to 71, wherein the binding site for binding to protein L has a less hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein that is monoclonal antibody G6-31.

[0314] 73. The method of any one of embodiments 69 to 71, wherein the binding site for binding to Protein L comprises a hydrophilic patch.

[0315] 74. The method of any one of embodiments 1 to 73, wherein the Protein L chromatography material is Pierce TM Protein L chromatography box, Capto TM L chromatography, Protein L chromatography, KanCap TM L chromatography, AF-rProtein L-650F chromatography or MabSelect TM VL chromatography.

[0316] 75. A composition comprising an antigen binding protein purified by a method comprising the method of any one of embodiments 1 to 74.

[0317] 76. The composition of embodiment 75, wherein the composition comprises one or more pharmaceutical excipients.

[0318] 77. A kit for purifying an antigen binding protein comprising a VL domain, wherein the kit comprises a Protein L chromatography material and a kosmotropic agent.

[0319] 78. The kit of embodiment 77, wherein the kit further comprises an equilibration buffer.

[0320] 79. The kit of embodiment 78, wherein the equilibration buffer comprises Tris, MES, MOPS, or EDTA.

[0321] 80. The kit of embodiment 78 or embodiment 79, wherein the equilibration buffer comprises Tris and NaCl.

[0322] 81. The kit of embodiment 79 or embodiment 80, wherein Tris is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM.

[0323] 82. The kit of embodiment 80 or embodiment 81, wherein NaCl is present in the equilibration buffer at a concentration of about 10 mM to about 50 mM, or about 25 mM.

[0324] 83. The kit of any one of embodiments 78 to 82, wherein the equilibration buffer has a pH of about 4 to about 10.

[0325] 84. The kit of 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.

[0326] 85. The kit of any one of embodiments 78 to 84, wherein the equilibration buffer has a pH of about 7 to about 8.

[0327] 86. The kit of any one of embodiments 77 to 85, wherein the kit further comprises a wash buffer.

[0328] 87. The kit of embodiment 86, wherein the wash buffer has a pH of about 4 to about 10.

[0329] 88. The kit of embodiment 86 or embodiment 87, wherein 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.

[0330] 89. The kit of any one of embodiments 86 to 88, wherein 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.

[0331] 90. The kit of any one of embodiments 86 to 88, wherein 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.

[0332] 91. The kit of any one of embodiments 86 to 90, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent.

[0333] 92. The kit of any one of embodiments 78 to 91, wherein the equilibration buffer further comprises a kosmotropic agent.

[0334] 93. The kit of any one of embodiments 77 to 92, wherein the kosmotropic agent is a phosphate or sulfate salt.

[0335] 94. The kit of any one of embodiments 77 to 93, wherein the kosmotropic agent is potassium phosphate, sodium sulfate, or ammonium sulfate.

[0336] 95. The kit of any one of embodiments 78 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM.

[0337] 96. The kit of any one of embodiments 78 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM.

[0338] 97. The kit of any one of embodiments 78 to 96, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM.

[0339] 98. The kit of any one of embodiments 78 to 97, wherein the concentration of the kosmotropic agent 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.

[0340] 99. The kit of any one of embodiments 77 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM.

[0341] 100. The kit of any one of embodiments 77 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or 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.

[0342] 101. The kit of any one of embodiments 77 to 100, wherein the kosmotropic agent is sodium sulfate.

[0343] 102. The kit of any one of embodiments 77 to 100, wherein the kosmotropic agent is potassium phosphate.

[0344] 103. The kit of any one of embodiments 77 to 100, wherein the kosmotropic agent is ammonium sulfate.

[0345] 104. The kit of any one of embodiments 77 to 103, wherein the kit further comprises an elution buffer.

[0346] 105. The kit of embodiment 104, wherein the elution buffer has a lower pH than the equilibration buffer.

[0347] 106. The kit of embodiment 104 or embodiment 105, wherein the elution buffer has a pH of about 2.0 to about 4.0, or about 2.5 to about 3.0.

[0348] 107. The kit of any one of embodiments 104 to 106, wherein 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.

[0349] 108. The kit of any one of embodiments 104 to 106, wherein 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.

[0350] 109. The kit of embodiment 104, wherein the elution buffer has a higher pH than the equilibration buffer.

[0351] 110. The kit of embodiment 104 or embodiment 109, wherein the elution buffer has a pH of about 10.0 to about 12.0.

[0352] 111. The kit of any one of embodiments 104 to 110, wherein the elution buffer comprises acetic acid.

[0353] 112. The kit of embodiment 111, wherein the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid.

[0354] 113. The kit of embodiment 111 or embodiment 112, wherein the elution buffer comprises about 150 mM acetic acid at a pH of about 2.8.

[0355] 114. The kit of any one of embodiments 104 to 110, wherein the elution buffer comprises sodium acetate, citrate, or glycine.

[0356] 115. The kit of any one of embodiments 77 to 114 for purifying an antibody or immunoadhesin or fragment thereof.

[0357] 116. The kit of any one of embodiments 77 to 115, wherein the antigen binding protein is a monoclonal antibody.

[0358] 117. The kit of any one of embodiments 77 to 116, wherein the antigen binding protein is a human antibody, a chimeric antibody, or a humanized antibody.

[0359] 118. The kit of any one of embodiments 115 to 117, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0360] 119. The kit of embodiment 118, wherein the antibody fragment is Fab.

[0361] 120. The kit of 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.

[0362] 121. The kit of embodiment 120, wherein the binding site for binding to protein L lacks a hydrophobic patch.

[0363] 122. The kit of embodiment 120, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

[0364] 123. The kit of embodiment 120 or embodiment 122, wherein the binding site for binding to protein L has a less hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein that is monoclonal antibody G6-31.

[0365] 124. The kit of any one of embodiments 120 to 123, wherein the binding site for binding to Protein L comprises a hydrophilic patch.

[0366] 125. The kit of 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 that is 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.

[0367] 126. The kit of embodiment 125, wherein the binding affinity (EC50) and / or the dissociation constant of the antigen binding protein for protein L has a value that 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 value of the binding affinity (EC50) and / or the dissociation constant of a reference antigen binding protein that is monoclonal antibody G6-31 for protein L.

[0368] 127. The kit of any one of embodiments 115 to 126, wherein the antibody does not contain an Fc domain.

[0369] 128. The kit of any one of embodiments 115 to 127, wherein the antibody is a multispecific antibody.

[0370] 129. The kit of embodiment 128, wherein the multispecific antibody comprises a VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL.

[0371] 130. The kit of embodiment 129, wherein the VL domain is a κ2 VL.

[0372] 131. The kit of embodiment 129 or embodiment 130, wherein the VL domain binds poorly to protein L.

[0373] 132. The kit of any one of embodiments 129 to 131, wherein the VL domain binds poorly to protein L and the second VL domain does not bind to protein L.

[0374] 133. The kit of any one of embodiments 129 to 132, wherein the second VL domain is a lambda (λ) VL.

[0375] 134. The kit of any one of embodiments 115 to 127, wherein the antibody comprises a VL domain that is a lambda subtype VL or a kappa VL.

[0376] 135. The kit of embodiment 134, wherein the VL domain binds poorly to protein L. 136. The kit of any one of embodiments 129 to 135, wherein the VL domain of the antigen binding protein binds poorly to protein L compared to a reference antigen binding protein that is monoclonal antibody G6-31.

[0377] 137. The kit of any one of embodiments 129 to 136, wherein the VL domain of the antigen binding protein binds weaker to Protein L than a reference antigen binding protein that is monoclonal antibody G6-31.

[0378] 138. The kit of any one of embodiments 134 to 137, wherein the VL domain is a kappa VL and comprises a modification that weakens binding of the VL domain to protein L.

[0379] 139. The kit of embodiment 138, wherein the modification comprises one or more amino acid substitutions in the VL domain.

[0380] 140. The kit of any one of embodiments 134 to 139, wherein the VL domain is a κ2 VL.

[0381] 141. The kit of any one of embodiments 77 to 140, wherein the antigen binding protein comprises a binding site for binding to protein L that lacks a hydrophobic patch or has a weakly hydrophobic patch.

[0382] 142. The kit of embodiment 141, wherein the binding site for binding to protein L lacks a hydrophobic patch.

[0383] 143. The kit of embodiment 141, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

[0384] 144. The kit of embodiment 141 or embodiment 143, wherein the binding site for binding to protein L has a less hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein that is monoclonal antibody G6-31.

[0385] 145. The kit of any one of embodiments 141 to 144, wherein the binding site for binding to Protein L comprises a hydrophilic patch.

[0386] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The present disclosure is further illustrated by the following examples, which should not be construed as limiting the present disclosure in scope or spirit to the specific procedures described herein.

[0387] Examples

[0388] Example 1. Protein L chromatography of antibody fragments using lyophiles

[0389] Protein L was used while developing a purification process for the pipeline molecule referred to herein as Fab 1 (Fab antibody fragment) because this Fab molecule lacks the Fc region, the binding site required for use of Protein A.

[0390] In developing a Protein L chromatography run ( Figure 1), an increase in UV absorbance was observed during the wash phase after loading and before elution. Furthermore, during the phase containing potassium phosphate (a non-corrosive buffer salt used to remove ionic impurities such as host cell DNA), the UV reading decreased, but after the end of this wash phase, the UV baseline increased again. This result suggests that protein is shed from the column after the loading phase, but the inventors found that this protein efflux was temporarily interrupted and inhibited by the presence of phosphate (a kosmotropic agent).

[0391] In order to study the purposes that lyophiles are used to improve protein L chromatography, multiple studies have been carried out to purify Fab 1 by protein L chromatography, and this chromatography uses the washing buffer solution that is supplemented with potassium phosphate (a kind of lyophile), sodium sulfate (a kind of stronger lyophile) and sodium chloride (a kind of neutral salt with little or no lyophilicity characteristic) of different concentrations.The host cell culture fluid (HCCF) containing Fab 1 antibody fragment is loaded onto 7ml Capto L post with the density of 3.74g / L and the flow velocity of 2.23ml / min.With the equilibration buffer solution (25mM Tris, 25mM NaCl, pH 7.7) containing lyophiles (sodium sulfate or potassium phosphate) or with the NaCl washing column that concentration is 600mM, 480mM, 360mM, 240mM or 120mM.The equilibration buffer solution that does not contain lyophiles or extra NaCl is used as negative control.Then use 100mM acetic acid (pH 2.9) to elute antibody from post. The protein concentration, conductivity, and pH measured by A280 were monitored throughout the chromatographic run. The yield and quality of the chromatographic run were analyzed using SEC-HPLC (TSKGel 2000SW column). As shown below, the use of a lyophilic salt method increased yield and purity.

[0392] Study 1: Sodium sulfate (strong lyophile)

[0393] The antibody of the present invention is purified by chromatography on the post of Figure 5. Six purifications have been run, wherein the equilibrium buffer has used the sodium sulfate of five different concentrations to assess the loss of antibody from post. The host cell culture fluid (HCCF) containing the Fab 1 antibody fragment is loaded on the 7ml Capto L post with the density of 3.74g / L and the flow velocity of 2.23ml / min. With containing sodium sulfate (600mM, 480mM, 360mM, 240mM, 120mM) or not containing sulfate (0mM; As negative control) equilibrium buffer (25mM Tris, 25mM NaCl, pH 7.7) washing post. Then use 100mM acetic acid (pH 2.9) to elute the antibody from post. In whole chromatographic operation process, monitoring is measured by A280 protein concentration, specific conductivity and the pH.

[0394] Chromatogram ( Figure 2) showed that higher sulfate concentrations were associated with less protein leaving the column during the wash phase and more protein recovered during the elution phase.

[0395] The pool recovered from the elution stage was collected and analyzed by concentration and size exclusion HPLC ( Figure 3 As shown in Table 2, both yield and purity improved when the kosmotropic agent was included in the wash buffer. Without being bound by theory, this result suggests that the kosmotropic agent is enhancing the binding of the main species to Protein L. It is likely that impurities (such as HMWF and LC-dimer) may bind more tightly to Protein L than the main species due to their two or more binding sites, which may result in poorer yield and quality of the main species.

[0396] Table 2. Yield and purity calculated from analysis of the elution pool.

[0397]

[0398] Study 2: Potassium Phosphate (a lyophile)

[0399] In this study, different salts with common lyophilic properties were evaluated. Six purification runs were performed with the equilibration buffer using five different concentrations of potassium sulfate (600 mM, 480 mM, 360 mM, 240 mM, 120 mM) or without sulfate (0 mM; as a negative control). Other chromatographic parameters were the same as for the sodium sulfate runs. Figure 4 As shown in Table 3, although the effect was not obvious in the previous study, the results were similar. Figure 4 ), the UV absorbance signal in the post-load wash was gradually suppressed with increasing potassium phosphate concentration. The yield and purity also improved with increasing potassium phosphate concentration ( Figure 5 , Table 3), although not as much of an improvement as an equivalent concentration of sodium sulfate.

[0400] Table 3. Yield and purity calculated from analysis of the elution pool.

[0401]

[0402] Study 3: Sodium chloride (neutral salt, control)

[0403] Adding sodium chloride to the wash buffer had minimal effect in rescuing the performance of this step. Figure 6 ), the UV absorbance after washing remained high even at high sodium chloride concentrations, and was accompanied by a small increase in yield and purity compared to equivalent concentrations of kosmotropic salts during the study ( Figure 7 , Table 4).

[0404] Table 4. Yield and purity calculated from analysis of the elution pool.

[0405] Wash buffer additives Calculated yield (%) Main peak (by SEC-HPLC) (%) 0 mM sodium chloride 21.91 78.86 120 mM sodium chloride 26.41 81.09 240 mM sodium chloride 28.60 83.70 360 mM sodium chloride 30.79 84.48 480 mM sodium chloride 32.15 84.19 600 mM sodium chloride 33.43 84.88

[0406] Example 2. Protein L Chromatography of Bispecific Antibodies Using a Lyophile

[0407] Poor binding of antibodies to protein L in another molecule was observed for a bispecific antibody with one light chain directed against target antigen A ("anti-A light chain") and one light chain directed against target antigen B ("anti-B light chain"), referred to herein as bispecific antibody 1. By engineering S12P in the VL region, the anti-B light chain was designed not to bind to protein L, leaving only the anti-A light chain to act as an "anchor" for protein L. The anti-A LC has a VK2κ framework, which is a poor binder to protein L.

[0408] The purposes of the protein L chromatogram that is used to improve bispecific antibody 1 in order to study lyophile, in the absence or presence of lyophile, chromatographic operation is carried out.Use 0.22 μ to filter the host cell culture fluid (HCCF) containing bispecific antibody 1 fragment and be diluted to 0.345g / L in equilibrium buffer (25mM Tris, 25mM NaCl, pH 7.7), and be loaded on 1ml HiTrap protein L post with the flow velocity of 0.4ml / min.With the equilibrium buffer without lyophile or with 600mM sodium sulfate washing column.Then use 170mM acetic acid (pH 2.75) to elute antibody from post.In whole chromatographic operation process, monitor the protein concentration, specific conductivity and pH measured by A280.

[0409] The results of the chromatographic runs in which the wash buffer did not contain a lyophile were Figure 8 and the results of the chromatographic run in which the wash buffer contained a kosmotropic agent (600 mM sodium sulfate) are shown in Figure 9 In the experiments with sulfate, protein damage and washout decreased, which resulted in protein retention and the recovery of a larger elution peak in the presence of sulfate compared to the absence of sulfate. Figure 9 and Figure 8 .

[0410] The step yield for the non-sulfate run was calculated to be 10.11%, but in the experiment using sulfate it was calculated to be 31.49%, a threefold increase in yield.

Claims

1. 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, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and c) eluting the antigen binding protein from the Protein L chromatography material with an elution buffer.

2. A method for improving 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, wherein the wash buffer comprises an equilibration buffer and a kosmotropic agent, and c) eluting the antigen binding protein from the Protein L chromatography material with an elution buffer.

3. The method according to claim 2, wherein: The yield and / or purity of the antigen binding protein is increased compared to Protein L chromatography wherein the wash buffer does not comprise a kosmotropic agent.

4. The method according to any one of claims 1 to 3, wherein the equilibration buffer comprises Tris, MES, MOPS or EDTA.

5. The method according to any one of claims 1 to 4, wherein the equilibration buffer comprises Tris and NaCl.

6. The method of claim 4 or claim 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 method of claim 5 or claim 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 of any one of claims 1 to 7, wherein the equilibration buffer has a pH of about 4 to about 10.

9. The method of any one of claims 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 claim 1 , wherein the equilibration buffer has a pH of about 7 to about 8.

11. The method of any one of claims 1 to 10, wherein the wash buffer has a pH of about 4 to about 10.

12. The method of any one of claims 1 to 11, wherein 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.

13. The method of any one of claims 1 to 12, wherein 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.

14. The method of any one of claims 1 to 12, wherein 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.

15. The method according to any one of claims 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a host cell culture supernatant.

16. The method according to any one of claims 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a purified polypeptide solution.

17. The method according to any one of claims 1 to 14, wherein the antigen binding protein is loaded onto the Protein L chromatography material in step a) as a mixture from a previous purification step.

18. The method of any one of claims 1 to 14, wherein the antigen binding protein is formulated in an equilibration buffer before being loaded onto the Protein L chromatography material in step a).

19. The method according to any one of claims 1 to 14 and 18, wherein the antigen binding protein is formulated in an equilibration buffer and the kosmotropic agent before being loaded onto the Protein L chromatography material in step a).

20. The method according to any one of claims 1 to 19, wherein the kosmotropic agent is a phosphate or a sulfate.

21. The method according to any one of claims 1 to 20, wherein the kosmotropic agent is potassium phosphate, sodium sulfate or ammonium sulfate.

22. The method of any one of claims 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM.

23. The method of any one of claims 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM.

24. The method of any one of claims 1 to 23, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM.

25. The method of any one of claims 1 to 24, wherein the concentration of the kosmotropic agent 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.

26. The method of any one of claims 1 to 21, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or the wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM.

27. The method of any one of claims 1 to 21, wherein the concentration of the kosmotropic agent 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.

28. The method of any one of claims 1 to 27, wherein the kosmotropic agent is sodium sulfate.

29. The method of any one of claims 1 to 27, wherein the kosmotropic agent is potassium phosphate.

30. The method of any one of claims 1 to 27, wherein the kosmotropic agent is ammonium sulfate.

31. The method of any one of claims 1 to 30, wherein the elution buffer has a lower pH than the equilibration buffer.

32. The method of any one of claims 1 to 31, wherein the elution buffer has a pH of about 2.0 to about 4.0, or about 2.5 to about 3.

0.

33. The method of any one of claims 1 to 32, wherein 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.

34. The method of any one of claims 1 to 32, wherein 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.

35. The method of any one of claims 1 to 30, wherein the elution buffer has a higher pH than the equilibration buffer.

36. The method of claim 35, wherein the elution buffer has a pH of about 10.0 to about 12.

0.

37. The method of any one of claims 1 to 36, wherein the elution buffer comprises acetic acid.

38. The method of claim 37, wherein the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid.

39. The method of claim 37 or claim 38, wherein the elution buffer comprises about 150 mM acetic acid at a pH of about 2.

8.

40. The method of any one of claims 1 to 36, wherein the elution buffer comprises sodium acetate, citrate or glycine.

41. The method of any one of claims 1 to 40, wherein the antigen binding protein binds poorly to Protein L.

42. The method of any one of claims 1 to 41, wherein the antigen binding protein binds poorly to Protein L compared to a reference antigen binding protein which is monoclonal antibody G6-31.

43. The method of any one of claims 1 to 41, wherein the antigen binding protein binds to Protein L more weakly than a reference antigen binding protein which is monoclonal antibody G6-31.

44. The method of any one of claims 1 to 43, wherein the antigen binding protein is an antibody or an immunoadhesin or a fragment thereof.

45. The method of any one of claims 1 to 44, wherein the antigen binding protein is a monoclonal antibody.

46. ​​The method of any one of claims 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 claims 44 to 46, wherein the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments.

48. The method of claim 47, wherein the antibody fragment is a Fab.

49. The method of claim 48, wherein the Fab comprises a binding site for binding to Protein L that lacks a hydrophobic patch or has a weak hydrophobic patch.

50. The method of claim 49, wherein the binding site for binding to protein L lacks a hydrophobic patch.

51. The method of claim 49, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

52. The method of any one of claims 49 to 51, wherein the binding site for binding to protein L has a weakly hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein which is monoclonal antibody G6-31.

53. A method according to any one of claims 48 to 52, wherein the binding site for binding to protein L comprises a hydrophilic patch.

54. according to the method described in any one of claims 1 to 53, wherein the binding affinity (EC50) and / or the dissociation constant of the antigen-binding protein to protein L have such values, and the values ​​are higher than the binding affinity (EC50) and / or the dissociation constant of the reference antigen-binding protein to protein L which is monoclonal antibody G6-31.

55. A method according to claim 54, wherein the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for protein L has the following values, which are 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 for protein L, which is monoclonal antibody G6-31.

56. The method of any one of claims 44 to 55, wherein the antibody does not contain an Fc domain.

57. The method of any one of claims 44 to 46, wherein the antibody is a multispecific antibody.

58. The method of claim 57, wherein the multispecific antibody comprises the VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL.

59. The method of claim 58, wherein the VL domain is a κ2 VL.

60. The method of any one of claims 57 to 59, wherein the VL domain binds poorly to protein L.

61. The method of any one of claims 58 to 60, wherein the VL domain binds poorly to protein L and the second VL domain does not bind to protein L.

62. The method of any one of claims 58 to 61, wherein the second VL domain is a lambda VL.

63. The method of any one of claims 1 to 56, wherein the VL domain is a lambda subtype VL or a kappa VL.

64. The method of claim 63, wherein the VL domain binds poorly to protein L.

65. The method of any one of claims 60 to 64, wherein the VL domain binds poorly to Protein L compared to the binding of the VL domain of a reference antigen binding protein that is monoclonal antibody G6-31 to Protein L.

66. The method of any one of claims 63 to 65, wherein the VL domain is a kappa VL and comprises a modification that weakens binding of the VL domain to protein L.

67. The method of claim 66, wherein the modification comprises one or more amino acid substitutions in the VL domain.

68. The method of any one of claims 63 to 67, wherein the VL domain is a κ2 VL.

69. The method of any one of claims 1 to 48 and 56 to 68, wherein the antigen binding protein comprises a binding site for binding to protein L that lacks a hydrophobic patch or has a weakly hydrophobic patch.

70. The method of claim 69, wherein the binding site for binding to protein L lacks a hydrophobic patch.

71. The method of claim 69, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

72. The method of any one of claims 69 to 71, wherein the binding site for binding to protein L has a weakly hydrophobic patch compared to the binding site for binding to protein L of a reference antigen binding protein which is monoclonal antibody G6-31.

73. A method according to any one of claims 69 to 71, wherein the binding site for binding to protein L comprises a hydrophilic patch.

74. The method of any one of claims 1 to 73, wherein the Protein L chromatography material is Pierce TM Protein L chromatography box, Capto TM L Chromatography, Protein L chromatography, KanCap TM L Chromatography, AF-rProtein L-650F chromatography or MabSelect TM VL Chromatography.

75. A composition comprising an antigen binding protein purified by a method comprising the method of any one of claims 1 to 74.

76. The composition of claim 75, wherein the composition comprises one or more pharmaceutical excipients.

77. A kit for purifying an antigen binding protein comprising a VL domain, wherein the kit comprises a protein L chromatography material and a lyophile.

78. The kit of claim 77, wherein the kit further comprises an equilibration buffer.

79. The kit of claim 78, wherein the equilibration buffer comprises Tris, MES, MOPS or EDTA.

80. The kit of claim 78 or claim 79, wherein the equilibration buffer comprises Tris and NaCl.

81. The kit of claim 79 or claim 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 of claim 80 or claim 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 of any one of claims 78 to 82, wherein the equilibration buffer has a pH of about 4 to about 10.

84. The kit of any one of claims 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 of any one of claims 78 to 84, wherein the equilibration buffer has a pH of about 7 to about 8.

86. The kit of any one of claims 77 to 85, wherein the kit further comprises a wash buffer.

87. The kit of claim 86, wherein the wash buffer has a pH of about 4 to about 10.

88. according to claim 86 or the kit of claim 87, wherein 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.

89. A kit according to any one of claims 86 to 88, wherein 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.

90. The kit of any one of claims 86 to 88, wherein 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.

91. The kit of any one of claims 86 to 90, wherein the wash buffer comprises the equilibration buffer and the kosmotropic agent.

92. The kit of any one of claims 78 to 91, wherein the equilibration buffer further comprises the kosmotropic agent.

93. A kit according to any one of claims 77 to 92, wherein the kosmotropic agent is a phosphate or sulfate salt.

94. A kit according to any one of claims 77 to 93, wherein the kosmotropic agent is potassium phosphate, sodium sulfate or ammonium sulfate.

95. The kit of any one of claims 78 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 100 mM to about 800 mM.

96. The kit of any one of claims 78 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer and the wash buffer is about 100 mM to about 800 mM.

97. The kit of any one of claims 78 to 96, wherein the concentration of the kosmotropic agent in the equilibration buffer or the wash buffer is about 120 mM to about 600 mM.

98. The kit of any one of claims 78 to 97, wherein the concentration of the kosmotropic agent 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.

99. A kit according to any one of claims 77 to 94, wherein the concentration of the kosmotropic agent in the equilibration buffer and / or the wash buffer is about 100 mM to about 800 mM, about 100 mM to about 700 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, or about 300 mM to about 500 mM.

100. The kit of any one of claims 77 to 94, wherein the concentration of the lyophile 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.

101. The kit of any one of claims 77 to 100, wherein the kosmotropic agent is sodium sulfate.

102. The kit of any one of claims 77 to 100, wherein the kosmotropic agent is potassium phosphate.

103. The kit of any one of claims 77 to 100, wherein the kosmotropic agent is ammonium sulfate.

104. The kit of any one of claims 77 to 103, wherein the kit further comprises an elution buffer.

105. The kit of claim 104, wherein the elution buffer has a lower pH than the equilibration buffer.

106. The kit of claim 104 or claim 105, wherein the elution buffer has a pH of about 2.0 to about 4.0, or about 2.5 to about 3.

0.

107. A kit according to any one of claims 104 to 106, wherein 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.

108. The kit of any one of claims 104 to 106, wherein 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.

109. The kit of claim 104, wherein the elution buffer has a higher pH than the equilibration buffer.

110. The kit of claim 104 or claim 109, wherein the elution buffer has a pH of about 10.0 to about 12.

0.

111. The kit of any one of claims 104 to 110, wherein the elution buffer comprises acetic acid.

112. The kit of claim 111, wherein the elution buffer comprises about 50 mM acetic acid to about 250 mM acetic acid.

113. The kit of claim 111 or claim 112, wherein the elution buffer comprises about 150 mM acetic acid at a pH of about 2.

8.

114. The kit of any one of claims 104 to 110, wherein the elution buffer comprises sodium acetate, citrate or glycine.

115. A kit according to any one of claims 77 to 114 for purifying an antibody or immunoadhesin or a fragment thereof.

116. The kit of any one of claims 77 to 115, wherein the antigen binding protein is a monoclonal antibody.

117. The kit of any one of claims 77 to 116, wherein the antigen binding protein is a human antibody, a chimeric antibody or a humanized antibody.

118. The kit according to any one of claims 115 to 117, wherein the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments.

119. The kit of claim 118, wherein the antibody fragment is a Fab.

120. The kit of claim 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 of claim 120, wherein the binding site for binding to protein L lacks a hydrophobic patch.

122. The kit of claim 120, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

123. A kit according to claim 120 or claim 122, wherein 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 which is monoclonal antibody G6-31.

124. A kit according to any one of claims 120 to 123, wherein the binding site for binding to protein L comprises a hydrophilic patch.

125. A kit according to any one of claims 77 to 124, wherein the binding affinity (EC50) and / or the dissociation constant of the antigen-binding protein for protein L has a value that is higher than the value of the binding affinity (EC50) and / or the dissociation constant of a reference antigen-binding protein for protein L which is monoclonal antibody G6-31.

126. A kit according to claim 125, wherein the binding affinity (EC50) and / or dissociation constant of the antigen binding protein for protein L has a value that 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 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.

127. The kit of any one of claims 115 to 126, wherein the antibody does not contain an Fc domain.

128. The kit of any one of claims 115 to 127, wherein the antibody is a multispecific antibody.

129. The kit of claim 128, wherein the multispecific antibody comprises a VL domain and a second VL domain, and wherein the VL domain is a kappa (κ) VL.

130. The kit of claim 129, wherein the VL domain is a κ2 VL.

131. A kit according to claim 129 or claim 130, wherein the VL domain binds poorly to protein L.

132. The kit of any one of claims 129 to 131, wherein the VL domain binds poorly to protein L and the second VL domain does not bind to protein L.

133. The kit of any one of claims 129 to 132, wherein the second VL domain is a lambda VL.

134. The kit of any one of claims 115 to 127, wherein the antibody comprises a VL domain that is a lambda subtype VL or a kappa VL.

135. The kit of claim 134, wherein the VL domain binds poorly to protein L.

136. The kit of any one of claims 129 to 135, wherein the VL domain of the antigen binding protein binds poorly to Protein L compared to a reference antigen binding protein which is monoclonal antibody G6-31.

137. The kit of any one of claims 129 to 136, wherein the VL domain of the antigen binding protein binds more weakly to Protein L than a reference antigen binding protein which is monoclonal antibody G6-31.

138. A kit according to any one of claims 134 to 137, wherein the VL domain is a kappa VL and comprises a modification that weakens the binding of the VL domain to protein L.

139. The kit of claim 138, wherein the modification comprises one or more amino acid substitutions in the VL domain.

140. The kit of any one of claims 134 to 139, wherein the VL domain is a κ2 VL.

141. A kit according to any one of claims 77 to 140, wherein the antigen binding protein comprises a binding site for binding to protein L that lacks a hydrophobic patch or has a weakly hydrophobic patch.

142. The kit of claim 141, wherein the binding site for binding to protein L lacks a hydrophobic patch.

143. The kit of claim 141, wherein the binding site for binding to protein L has a weakly hydrophobic patch.

144. A kit according to claim 141 or claim 143, wherein 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 which is monoclonal antibody G6-31.

145. A kit according to any one of claims 141 to 144, wherein the binding site for binding to protein L comprises a hydrophilic patch.

Images