Purification of multispecific antibodies

Abstract

To provide methods of purifying multispecific antibodies, and compositions comprising multispecific antibodies.SOLUTION: A method for purifying a multispecific antibody from a composition comprising the multispecific antibody and an impurity is provided, wherein the multispecific antibody comprises multiple arms, each arm comprising a VH / VL unit, the method comprising the sequential steps of: a) subjecting the composition to a capture chromatography to produce a capture chromatography eluate; b) subjecting the capture chromatography eluate to a first mixed mode chromatography to generate a first mixed mode eluate; and c) subjecting the first mixed mode eluate to a second mixed mode chromatography to generate a second mixed mode eluate; and d) collecting a fraction comprising the multispecific antibody, wherein the method reduces the amount of a product-specific impurity from the composition.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 351,908, filed June 17, 2016, which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings in ASCII text files. The following submission in ASCII text file is incorporated herein by reference in its entirety: a computer-readable format (CRF) sequence listing (filename: 146392036340SEQLIST.TXT, date: June 9, 2017, size: 32KB).

[0003] A method is provided for purifying multispecific antibodies from compositions containing at least one product-specific impurity. In some embodiments, the product-specific impurity is, for example, a precursor, aggregate, and / or variant of the multispecific antibody. The multispecific antibody purified according to the method, and compositions and formulations containing such multispecific antibodies are also provided. [Background technology]

[0004] For recombinant biopharmaceutical proteins acceptable for administration to human patients, it is crucial that residual impurities resulting from the manufacturing and purification processes are removed from the final biological product. These process components include culture medium proteins, immunoglobulin affinity ligands, viruses, endotoxins, DNA, and host cell proteins (HCPs). The development of new antibody forms, such as multispecific antibodies, presents new challenges because conventional manufacturing and purification processes are insufficient to adequately remove product-specific impurities, including non-paired antibody arms and misassembled antibodies.

[0005] When compared to the purification of standard antibodies, the purification of multispecific antibodies from production media presents unique challenges. While standard single-specificity bivalent antibodies result from the dimerization of identical heavy chain / light chain subunits, the production of multispecific antibodies requires the dimerization of at least two different heavy chain / light chain subunits, each containing a different heavy chain as well as a different light chain. The production and purification of final and complete multispecific antibodies with minimal amounts of mispaired, misassembled, or incomplete molecules presents different challenges. Chain mispairing (e.g., homodimerization of the same heavy chain peptide or inappropriate heavy chain / light chain associations) is often observed as an incomplete protein assembly resulting from unbalanced host cell expression of different antibody chains. Commonly observed product-specific impurities include half (1 / 2) antibodies (containing a single heavy chain / light chain pair), three-quarters (3 / 4) antibodies (containing a complete antibody lacking a single light chain), and homodimers. Additional product-specific impurities can be observed depending on the format of the multispecificity used. For example, when the variable domains of a multispecific antibody are constructed as single-chain Fabs (scFabs), 5 / 4 antibodies (containing an additional heavy or light chain variable domain) can be observed in the product. Such corresponding product-specific impurities do not occur in standard antibody production.

[0006] Conventional purification techniques designed to remove process-related impurities such as HCP, DNA, endotoxin, and other substances with very different characteristics and properties from antibodies can be insufficient when carried out to remove impurities similar to those of multispecific antibodies. Thus, there is a need to develop manufacturing and purification schemes that effectively remove product-specific impurities and produce sufficient amounts of correct and complete multispecific antibodies.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0008] As described and illustrated herein, the applicant has found that the use of at least two mixed-mode (also referred herein as multi-modal) chromatography steps after the initial capture chromatography step results in greater removal of product-specific impurities and improves the process for purifying multispecific antibodies. Accordingly, in certain embodiments, a method is provided for purifying a multispecific antibody from a composition comprising the multispecific antibody and impurities, wherein the multispecific antibody comprises multiple arms, each arm comprising VH / VL units, and the method comprises the steps of sequentially: a) subjecting the composition to capture chromatography to produce a capture chromatography eluate; b) subjecting the capture chromatography eluate to a first mixed-mode chromatography to produce a first mixed-mode eluate; c) subjecting the first mixed-mode eluate to a second mixed-mode chromatography to produce a second mixed-mode eluate; and d) collecting a fraction containing the multispecific antibody, thereby reducing the amount of product-specific impurities in the composition. In some embodiments according to any of the above embodiments (or applied thereto), the capture chromatography eluate is subjected to ion exchange chromatography (e.g., anion exchange) or hydrophobic interaction chromatography before the first mixed-mode chromatography. In some embodiments according to any of the above embodiments (or applied thereto), the second mixed-mode eluate is subjected to ion exchange chromatography (e.g., anion exchange) or hydrophobic interaction chromatography.

[0009] In a particular embodiment, a method is provided for purifying a multispecific antibody from a composition comprising the multispecific antibody and impurities, wherein the multispecific antibody comprises multiple arms, each arm comprising VH / VL units, and each arm of the multispecific antibody is produced separately, and the method comprises the steps of: a) subjecting each arm of the multispecific antibody to capture chromatography to produce a capture eluate for each arm of the multispecific antibody; b) forming a mixture containing the capture eluates of each arm of the multispecific antibody under conditions sufficient to produce a composition comprising the multispecific antibody; c) subjecting the composition comprising the multispecific antibody to a first mixed-mode chromatography to produce a first mixed-mode eluate; and d) subjecting the first mixed-mode eluate to a second mixed-mode chromatography to produce a second mixed-mode eluate; and e) collecting a fraction comprising the multispecific antibody, wherein the method reduces the amount of product-specific impurities in the composition. In some embodiments according to (or applied to) any of the above embodiments, the capture chromatography eluates are subjected to ion exchange chromatography (e.g., anion exchange) or hydrophobic interaction chromatography prior to the first mixed-mode chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the second mixed-mode eluate is subjected to ion exchange chromatography (e.g., anion exchange) or hydrophobic interaction chromatography.

[0010] In some embodiments according to any of the above embodiments (or applicable thereto), the capture chromatography is affinity chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the affinity chromatography is protein L chromatography, protein A chromatography, protein G chromatography, protein A and protein G chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the affinity chromatography is protein A chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the capture chromatography is performed in binding and elution modes.

[0011] In some embodiments according to any of the above embodiments (or applicable thereto), the first mixed-mode chromatography and the second mixed-mode chromatography are continuous. In some embodiments according to any of the above embodiments (or applicable thereto), the first mixed-mode chromatography is mixed-mode anion exchange chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the second mixed-mode chromatography is mixed-mode cation exchange chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the first mixed-mode chromatography is mixed-mode cation exchange chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the second mixed-mode chromatography is mixed-mode anion exchange chromatography.

[0012] In some embodiments according to any of the above embodiments (or applicable thereto), the first mixed-mode chromatography is performed in binding and elution mode or flow-through mode. In some embodiments according to any of the embodiments in which the first mixed-mode chromatography is performed in binding and elution mode (or applicable thereto), elution is gradient elution.

[0013] In some embodiments according to (or applied to) any of the above embodiments, the second mixed-mode chromatography is performed in binding and elution mode or flow-through mode. In some embodiments according to (or applied to) any of the embodiments in which the second mixed-mode chromatography is performed in binding and elution mode, elution is gradient elution.

[0014] In some embodiments according to any of the above embodiments (or applicable thereto), the method further includes the step of ultrafiltration of the second mixed-mode eluate. In some embodiments according to any of the above embodiments (or applicable thereto), the ultrafiltration includes sequentially a first ultrafiltration, diafiltration, and a second ultrafiltration.

[0015] In some embodiments according to any of the above embodiments (or applicable thereto), the protein A chromatography includes protein A linked to agarose. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A chromatography is MAbSelect®, MAbSelect® SuRe and MAbSelect® SuRe LX, Prosep-VA, Prosep-VA Ultra Plus, protein A Sepharose Fast Flow, or Toyopearl protein A chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A chromatography uses one or more of the protein A equilibration buffer, protein A loading buffer, or protein A washing buffer, and the equilibration buffer, loading buffer, and / or washing buffer are about pH 7 to about pH 8. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A equilibration buffer is about pH 7.7. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A equilibration buffer includes about 25 mM Tris and about 25 mM NaCl. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A chromatograph is washed with equilibration buffer after loading. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the protein A chromatograph by applying a protein A elution buffer having a low pH to the protein A chromatograph. In some embodiments according to any of the above embodiments (or applicable thereto), the protein A elution buffer contains about 150 mM acetic acid, about pH 2.9. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the protein A chromatograph by a pH gradient.

[0016] In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange mixed-mode chromatography includes a quaternary amine and a hydrophobic moiety. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange mixed-mode chromatography includes a quaternary amine linked to agarose and a highly crosslinked hydrophobic moiety. In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode chromatography is Capto® Adhere chromatography or Capto® Adhere ImpRes chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the cation exchange mixed-mode chromatography includes N-benzyl-n-methylethanolamine. In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode chromatography is Capto® MMC chromatography or Capto® MMC ImpRes chromatography.

[0017] In some embodiments according to any of the above embodiments (or applied thereto), the first mixed-mode chromatography uses one or more of the mixed-mode pre-equilibrium buffer, mixed-mode equilibration buffer, mixed-mode loading buffer, and mixed-mode washing buffer, where the mixed-mode pre-equilibrium buffer, mixed-mode equilibration buffer, mixed-mode loading buffer, and / or mixed-mode washing buffer are approximately pH 6 to approximately pH 7. In some embodiments, the anionic mixed-mode equilibration buffer is approximately pH 6.5 to approximately pH 8.

[0018] In some embodiments according to any of the above embodiments (or applicable thereto), the second mixed-mode chromatography uses a mixed-mode pre-equilibrium buffer, a mixed-mode equilibration buffer, a mixed-mode loading buffer, or a mixed-mode wash buffer, wherein the mixed-mode pre-equilibrium buffer, the mixed-mode equilibration buffer, and / or the mixed-mode wash buffer are approximately pH 5 to approximately pH 8, optionally approximately pH 5 to approximately pH 7, or approximately pH 5 to approximately pH 6, or approximately pH 6 to approximately pH 7.

[0019] In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode pre-equilibrium buffer, the mixed-mode equilibration buffer, and / or the mixed-mode wash buffer are at approximately pH 5.5. In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode pre-equilibrium buffer contains approximately 500 mM acetate. In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode equilibration buffer contains approximately 50 mM acetate.

[0020] In some embodiments according to any of the above embodiments (or applicable thereto), the first mixed-mode chromatography is washed with a wash buffer after loading. In some embodiments according to any of the above embodiments (or applicable thereto), the second mixed-mode chromatography is washed with a wash buffer after loading. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the first mixed-mode chromatography by a salt gradient and / or pH gradient or pH step elution. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the first mixed-mode chromatography by applying a mixed-mode elution buffer having a low pH to the mixed-mode exchange chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the second mixed-mode chromatography by a salt gradient and / or pH gradient or pH step elution. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the second mixed-mode chromatography by applying a mixed-mode elution buffer having a low pH to the mixed-mode exchange chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the mixed-mode elution buffer contains about 25 mM acetate and about pH 5.5.

[0021] In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange chromatography comprises a quaternary amine. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange chromatography comprises a quaternary amine linked to a crosslinked agarose. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange chromatography is QSFF chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange chromatography uses one or more of the anion exchange pre-equilibrium buffer, anion exchange equilibration buffer, or anion exchange loading buffer, and the anion exchange pre-equilibrium buffer, anion exchange equilibration buffer, and / or anion exchange loading buffer have a pH of approximately 8 to approximately 9. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange pre-equilibrium buffer, anion exchange equilibration buffer, and / or anion exchange loading buffer have a pH of approximately 8.5. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange pre-equilibrium buffer contains about 50 mM Tris and 500 mM sodium acetate. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange equilibrium buffer contains about 50 mM Tris. In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange chromatograph is washed with the anion exchange equilibrium buffer after loading. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the anion exchange chromatograph by a salt gradient. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is eluted from the anion exchange chromatograph by applying an anion exchange elution buffer having an increased salt concentration to the anion exchange chromatograph.In some embodiments according to any of the above embodiments (or applicable thereto), the anion exchange elution buffer contains about 50 mM Tris and 100 mM sodium acetate at about pH 8.5.

[0022] In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody arm is produced in cells. In some embodiments according to any of the above embodiments (or applicable thereto), the cells are prokaryotic cells. In some embodiments according to any of the above embodiments (or applicable thereto), the prokaryotic cells are E. coli cells. In some embodiments according to any of the above embodiments (or applicable thereto), the cells are engineered to express one or more chaperones. In some embodiments according to any of the above embodiments (or applicable thereto), the chaperone is one or more of FkpA, DsbA, or DsbC. In some embodiments according to any of the above embodiments (or applicable thereto), the chaperone is an E. coli chaperone. In some embodiments according to any of the above embodiments (or applicable thereto), the cells are eukaryotic cells. In some embodiments according to any of the above embodiments (or applicable thereto), the eukaryotic cells are yeast cells, insect cells, or mammalian cells. In some embodiments according to any of the above embodiments (or applicable thereto), the eukaryotic cells are CHO cells.

[0023] In some embodiments according to any of the above embodiments (or applicable thereto), cells are lysed before capture chromatography to produce a cell lysate containing multispecific antibodies or an arm of multispecific antibodies. In some embodiments according to any of the above embodiments (or applicable thereto), cells are lysed using a microfluidic agent. In some embodiments according to any of the above embodiments (or applicable thereto), polyethyleneimine (PEI) is added to the cell lysate before chromatography. In some embodiments according to any of the above embodiments (or applicable thereto), PEI is added to the lysate to a final concentration of about 0.4%. In some embodiments according to any of the above embodiments (or applicable thereto), the cell lysate is purified by centrifugation. In some embodiments according to any of the above embodiments (or applicable thereto), the cell lysate of mammalian cells, e.g., CHO cells, undergoes one or more of the following treatments by the addition of a drug: thermal inactivation, low pH inactivation, and viral inactivation.

[0024] In some embodiments according to any of the above embodiments (or applicable thereto), the method reduces the amount of process-specific impurities in the composition, such as host cell proteins (HCPs), leached protein A, nucleic acids, cell culture medium components, or viral impurities.

[0025] In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody is a bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody is a knob-in-hole (KiH) antibody, for example, a KiH bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody is a CrossMab bispecific antibody.

[0026] In some embodiments according to any of the above methods (or applied thereto), the fraction is collected after the last chromatographic step and contains at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of multispecific antibodies. In some embodiments according to any of the above embodiments (or applied thereto), the fraction is collected after the last chromatographic step and contains a reduced amount of product-specific impurities, the product-specific impurities being one or more of unpaired antibody arms, antibody homodimers, high molecular weight species (HMWS), low molecular weight species (LMWS), or 3 / 4 antibodies. In some embodiments according to any of the above embodiments (or applied thereto), the fraction contains less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% of unpaired antibody arms. In some embodiments according to any of the above embodiments (or applied thereto), the fraction contains less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% of antibody homodimers. In some embodiments according to any of the above embodiments (or applicable thereto), the fraction contains about 1% or less of HMWS or about 2% or less. In some embodiments according to any of the above embodiments (or applicable thereto), the fraction contains about 2% or less of LMWS or about 1% or less. In some embodiments according to any of the above embodiments (or applicable thereto), the fraction contains about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of 3 / 4 antibody.

[0027] In some embodiments according to any of the above embodiments (or applicable thereto), the fraction is: a) At least approximately 95% to approximately 100% multispecific antibodies, b) Arms with approximately 1% to less than 5% non-counterantibody c) Antibody homodimers of approximately 1% to less than 5% d) HMWS of approximately 1% or 2% or less, e) LMWS of approximately 1% or less of approximately 2%, and / or f) Contains 3 / 4 antibodies of approximately 5% or less.

[0028] In a particular embodiment, a composition is provided comprising a multispecific antibody purified by any one of the methods described above.

[0029] In some embodiments according to any of the above methods (or applied thereto), a composition comprising a multispecific antibody is provided, the composition comprising at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the multispecific antibody. In some embodiments according to any of the above embodiments (or applied thereto), a composition comprising a multispecific antibody is provided, the composition comprising a reduced amount of product-specific impurities, the product-specific impurities being one or more of unpaired antibody arms, antibody homodimers, high molecular weight species (HMWS), low molecular weight species (LMWS), or 3 / 4 antibodies. In some embodiments according to any of the above embodiments (or applied thereto), the composition comprising less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% of unpaired antibody arms. In some embodiments according to any of the above embodiments (or applied thereto), the composition comprising less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% of antibody homodimers. In some embodiments according to any of the above embodiments (or applicable thereto), the composition contains about 1% or less of HMWS or about 2% or less. In some embodiments according to any of the above embodiments (or applicable thereto), the composition contains about 2% or less of LMWS or about 1% or less. In some embodiments according to any of the above embodiments (or applicable thereto), the composition contains about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of 3 / 4 antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibody in the composition is a bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody is a nob-in-hole (KiH) antibody, for example, a KiH bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody is a CrossMab bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody binds ANG-2 and VEGF.In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody binding ANG-2 and VEGF comprises a) a heavy and light chain of a first full-length antibody containing a first antigen-binding site, and b) a modified heavy and modified light chain of a full-length antibody containing a second antigen-binding site, wherein the constant domains CL and CH1 are substituted for each other.

[0030] In some embodiments according to any of the above embodiments (or applicable thereto), the composition comprises a) at least about 95% to about 100% of multispecific antibodies, b) an arm of about 1% to less than 5% of unpaired antibodies, c) about 1% to less than 5% of antibody homodimers, d) about 1% or 2% or less of HMWS, e) about 1% or 2% or less of LMWS, and / or f) about 5% or less of 3 / 4 antibodies. In some embodiments according to any of the above embodiments (or applicable thereto), the multispecific antibodies in the composition are bispecific antibodies. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibodies are knob-in-hole (KiH) antibodies, e.g., KiH bispecific antibodies. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibodies are CrossMab bispecific antibodies. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibodies conjugate ANG-2 and VEGF. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody binding ANG-2 and VEGF comprises a) a heavy and light chain of a first full-length antibody containing a first antigen-binding site, and b) a modified heavy and modified light chain of a full-length antibody containing a second antigen-binding site, wherein the constant domains CL and CH1 are substituted for each other.

[0031] In some embodiments according to any of the above embodiments (or applicable thereto), a composition is provided comprising a multispecific or bispecific antibody (such as a bispecific antibody binding to ANG2 and VEGF) purified by any one of the methods described above, for the treatment of cancer or eye disease.

[0032] In some embodiments according to any of the above embodiments (or applicable thereto), a use is provided which includes a multispecific or bispecific antibody (such as a bispecific antibody binding to ANG2 and VEGF) purified by any one of the methods described above, for the manufacture of a pharmaceutical product for the treatment of cancer or eye disease.

[0033] In some embodiments according to any of the above embodiments (or applicable thereto), the method provided herein is used for the purification of Fc-containing heterodimer polypeptides.

[0034] In some embodiments according to any of the above embodiments (or applicable thereto), the use of any of the methods provided herein for reducing Fc-containing heterodimer polypeptide-related impurities in the composition is provided.

[0035] In certain embodiments, a method is provided for purifying an Fc region-containing heterodimer polypeptide by a multi-step chromatography method, the method comprising an affinity chromatography step, followed by two different multimodal ion exchange chromatography steps, thereby purifying the Fc region-containing heterodimer polypeptide. In some embodiments according to any of the above embodiments (or applicable thereto), the multi-step chromatography method comprises (i) an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step, or (ii) an affinity chromatography step, followed by a multimodal cation exchange chromatography step, followed by a multimodal anion exchange chromatography step.

[0036] In some embodiments according to any of the above embodiments (or applicable thereto), the multi-step chromatography method includes an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step. In some embodiments according to any of the above embodiments (or applicable thereto), the multi-step chromatography method includes exactly three chromatography steps. In some embodiments according to any of the above embodiments (or applicable thereto), the multimodal anion exchange chromatography step is performed in flow-through mode. In some embodiments according to any of the above embodiments (or applicable thereto), in the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value of less than 7 mS / cm. In some embodiments according to any of the above embodiments (or applicable thereto), in the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value in the range of about 6 mS / cm to about 2 mS / cm. In some embodiments according to any of the above embodiments (or applicable thereto), in the step of multimodal anion exchange chromatography, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity of about 4.5 mS / cm. In some embodiments according to any of the above embodiments (or applicable thereto), the step of multimodal anion exchange chromatography is carried out at a pH of about 7. In some embodiments according to any of the above embodiments (or applicable thereto), in the step of multimodal anion exchange chromatography, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity of about 4.5 mS / cm and a pH of about 7.In some embodiments according to (or applied to) any of the above embodiments, the Fc region-containing heterodimer polypeptide is applied in the step of multimodal anion exchange chromatography in an amount ranging from about 100 g to about 300 g per liter of chromatography material.

[0037] In some embodiments according to any of the above embodiments (or applicable thereto), the multimodal anion exchange chromatography material is a multimodal strong anion exchange chromatography material. In some embodiments according to any of the above embodiments (or applicable thereto), the multimodal anion exchange chromatography material has a high-flow agarose matrix, multimodal strong anion exchange as a ligand, an average particle size of 36-44 μm, and an ion capacity of 0.08-0.11 mmolCl- / mL of the medium. In some embodiments according to any of the above embodiments (or applicable thereto), the multimodal cation exchange chromatography medium is a multimodal weak cation exchange chromatography medium. In some embodiments according to any of the above embodiments (or applicable thereto), the multimodal cation exchange chromatography medium has a high-flow agarose matrix, multimodal weak cation exchange as a ligand, an average particle size of 36-44 μm, and an ion capacity of 25-39 μmol / mL. In some embodiments according to any of the above embodiments (or applicable thereto), the steps of multimodal anion exchange chromatography are carried out in binding and elution modes. In some embodiments according to (or applicable to) any of the above embodiments, the capture chromatography is performed by affinity chromatography. In some embodiments, the affinity chromatography is a step of chromatography using protein A affinity chromatography, or protein G affinity chromatography, or single-stranded Fv ligand affinity chromatography, or CaptureSelect chromatography material, or CaptureSelect FcXL chromatography material. In some embodiments according to (or applicable to) any of the above embodiments, the affinity chromatography step is a step of protein A chromatography.In some embodiments according to any of the above embodiments (or applicable thereto), the affinity chromatography step is a chromatography step using CaptureSelect® chromatography material.

[0038] In some embodiments according to any of the above embodiments (or applicable thereto), the Fc-domain-containing heterodimer polypeptide is an antibody, a bispecific antibody, or an Fc-fusion protein. In some embodiments according to any of the above embodiments (or applicable thereto), the Fc-domain-containing heterodimer polypeptide is a bispecific antibody. In some embodiments according to any of the above embodiments (or applicable thereto), the Fc-domain-containing heterodimer polypeptide is CrossMab. In some embodiments according to any of the above embodiments (or applicable thereto), the Fc-domain-containing heterodimer polypeptide comprises a) a heavy and light chain of a first full-length antibody that specifically binds a first antigen, and b) a modified heavy and modified light chain of a full-length antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are substituted for each other.

[0039] In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody binds ANG2 and VEGF. In some embodiments according to any of the above embodiments (or applicable thereto), CrossMab binds ANG2 and VEGF. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody is vanucizumab. In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody includes a first antigen-binding site comprising SEQ ID NO: 1 as the heavy chain variable domain (VH) and SEQ ID NO: 2 as the light chain variable domain (VL), and a second antigen-binding site comprising SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL). In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody includes a first heavy chain having the amino acid sequence of SEQ ID NO: 9, a second heavy chain having the amino acid sequence of SEQ ID NO: 10, a first light chain having the amino acid sequence of SEQ ID NO: 11, and a second light chain having the amino acid sequence of SEQ ID NO: 12.

[0040] In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody includes a first antigen-binding site comprising SEQ ID NO: 5 as the heavy chain variable domain (VH) and SEQ ID NO: 6 as the light chain variable domain (VL), and a second antigen-binding site comprising SEQ ID NO: 7 as the heavy chain variable domain (VH) and SEQ ID NO: 8 as the light chain variable domain (VL). In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody includes a first heavy chain having the amino acid sequence of SEQ ID NO: 13, a second heavy chain having the amino acid sequence of SEQ ID NO: 14, a first light chain having the amino acid sequence of SEQ ID NO: 15, and a second light chain having the amino acid sequence of SEQ ID NO: 16.

[0041] In any of the embodiments described above (or in some embodiments applied thereto), the purified Fc region-containing heterodimer polypeptide contains approximately 5% or less of 3 / 4 antibody.

[0042] In a particular embodiment, a method is provided for purifying a bispecific antibody that binds ANG2 and VEGF by a multi-step chromatography method, the method comprising an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step, thereby purifying a bispecific antibody that binds ANG2 and VEGF, wherein the bispecific antibody that binds ANG2 and VEGF comprises a first antigen-binding site including SEQ ID NO: 1 as the heavy chain variable domain (VH) and SEQ ID NO: 2 as the light chain variable domain (VL), and a second antigen-binding site including SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL), or a first antigen-binding site including SEQ ID NO: 5 as the heavy chain variable domain (VH) and SEQ ID NO: 6 as the light chain variable domain (VL), and a second antigen-binding site including SEQ ID NO: 7 as the heavy chain variable domain (VH) and SEQ ID NO: 8 as the light chain variable domain (VL). In some embodiments according to any of the above embodiments (or applicable thereto), the bispecific antibody binding ANG2 and VEGF comprises a) a heavy and light chain of a first full-length antibody containing a first antigen-binding site, and b) a modified heavy and modified light chain of a full-length antibody containing a second antigen-binding site, wherein the constant domains CL and CH1 are substituted for each other.

[0043] In some embodiments, the use of any of the methods described in any of the above embodiments (or applicable thereto) is provided for reducing Fc-containing heterodimer polypeptide-related impurities.

[0044] In some embodiments, Fc-containing heterodimer polypeptides obtained by any of the above embodiments (or applicable thereto) are provided for the manufacture of pharmaceuticals for the treatment of cancer or eye diseases.

[0045] In some embodiments, Fc-containing heterodimer polypeptides obtained by any of the above embodiments (or applicable thereto) are provided for use in the treatment of cancer or eye disease.

[0046] In a particular embodiment, a method is provided for producing an Fc-containing heterodimer polypeptide, comprising the steps of (i) culturing cells containing nucleic acids encoding an Fc-containing heterodimer polypeptide, (ii) recovering an Fc-containing heterodimer protein from the cells or culture medium, and (iii) purifying the Fc-containing heterodimer polypeptide using a method according to (or applicable to) any of the above embodiments, thereby producing an Fc-containing heterodimer polypeptide.

[0047] In a particular embodiment, a method is provided for producing a bispecific antibody that binds ANG-2 and VEGF, comprising the steps of (i) culturing cells containing nucleic acids encoding a bispecific antibody, (ii) recovering the bispecific antibody from the cells or culture medium, and (iii) purifying the bispecific antibody using a method according to (or applicable to) any of the above embodiments, thereby producing a bispecific antibody that binds ANG-2 and VEGF. [Brief explanation of the drawing]

[0048]

Figure 1A

Figure 1B

Figure 1C

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 7C

[0049] A method for purifying a multispecific antibody (such as a bispecific antibody or a divalent F(ab')2) comprising the sequential steps of a) subjecting a composition containing the multispecific antibody to capture chromatography, b) subjecting it to a first mixed-mode chromatography, and c) subjecting it to a second mixed-mode chromatography is provided herein. In some embodiments, a method for purifying a multispecific antibody is provided, wherein each individual arm of the multispecific antibody is generated in a separate culture and purified separately by capture chromatography. The purified antibody arms are then assembled to produce a multispecific antibody. The assembled multispecific antibody is then subjected to first mixed-mode anion exchange chromatography, followed by second mixed-mode chromatography. Each of the capture chromatography, first mixed-mode chromatography, and / or second mixed-mode chromatography may optionally precede and / or be followed by one or more additional chromatographic steps. The terms mixed-mode chromatography and multimodal chromatography are used interchangeably herein.

[0050] In some embodiments, compositions are provided comprising multispecific antibodies having reduced levels of one or more process-specific and / or product-specific impurities, such as non-counter-antibody arms, homodimers, aggregates, low molecular weight species, and acidic and basic variants. In some embodiments, compositions are provided comprising multispecific antibodies having reduced levels of one or more process-specific impurities, such as prokaryotic host cell proteins, eukaryotic host cell proteins (such as CHO proteins or "CHOP"), nucleic acids, and chaperones (such as prokaryotic chaperones, e.g., FkpA, DsbA, and DsbC). In certain embodiments, the compositions provided herein are obtained using methods provided herein. In certain embodiments, the compositions provided herein have reduced levels of one or more process-specific and / or product-specific impurities than compositions obtained using methods known in the art.

[0051] In some embodiments, the use of the methods reported herein is provided for the purification of Fc-containing heterodimer polypeptides and the reduction of Fc-containing heterodimer polypeptide-related impurities. Improved reduction of product-specific impurities is achieved. In the case of CrossMab-specific impurities, for example, reduction of 3 / 4 antibody is achieved.

[0052] definition The terms “polypeptide” and “protein” are used herein synonymously to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphate chlorination, or any other operation or modification, e.g., conjugation with a labeling component. For example, polypeptides containing one or more analogues of amino acids (including, e.g., non-natural amino acids), and other modifications known in the art are also included in this definition. As used herein, the terms “polypeptide” and “protein” specifically encompass antibodies.

[0053] A “purified” polypeptide (e.g., an antibody or immunoadhesin) means that the polypeptide exists in a purer form than it would in its natural environment, and / or that the purity of the polypeptide is increased when it is first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity. The terms “purify,” “separate,” or “isolate,” as used interchangeably herein, refer to increasing the purity of a desired molecule (e.g., a multispecific antibody, or a bispecific antibody) in a composition or sample containing the desired molecule and one or more impurities. Typically, the purity of a desired molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0054] A "bispecific antibody that binds to a target antigen" is one that is useful as a diagnostic and / or therapeutic agent for targeting a protein, or cells or tissues that express a protein, and that binds the antigen with sufficient affinity so as not to cross-react significantly with other proteins. In such embodiments, the degree of binding of the bispecific antibody to "non-target" proteins is less than about 10% of the binding of the bispecific antibody to its particular target protein, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), or ELISA. With respect to the binding of a bispecific antibody to a target molecule, the terms "specific binding," "specifically binding," or "specific" to a particular polypeptide or epitope on a particular polypeptide target mean a binding that is somewhat different from nonspecific interactions (for example, nonspecific interactions may be the binding of bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of the molecule by comparing it to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled targets. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled targets. The terms “specific binding,” “specifically binding,” or “specific” to an epitope on a particular polypeptide or a particular polypeptide target as used herein may be indicated by a molecule having a Kd of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater affinity to a target.In one embodiment, the term “specific binding” refers to a binding in which a multispecific antigen-binding protein binds to a specific polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0055] "Binding affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., a multispecific antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). For example, Kd may be about 200 nM or less, about 150 nM or less, about 100 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, or about 1 nM or less. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any of these may be used for the purposes of the methods and compositions provided herein.

[0056] In one embodiment, the “Kd” or “Kd value” according to the present invention is measured at 25°C using a surface plasmon resonance assay with BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ) using a CM5 chip with an immobilized target (e.g., antigen) of -10 response units (RUs). Briefly, the carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate (pH 4.8) to 5 μg / mL (approximately 0.2 μM) and then injected at a flow rate of 5 μL / min to obtain a coupled protein of approximately 10 response units (RUs). After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic analysis, 2-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) are injected into PBS (PBST) containing 0.05% Tween20 at a flow rate of approximately 25 uL / min at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated using a simple one-to-one Langmuir coupled model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. off / k on It is calculated as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The binding rate obtained by the above surface plasmon resonance assay is 10 6 M -1 s -1If it exceeds this, the binding rate can be measured using a spectrophotometer such as an Aviv Instruments spectrophotometer with stopped flow or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred red cuvette, and by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band passthrough) of 20 nM anti-antigen antibody (Fab form) (pH 7.2) in PBS at 25°C in the presence of increasing antigen concentrations.

[0057] In this specification, “active” or “active” means the form(s) of a polypeptide that retains the biological and / or immunological activity of a natural or naturally occurring polypeptide; “biological” activity means any biological function (either inhibition or stimulation) caused by a natural or naturally occurring polypeptide other than its ability to induce the production of antibodies against an antigen epitope; and “immunological” activity means the ability of a natural or naturally occurring polypeptide to induce the production of antibodies against an antigen epitope.

[0058] With respect to multispecific antigen-binding proteins provided herein, such as antibodies, fragments, or derivatives thereof, “biologically active,” “biological activity,” and “biological characteristics” mean having the ability to bind biological molecules, unless otherwise specified.

[0059] The term “antibody” as used herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used herein as synonymous with antibody.

[0060] Antibodies are naturally occurring immunoglobulin molecules with diverse structures, all based on immunoglobulin folding. For example, an IgG antibody has two "heavy" chains and two "light" chains that are disulfide-bonded to form a functional antibody. Each heavy and light chain contains a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immune effectors. Antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the antibody's ability to specifically bind to a particular antigen.

[0061] The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. The variability is not evenly distributed across the 110-amino acid length of the variable domain. Instead, the V region consists of relatively invariant extensions called framework regions (FRs) of 15-30 amino acids, separated by shorter, highly variable regions called "hypervariable regions" of 9-12 amino acids each. The variable domains of the natural heavy and light chains each primarily employ a β-sheet configuration, are linked by three hypervariable regions, and contain four FRs that form loops, linking to and sometimes forming part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable region of the other chain by the FRs, contributing to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain is not directly involved in antibody binding to antigens, but it exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC).

[0062] Each V region typically includes three complementarity-determining regions (each a "CDR" containing a "hypervariable loop") and four framework regions. Thus, the antibody binding site, the minimal structural unit required to bind with substantial affinity to a particular desired antigen, typically includes three CDRs and at least three, preferably four, framework regions that intersperse among them to hold and present the CDRs in an appropriate conformation. Classical four-chain antibodies have an antigen binding site defined by the cooperation of a V H domain and a V L domain. Certain antibodies, such as camel and shark antibodies, lack a light chain and rely on a binding site formed by only the heavy chain. Engineered single-domain immunoglobulins can be prepared such that the binding site is formed by only the heavy or light chain if there is no cooperation between the V H and the V L and.

[0063] The term "variable" refers to the fact that certain parts of the variable domain differ significantly within an antibody, and these differences are used to determine the binding affinity and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions within the variable domains of both the light and heavy chains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each primarily employ a β-sheet configuration and contain four FRs that link to the β-sheet structure, forming loops that, in some cases, form part of it, linked by three hypervariable regions. The hypervariable regions in each chain are held in close proximity to the hypervariable region of the other chain by the FRs, contributing to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain is not directly involved in antibody binding to antigens, but it exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC).

[0064] The term "hypervariable region," as used herein, refers to an amino acid residue of an antibody involved in antigen binding. The hypervariable region is also known as the "complementarity-determining region" or "CDR" (e.g., V L So, approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), as well as V H So, roughly speaking, 31-35B(H1), 50-65(H2), and 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or those residues from the "hypervariable loop" (e.g., V LThen, residues 26-32 (L1), 50-52 (L2), and 91-96 (L3), and V H Therefore, it may contain amino acid residues from 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0065] A “framework” or “FR” residue is a variable domain residue other than a hypervariable region residue as defined herein.

[0066] The “hinge region” in relation to an antibody or semi-antibody is generally defined as the extension of human IgG1 from Glu216 to Pro230 (Burton, Molec. Immunol. 22:161-206 (1985). Hinge regions of other IgG isotypes may align with the IgG1 sequence by placing the first and last cysteine ​​residues that form the intra-heavy chain disulfide bond in the same position.

[0067] The "lower hinge region" of the Fc domain is typically defined as the extension of the residue immediately C-terminus of the hinge region, i.e., residues 233-239 of the Fc domain. Prior to this application, FcγR binding was generally attributed to amino acid residues in the lower hinge region of the IgG Fc domain.

[0068] The "CH2 domain" in the human IgG Fc region typically extends from approximately IgG residues 231 to 340. The CH2 domain is unique in that it does not closely pair with another domain. Rather, two N-linked branched carbohydrate chains interpose between the two CH2 domains in an intact, natural IgG molecule. It is hypothesized that the carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985))

[0069] The "CH3 domain" includes an extension of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from approximately amino acid residues 341 to approximately amino acid residues 447 of IgG).

[0070] An "antibody fragment" comprises a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb); linear antibodies (e.g., Example 2 of U.S. Patent No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995); single-arm antibodies, single-variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0071] Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments and one residual "Fc" fragment, denoted to reflect its readily crystallizable ability. The Fab fragment consists of the entire light chain with a variable region domain (VH) of the heavy chain, and a first constant domain (CH1) of one heavy chain. Pepsin treatment of the antibody produces a single large F(ab')2 fragment, which generally corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and can still crosslink to an antigen. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the heretical notation for Fab' in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with a Fab' fragment that has a hinge cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0072] "Fv" is the smallest antibody fragment containing a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form V H -V L The antigen-binding site is defined on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, but with lower affinity than the entire binding site.

[0073] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is the herein notation for Fab' in which the cysteine ​​residue(s) of the constant domain have at least one free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with the Fab' fragment, which has a hinge cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0074] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0075] Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant heavy chain domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0076] "Single-stranded Fv" or "scFv" antibody fragments are the V of the antibody. H and V L It includes domains, where these domains are present in a single polypeptide. In some embodiments, the Fv polypeptide is V H Domain and V L The scFv further contains a polypeptide linker between the domain and the scFv, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0077] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, and these fragments have a light chain variable domain (V) within the same polypeptide chain. L ) connected to a heavy chain variable domain (V H ) including (V H -V LBy using a linker that is too short to allow pairing between two domains on the same chain, a domain can be paired with a complementary domain on another chain, generating two antigen-binding sites. Diabodies are described in more detail, for example, EP404,097, WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0078] As used herein, “hemiantibody” or “hemimer” refers to a monovalent antigen-binding polypeptide. In certain embodiments, the hemiantibody or hemimer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In certain embodiments, the hemiantibody or hemimer comprises one immunoglobulin heavy chain associated with one immunoglobulin light chain, or its antigen-binding fragment. In certain embodiments, the hemiantibody or hemimer is mono-specific, i.e., binds to a single antigen or epitope. Those skilled in the art will readily understand that the hemiantibody may have an antigen-binding domain consisting of a single variable domain, for example, derived from a camelid.

[0079] The term "VH / VL unit" refers to the antigen-binding region of an antibody that includes at least one VH HVR and at least one VL HVR. In certain embodiments, a VH / VL unit includes at least one, at least two, or all three VH HVRs and at least one, at least two, or all three VL HVRs. In certain embodiments, a VH / VL unit further includes at least a portion of the framework region (FR). In some embodiments, a VH / VL unit includes three VH HVRs and three VL HVRs. In some such embodiments, a VH / VL unit includes at least one, at least two, at least three, or all four VH FRs and at least one, at least two, at least three, or all four VL FRs.

[0080] The term "multispecific antibody" is used most broadly to specifically encompass antibodies that contain an antigen-binding domain that has multiple epitope specificity (i.e., can specifically bind to two or more different epitopes on one biological molecule, or can specifically bind to epitopes on two or more different biological molecules). In some embodiments, the antigen-binding domain of a multispecific antibody (such as a bispecific antibody or bivalent F(ab')2) comprises two VH / VL units, where the first VH / VL unit specifically binds to a first epitope, and the second VH / VL unit specifically binds to a second epitope, and each VH / VL unit contains a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies), and antibody fragments that are covalently or noncovalently linked. A VH / VL unit further comprising at least a portion of the heavy chain constant region and / or at least a portion of the light chain constant region may also be referred to as a "hemimer" or "half-antibody." In some embodiments, a half-antibody comprises at least a portion of a single heavy chain variable region and at least a portion of a single light chain variable region. In some such embodiments, a bispecific antibody comprising two half-antibodies and binding to two antigens includes a first half-antibody that binds to a first antigen or first epitope but not to a second antigen or second epitope, and a second half-antibody that binds to a second antigen or second epitope but not to the first antigen or first epitope. According to some embodiments, the multispecific antibody is an IgG antibody that binds to each antigen or epitope with affinity of 5M to 0.001pM, 3M to 0.001pM, 1M to 0.001pM, 0.5M to 0.001pM, or 0.1M to 0.001pM. In some embodiments, the hemimer includes a portion of the heavy chain variable region sufficient to allow the formation of an intramolecular disulfide bond with a second hemimer.In some embodiments, the hemimer includes a knob mutation or a hole mutation that enables heterodimerization with a second hemimer or half-antibody, for example, including a complementary hole mutation or a knob mutation. Knob mutations and hole mutations are discussed further below.

[0081] A “bispecific antibody” is a multispecific antibody that contains an antigen-binding domain capable of specifically binding to two different epitopes on one biological molecule, or to two different epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having “bispecificity” or being “bispecific.” Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody are listed by the bispecific antibody name is random. In some embodiments, a bispecific antibody comprises two half-antibodies, each half-antibody comprising a single heavy chain variable region and optionally at least a portion of the heavy chain constant region, and a single light chain variable region and optionally at least a portion of the light chain constant region. In certain embodiments, a bispecific antibody comprises two half-antibodies, each half-antibody comprising a single heavy chain variable region and a single light chain variable region, but not more than one single heavy chain variable region, and not more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, each half-antibodycete comprising a single heavy chain variable region and a single light chain variable region, wherein the first half-antibodycete binds to the first antigen but not to the second antigen, and the second half-antibodycete binds to the second antigen but not to the first antigen.

[0082] As used herein, the terms “knob-into-hole” or “KiH” technology refer to a technology that aims for in vitro or in vivo pairing of two polypeptides by introducing a bump (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface in which they interact. For example, KiH is introduced at the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, KiH drives the pairing of two different heavy chains during the production of multispecific antibodies. For example, multispecific antibodies having KiH within their Fc regions may further contain a single variable domain linked to each Fc region, or different heavy chain variable domains paired with similar or different light chain variable domains. KiH technology can also be used to pair two different receptor extracellular domains together, or with any other polypeptide sequence containing different target recognition sequences (e.g., including affibodies, peptidebodies, and other Fc fusions).

[0083] As used herein, the term “knob mutation” refers to a mutation that introduces a bump (knob) into a polypeptide at an interface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a whole mutation (see, for example, US5,731,168, US5,807,706, US5,821,333, US7,695,936, and US8,216,805, each of which is incorporated herein by reference in whole).

[0084] As used herein, the term “hole mutation” refers to a mutation that introduces a cavity (hole) into a polypeptide at an interface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation (see, for example, US5,731,168, US5,807,706, US5,821,333, US7,695,936, and US8,216,805, each of which is incorporated herein by reference in whole).

[0085] The terms "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refer to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, a single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as antibodies obtained from recombinant methods using human and mouse antibodies (Nature (1989) 341:544-546, Dev Comp Immunol (2006) 30:43-56, Trend Biochem Sci (2001) 26:230-235, Trends Biotechnol (2003): 21:484-490, WO2005 / 035572, WO03 / 035694, FEBS Lett (1994) 339:285-290, WO00 / 29004, WO02 / 051870).

[0086] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within that population are identical and / or bind to the same epitope, except for any hypothetical variants that may occur during the production of the monoclonal antibody, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies also have the advantage of not being contaminated by other immunoglobulins. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used according to the methods provided herein may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0087] The term "monoclonal antibody" as used herein specifically includes "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody of a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody of a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). The term "chimeric antibody" as used herein includes "primatized" antibodies that include a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys such as baboons, rhesus macaques, or cynomolgus macaques) and a human constant region sequence (U.S. Patent No. 5,693,780).

[0088] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing the minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, with the desired specificity, affinity, and capability. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody contains at least one, typically two, variable domains in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence, except for the aforementioned FR substitutions. Humanized antibodies also optionally contain immunoglobulin constant regions, typically at least a portion of the human immunoglobulin constant region. For further details, see, for example, 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).

[0089] In this specification, "intact antibody" includes heavy and light variable domains, as well as an Fc region. The constant domain may be the constant domain of the natural sequence (e.g., the constant domain of the human natural sequence) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0090] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is attached to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H ) has several constant domains followed by each light chain, with a variable domain (V) at one end. L It has a constant domain at the other end, the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the variable domain of the light chain and the variable domain of the heavy chain.

[0091] A "naked antibody" is an antibody (as defined herein) that is not bound to a heterologous molecule such as a cytotoxic moiety or to a radiolabel.

[0092] As used herein, the term “immunoadhesin” refers to a molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin is an amino acid sequence with desired binding specificity (this amino acid sequence is other than the antigen recognition and binding site of the antibody (i.e., “heterologous” compared to the constant region of the antibody)) and an immunoglobulin constant domain sequence (e.g., the CH2 and / or CH3 sequence of IgG). An exemplary adhesin sequence is a contiguous amino acid sequence containing a portion of a receptor or ligand that binds to the target protein. An adhesin sequence may also be a sequence that binds to the target protein but is not a receptor or ligand sequence (e.g., an adhesin sequence in a peptide body). Such polypeptide sequences can be selected or identified by various methods, including phage display techniques and high-throughput sorting methods. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0093] In certain embodiments, the Fc-domain-containing heterodimer polypeptide is an antibody, a bispecific antibody, or an Fc-fusion protein.

[0094] In certain embodiments, the Fc-fusion protein produced by the method provided herein is a targeted immune cytokine. In certain embodiments, the targeted immune cytokine is a CEA-IL2v immune cytokine. In certain embodiments, the CEA-IL2v immune cytokine is RG7813. In certain embodiments, the targeted immune cytokine is an FAP-IL2v immune cytokine. In certain embodiments, the FAP-IL2v immune cytokine is RG7461.

[0095] In certain embodiments, the multispecific antibody (such as a bispecific antibody) produced by the method provided herein conjugates CEA and at least one additional target molecule. In certain embodiments, the multispecific antibody (such as a bispecific antibody) produced by the method provided herein conjugates a tumor-targeting cytokine and at least one additional target molecule. In certain embodiments, the multispecific antibody produced by the method provided herein is fused to IL2v (i.e., divalent interleukin) and at least one additional target molecule. In certain embodiments, the multispecific antibody produced by the method provided herein is a T-cell bispecific antibody (i.e., a bispecific T-cell engager or BiTE).

[0096] In some embodiments, the "effector function" of an antibody refers to the biological activity resulting from the antibody's Fc region (either the native sequence Fc region or the amino acid sequence variant Fc region), which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors.

[0097] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide, e.g., an antibody) complexed with an alloantigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J.Immunol.Methods202:163 (1996), may be performed.

[0098] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of the target cells. While NK cells, the primary cells mediating ADCC, express only FcγRIII, monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the target molecule, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the target molecule may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0099] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, these cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred.

[0100] The terms “Fc receptor” or “FcR” are used to describe receptors that bind to the Fc region of an antibody. In some embodiments, the FcR is the natural sequence human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and include the FcγRI, FcγRII, and FcγRIII subclass receptors, which include allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine system activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine system inhibitory motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR has been described by Ravetch and Kinet, Annu.Rev.Immunol9:457-92(1991), Capel et al., Immunomethods4:25-34(1994), and de Haas et al., J.Lab.Clin.Med. This is outlined in 126:330-41 (1995). Other FcRs, including those to be identified in the future, are included in the term “FcR” as used herein. This term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)).

[0101] The terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Examples of host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. The offspring may contain mutations, although they may not be entirely identical to the parent cells in terms of nucleic acid content. Mutant offspring having the same function or biological activity as those screened or selected from the initially transformed cells are included herein.

[0102] "Impurities" refer to substances different from the desired polypeptide product. Impurities may include product-specific polypeptides such as one-arm antibodies and misassembled antibodies, antibody variants including basic and acidic variants, and aggregates. Other impurities, though not limited to these, include process-specific impurities such as host cell material (HCPs), leached protein A, nucleic acids, other polypeptides, endotoxins, viral contaminants, and components of cell culture media. In some cases, impurities may be HCPs from bacterial cells, e.g., E. coli cells (ECPs), insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, and fungal cells, for example. In some cases, impurities may be HCPs from mammalian cells, i.e., CHO cells, i.e., CHO cell protein (CHOP). Impurities may also refer to accessory proteins used to facilitate the expression, folding, or assembly of multispecific antibodies, such as prokaryotic chaperones like FkpA, DsbA, and DsbC.

[0103] As used herein, “complex” or “complexed” refers to the association of two or more molecules that interact with each other through non-peptide bonds and / or forces (e.g., van der Waals forces, hydrophobic forces, hydrophilic forces). In one embodiment, the complex is a heteropolymer. As used herein, the terms “protein complex” or “polypeptide complex” should be understood to include complexes having non-protein subjects that are protein-conjugated in a protein complex (e.g., chemical molecules such as toxins or detection agents, but not limited to these).

[0104] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components in their natural environment. Isolated nucleic acids typically contain nucleic acid molecules found within cells that contain nucleic acid molecules, but these nucleic acid molecules exist outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0105] The "amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to achieve the maximum possible sequence identity percentage, gaps have been introduced as necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. In certain embodiments, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559) and is registered under U.S. copyright number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0106] When ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or alternatively, a given amino acid sequence A that has or contains a certain amino acid sequence identity percentage to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y

[0107] In the formula, X is the number of amino acid residues scored as a perfect match in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically indicated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0108] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the antibody's binding to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated from antigen-binding antibodies using the VH or VL domain, and libraries of complementary VL or VH domains can be screened, respectively. For example, see Portolano et al., J.Immunol 150:880-887 (1993) and Clarkson et al., Nature 352:624-628 (1991).

[0109] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is bound. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of the nucleic acid to which they are manipulably linked. Such vectors are referred to herein as “expression vectors.”

[0110] As used herein with respect to chromatography, the term “sequential” refers to the steps of chromatography in a particular sequence, for example, a first chromatographic step, followed by a second chromatographic step, followed by a third chromatographic step. Further steps may be included between the sequential chromatographic steps.

[0111] As used herein with respect to chromatography, the term “continuous” refers to having two chromatographic materials connected directly or via some other mechanism that allows for continuous flow between the first and second chromatographic materials.

[0112] "Loading density" refers to the amount of composition in contact with the volume of chromatography material (e.g., liters) (e.g., grams). In some cases, loading density is expressed in g / L.

[0113] A “sample” refers to a small portion of a larger quantity of material. Generally, tests performed according to the methods described herein are performed on a sample. Samples are typically obtained, for example, from a cultured recombinant polypeptide-expressing cell line (also referred to herein as a “product cell line”) or from a recombinant polypeptide preparation obtained from a cultured host cell. When used herein, “host cell” does not contain the gene for the expression of the recombinant polypeptide or product of interest. Samples may, but are not limited to, a collected cell culture medium, an in-process pool at a particular step of the purification process, or the final purified product. Samples also include diluents, buffers, detergents, and residues found when mixed with contaminant species and the desired molecule (multispecific antibodies, e.g., bispecific antibodies).

[0114] In this specification, references to values ​​or parameters "about" include (and describe) variations relating to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

[0115] It is understood that the aspects and embodiments of the present invention described herein include "including," "consisting of," and "essentially consisting of."

[0116] When used herein and in the appended claims, the singular forms “a,” “or,” and “the” refer to multiple subjects unless the context otherwise explicitly indicates otherwise. The aspects and variations of the invention described herein are understood to include “consisting of” and / or “essentially consisting of” aspects and variations.

[0117] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0118] Method for purifying multispecific antibodies Methods for purifying multispecific antibodies are provided herein. In certain embodiments, the multispecific antibody is a bispecific antibody. In certain embodiments, the multispecific antibody is a bivalent F(ab')2 comprising a first F(ab) that binds to a first target and a second F(ab) that binds to a second target. In certain embodiments, the multispecific antibody is a bispecific antibody, i.e., an antibody having two antigen-binding arms that are identical in amino acid sequence, where each Fab arm is capable of recognizing two antigens (e.g., a dual-acting Fab antibody).

[0119] In some embodiments, the purification of multispecific antibodies includes sequential steps of capture chromatography, a first mixed-mode chromatography, and a second mixed-mode chromatography. In some embodiments, the multispecific antibodies are assembled before capture chromatography. In some embodiments, the multispecific antibodies are assembled after capture chromatography.

[0120] In some embodiments, a multispecific antibody (such as a bispecific antibody or a bivalent F(ab')2) comprises two or more antibody arms, where different antibody arms bind to different epitopes. In certain embodiments, the different epitopes lie on the same antigen. In certain embodiments, each epitope lies on a different antigen. In certain embodiments, the antibody arms contain VH / VL units. In certain embodiments, the antibody arms contain hemimers also known as half-antibodies. To facilitate assembly, in certain embodiments, the heavy chain of one antibody arm is modified to contain a “knob,” and the heavy chain of another antibody arm contains a “hole” such that the knob of the first heavy chain fits into a hole in the second heavy chain.

[0121] In certain embodiments, each arm of the multispecific antibody is produced in a separate cell culture. After expression of the antibody arms in host cells, the whole cell broth is collected and homogenized, and the antibody arms are extracted. In certain embodiments, polyethyleneimine (PEI) is added to the cell lysate before chromatography. In some embodiments, the cell lysate is centrifuged before chromatography. Each arm of the multispecific antibody is then purified by capture chromatography (so that each arm is purified on a separate chromatography column or membrane). In certain embodiments, the capture chromatography is affinity chromatography. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the affinity chromatography is protein G chromatography. In certain embodiments, the affinity chromatography is protein A / G chromatography. In certain embodiments, the affinity chromatography is protein L chromatography. After capture chromatography, the purified antibody arms can be analyzed by, for example, SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody can then be combined and assembled, as will be discussed in more detail elsewhere herein.

[0122] In other embodiments, each arm of the multispecific antibody is produced in a separate cell culture. After expression of the antibody arms in the host cells, the whole cell broth is collected and homogenized. The cell homogenates from each culture are then mixed, and the combined antibody arms are extracted. In some embodiments, polyethyleneimine (PEI) is added to the cell lysate before chromatography. In some embodiments, the cell lysate is centrifuged before chromatography. The combined arms of the multispecific antibody are then purified by affinity chromatography. In some embodiments, the affinity chromatography is protein A chromatography. At this point, the purified antibody arms can be analyzed by, for example, SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody can then be combined and assembled by the methods described herein.

[0123] In other embodiments, each arm of the multispecific antibody is produced in the same cell culture. After the expression of the antibody arms in the host cells, the whole cell broth is collected and homogenized, and the antibody arms are extracted. In some embodiments, polyethyleneimine (PEI) is added to the cell lysate before chromatography. In some embodiments, the cell lysate is centrifuged before chromatography. The multispecific antibody arms are then purified by affinity chromatography. In some embodiments, the affinity chromatography is protein A chromatography. At this point, the purified antibody arms can be analyzed by, for example, SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody can then be assembled by the methods described herein.

[0124] In some embodiments, the final concentration of PEI in the cell lysate is at least about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0%. In some embodiments, the final concentration of PEI in the cell lysate is at least about 0.1% to about 5%, about 0.1% to about 1%, about 0.1% to about 0.5%, about 0.5% to about 5%, about 0.5% to about 1%, or about 1% to about 5%. In some embodiments, the cell lysate containing PEI is held for more than one of the following durations: approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 12 hours, approximately 14 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, or approximately 24 hours. In some embodiments, the cell lysate containing PEI is held for more than one of the following durations: approximately 1 hour to 24 hours, 1 hour to 6 hours, 6 hours to 12 hours, 12 hours to 18 hours, or 18 hours to 24 hours. In some embodiments, the cell lysate containing PEI is held for one of the following durations: approximately 10 hours to 14 hours. In some embodiments, the cell lysate containing PEI is held at approximately 4°C to 37°C. In some embodiments, the cell lysate containing PEI is held at approximately ambient temperature.

[0125] In some embodiments, the cell lysate is purified by centrifugation before chromatography. In some embodiments, the cell lysate is filtered before chromatography. In some embodiments, the cell lysate is filtered through a 0.22 μm filter before chromatography.

[0126] Examples of affinity chromatography include, but are not limited to, protein A chromatography, protein G chromatography, protein A / G chromatography, or protein L chromatography. Examples of affinity chromatography materials include, but are not limited to, ProSep®-vA, ProSep® Ultra Plus, Protein A Sepharose® Fastflow, Toyopearl® AF-r Protein A, MabSelect®, MabSelect SuRe®, MabSelect SuRe® LX, KappaSelect, CaptureSelect®, and CaptureSelect® FcXL. In certain embodiments, the affinity chromatography material is in a column. In certain embodiments, affinity chromatography is performed in a “binding and elution mode” (alternatively referred to as a “binding and elution process”). The “binding and elution mode” refers to a product separation technique in which a product in the sample (such as a multispecific antibody) is bound to the affinity chromatography material and sequentially eluted from the affinity chromatography material. In some embodiments, elution is step elution, in which the composition of the mobile phase is changed stepwise in one or more cases during the elution process. In certain embodiments, elution is gradient elution, in which the composition of the mobile phase is changed continuously during the elution process. In certain embodiments, the affinity chromatography material is a membrane. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the protein A chromatography is MAbSelect SuRe chromatography. In certain embodiments, the affinity chromatography is CaptureSelect chromatography. In certain embodiments, the affinity chromatography is CaptureSelect FcXL chromatography.

[0127] In certain embodiments, the elutes from the affinity chromatography step are sequentially applied to the first mixed-mode chromatography. In certain embodiments, the first mixed-mode material includes functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, pi-pi bond interaction, hydrophilic interaction, thiophilic interaction, and hydrophobic interaction. In certain embodiments, the first mixed-mode material includes functional groups capable of anion exchange and hydrophobic interaction. In certain embodiments, the first mixed-mode material includes functional groups capable of cation exchange and hydrophobic interaction. In certain embodiments, the first mixed-mode material includes N-benzyl-N-methylethanolamine, 4-mercapto-ethylpyridine, 2-benzamido-4-mercaptobutanoic acid, hexylamine, or phenylpropylamine, or crosslinked polyallylamine. Examples of mixed-mode materials include Capto® Adhere resin, Capto® MMC resin, MEP HyperCel® resin, HEA HyperCel® resin, PPA HyperCel® resin, Eshmuno® HCX, Capto® Adhere ImpRes, Capto® MMC ImpRes, and Nuvia® cPrime® membranes. In some embodiments, the first mixed-mode material is Capto® Adhere resin. In certain embodiments, the first mixed-mode material is Capto® Adhere resin. In certain embodiments, the first mixed-mode material is Capto® MMC. In certain embodiments, the first mixed-mode chromatography does not involve ceramic hydroxyapatite chromatography. In certain embodiments, the first mixed-mode chromatography is performed in "binding and elution" mode. In some embodiments, elution is step elution. In certain embodiments, elution is gradient elution. In certain embodiments, the first mixed-mode chromatography is performed in "flow-through" mode. In the above-described embodiments, the first mixed-mode material is in a column. In the above-described embodiments, the first mixed-mode material is in a membrane.

[0128] In certain embodiments, the capture chromatography and the first mixed-mode chromatography are continuous, for example, the capture chromatography material and the first mixed-mode material are either directly connected or connected by some other mechanism that allows for continuous flow between the capture chromatography material and the first mixed-mode material. In certain embodiments, the capture chromatography and the first mixed-mode chromatography are continuous, with the first mixed-mode chromatography performed immediately after the capture chromatography.

[0129] In certain embodiments, the eluent from capture chromatography is subjected to one or more additional chromatographic steps before being applied to the resin in the first mixed-mode chromatography. For example, the eluent from capture chromatography may be subjected to one or more of the following chromatographic steps in any order and / or combination before being subjected to the first mixed-mode chromatography: hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography, hydrophilic interaction liquid chromatography (HILIC), etc.

[0130] Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to their hydrophobicity. Examples of HIC chromatography materials, but not limited to these, include Toyopear® Hexyl-650, Toyopear® Butyl-650, Toyopear® Phenyle-650, Toyopear® I-Ether-650, HiTrap® Sepharose, Octyl Sepharose®, Phenyle Sepharose®, and Butyl Sepharose®. In some embodiments, the HIC chromatography material includes Phenyle Sepharose. In certain embodiments, HIC chromatography is performed in "binding and elution" mode. In some embodiments, HIC chromatography is performed in "flow-through" mode. In some embodiments above, the HIC chromatography material is in a column. In some embodiments above, the HIC chromatography material is in a membrane.

[0131] Anion exchange chromatography materials are solid phases that are positively charged and have free anions for exchange with anions in an aqueous solution (such as a composition containing multispecific antibodies and impurities) that passes through or through the solid phase. In some embodiments of the methods described herein, the anion exchange material may be a membrane, a monolith, or a resin. In one embodiment, the anion exchange material may be a resin. In some embodiments, the anion exchange material may contain a primary amine, secondary amine, tertiary amine, or a quaternary ammonium ion functional group, a polyamine functional group, or a diethylaminoethyl functional group. Examples of anion exchange materials that are known in the art, but are not limited to these, include Poros® HQ50, Poros® PI50, Poros® D, Mustang® Q, Q Sepharose® Fast Flow (QSFF), Accel® containing a quaternary methylamine (QMA) resin, Sartobind STIC®, and DEAE-Sepharose®. In some embodiments, anion exchange chromatography is performed in "binding and elution" mode. In some embodiments, anion exchange chromatography is performed in "flow-through" mode. In some of the above embodiments, the anion exchange chromatography material is in a column. In some of the above embodiments, the anion exchange chromatography material is in a membrane.

[0132] The cation exchange chromatography material is a solid phase that is negatively charged and has free anions for exchange with anions in an aqueous solution (such as a composition containing multispecific antibodies and impurities) that passes through or through the solid phase. In some embodiments of the methods described herein, the cation exchange material may be a membrane, a monolith, or a resin. In some embodiments, the cation exchange material may be a resin. The cation exchange material may include, but is not limited to, carboxylic acid functional groups or sulfonic acid functional groups such as sulfonates, carboxyls, carboxymethylsulfonic acid, sulfisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfoxyethyl, or orthophosphates. In some embodiments above, the cation exchange chromatography material is a cation exchange chromatography column. In some embodiments above, the cation exchange chromatography material is a cation exchange chromatography membrane. Examples of cation exchange materials known in the art include, but are not limited to, Mustang® S, Sartobind® S, SO3 Monolith (e.g., CIM®, CIMmultus®, and CIMac® SO3), S Ceramic HyperD®, Poros® XS, Poros® HS50, Poros® HS20, Sulfopropyl Sepharose® Fast Flow (SPSFF), SP-Sepharose® XL (SPXL), CM Sepharose® Fast Flow, Capto® S, Fractogel® EMD Se Hicap, and Fractogel® EMD SO3. - , or Fractogel® EMD COO -This includes: In some embodiments, cation exchange chromatography is performed in "binding and elution" mode. In some embodiments, cation exchange chromatography is performed in "flow-through" mode. In some embodiments above, the cation exchange chromatography material is in a column. In some embodiments above, the cation exchange chromatography material is in a membrane.

[0133] Hydroxyapatite (Ca 10 The functional groups of (PO4)6(OH)2) chromatography material include positively charged pairs of clusters of six negatively charged oxygen atoms associated with triplets of crystalline calcium ions (C-sites) and crystalline phosphate (P-sites). The C-sites, P-sites, and hydroxyls are distributed in a fixed pattern on the crystalline surface. Proteins are typically absorbed into hydroxyapatite in low concentrations (e.g., 10–25 mM) of phosphate buffer, although certain acidic proteins may be absorbed when loaded into water, saline, or non-phosphate buffers. Proteins are usually eluted by increasing the phosphate gradient, but Ca 2+ Mg 2+ , or Cl - Ion gradients can also be used for selective elution of basic proteins, etc. In some embodiments of the methods described herein, the hydroxyapatite chromatography material may be a resin. In some embodiments, the hydroxyapatite chromatography material may be a resin. In some embodiments described above, the hydroxyapatite chromatography material is a column. Examples of hydroxyapatite chromatography materials known in the art, but not limited to these, include CHT® ceramic hydroxyapatite, CHT ceramic hydroxyapatite type I support, and CHT ceramic hydroxyapatite type II support. In some embodiments, hydroxyapatite chromatography is performed in a "binding and elution" mode. In some embodiments, hydroxyapatite chromatography is performed in a "flow-through" mode.

[0134] In one embodiment, the method reported herein may further include a method for separating a bispecific antibody containing an Fc domain from a solution containing the bispecific antibody, the method comprising (a) contacting the solution with a hydroxyapatite chromatography medium, (b) absorbing the bispecific antibody into the hydroxyapatite chromatography medium, and (c) eluting the bispecific antibody from the hydroxyapatite chromatography medium in the presence of chloride ions, wherein the solution further comprises one or more fragments of a bispecific antibody in which one or more fragments contain an Fc domain, and / or the solution comprises one or more polypeptides having a molecular weight exceeding the molecular weight of the bispecific antibody, and one or more polypeptides further comprising at least one of two heavy chains of a bispecific antibody in which one or more polypeptides contain an Fc domain as referenced in WO2015 / 024896.

[0135] In certain embodiments, the eluted product from capture chromatography is subjected to anion exchange chromatography. In certain embodiments, the anion exchange chromatography material is Q Sepharose® Fast Flow (QSFF). In certain embodiments, the anion exchange chromatography is performed in "binding and elution" mode.

[0136] In certain embodiments, the elutes collected after the first mixed-mode chromatography are sequentially applied to the second mixed-mode chromatography. In certain embodiments, the second mixed-mode material includes functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, pi-pi bond interaction, hydrophilic interaction, thiophilic interaction, and hydrophobic interaction. In certain embodiments, the second mixed-mode material includes functional groups capable of anion exchange and hydrophobic interaction. In certain embodiments, the second mixed-mode material includes functional groups capable of cation exchange and hydrophobic interaction. In certain embodiments, the second mixed-mode material includes N-benzyl-N-methylethanolamine, 4-mercapto-ethylpyridine, 2-benzamido-4-mercaptobutanoic acid, hexylamine, or phenylpropylamine, or crosslinked polyallylamine. Examples of mixed-mode materials include Capto® Adhere resin, Capto® MMC resin, MEP HyperCel® resin, HEA HyperCel® resin, Eshmuno® HCX, Capto® Adhere ImpRes, Capto® MMC ImpRes, and Nuvia® cPrime® membranes. In some embodiments, the second mixed-mode material is Capto® Adhere resin. In certain embodiments, the twelfth mixed-mode material is Capto® Adhere resin. In certain embodiments, the second mixed-mode material is Capto® MMC. In certain embodiments, the second mixed-mode chromatography does not involve ceramic hydroxyapatite chromatography. In certain embodiments, the second mixed-mode chromatography is performed in "binding and elution" mode. In some embodiments, elution is step elution. In certain embodiments, elution is gradient elution. In certain embodiments, the first mixed-mode chromatography is performed in "flow-through" mode. In one particular embodiment described above, the second mixed-mode material is located in the column. In another particular embodiment described above, the second mixed-mode material is located in the membrane.

[0137] In certain embodiments, the first mixed-mode chromatography and the second mixed-mode chromatography are continuous, and for example, the capture chromatography material and the first mixed-mode material are either directly connected or connected by some other mechanism that allows for continuous flow between the capture chromatography material and the first mixed-mode material. In certain embodiments, the first mixed-mode chromatography and the second mixed-mode chromatography are continuous, and the second mixed-mode chromatography is performed immediately after the first mixed-mode chromatography.

[0138] In certain embodiments, the eluent from the first mixed-mode chromatography is subjected to one or more additional chromatographic operations before being applied to the resin in the second mixed-mode chromatography. For example, the eluent from the first mixed-mode chromatography may be subjected to one or more of the following chromatographic steps in any order and / or combination before being subjected to the second mixed-mode chromatography: hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography, hydrophilic interaction liquid chromatography (HILIC), etc.

[0139] In certain embodiments of any of the methods described herein, the eluent from the second mixed-mode chromatography is subjected to one or more additional chromatographic steps. For example, the eluent from the second mixed-mode chromatography may be subjected to one or more of the following chromatographic steps in any order and / or any combination: hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography, hydrophilic interaction liquid chromatography (HILIC), mixed-mode chromatography, etc.

[0140] In certain embodiments of any of the methods described herein, the method includes the use of a buffer. Various buffers may be used during the purification of multispecific antibodies, depending, for example, the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the multispecific antibody to be purified, and the purification method. The buffer may be a loading buffer, an equilibration buffer, or a washing buffer. In certain embodiments, one or more of the loading buffer, equilibration buffer, and / or washing buffers are the same. In certain embodiments, the loading buffer, equilibration buffer, and / or washing buffers are different. In certain embodiments of any of the methods described herein, the buffer contains a salt. In certain embodiments, the buffer contains sodium chloride, sodium acetate, Tris HCl, Tris acetate, sodium phosphate, potassium phosphate, MES, CHES, MOPS, Bistris, arginine, arginine HCl, or a mixture thereof. In certain embodiments, the buffer is a sodium chloride buffer. In some embodiments, the buffer is a sodium acetate buffer. In certain embodiments, the buffer is Tris, arginine, phosphate, MES, CHES, or MOPS buffer.

[0141] "Loading material" refers to the composition to be loaded into the chromatography material. The loading buffer is a buffer used to load the composition (e.g., a composition containing a multispecific antibody and impurities, or a composition containing antibody arms and impurities) into the chromatography material (such as any one of the chromatography materials described herein). The chromatography material may be equilibrated with an equilibration buffer before loading the composition to be purified. The washing buffer is used after the composition has been loaded into the chromatography material. The elution buffer is used to elute the polypeptide of interest from the solid phase.

[0142] The loading of a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) onto any of the chromatographic materials described herein may be optimized for the purification of the multispecific antibody from impurities. In some embodiments, the loading of a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) onto a chromatographic material is optimized for the binding of the multispecific antibody to the chromatographic material when the chromatography is performed in binding and elution modes (e.g., affinity chromatography, mixed-mode chromatography, and ion-exchange chromatography as designed herein).

[0143] Conductivity refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, as the amount of ions present in the aqueous solution increases, the solution will have higher conductivity. The basic unit of conductivity is Siemens (mS / cm) or ohm (mho), and it can be measured using a conductivity meter, such as various models of Orion conductivity meters. Since electrolyte conductivity is the ability of ions in a solution to carry electric current, the conductivity of a solution can be altered by changing the concentration of ions in it. For example, the concentration of buffers and / or salts (e.g., sodium chloride, sodium acetate, or potassium chloride) in a solution can be changed to achieve a desired conductivity. Preferably, the salt concentrations of various buffers are modified to achieve a desired conductivity.

[0144] For example, in a particular embodiment, a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) is loaded onto a chromatography column containing a chromatography material in a loading buffer with several different pH values, e.g., one of the chromatography materials described herein, while maintaining a constant conductivity of the loading buffer. Alternatively, a solution containing a multispecific antibody may be loaded onto a chromatography material in a loading buffer with several different conductivity values, while maintaining a constant pH of the loading buffer. When a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) is loaded onto a chromatography material and the product is eluted from the chromatography material into a pool fraction, the amount of impurities remaining in the pool fraction provides information about the separation of the multispecific antibody from the impurities for a given pH or conductivity. Similarly, for chromatography, if the multispecific antibody penetrates the chromatography material, the loading buffer is optimized for pH and conductivity such that the multispecific antibody penetrates the chromatography, but the impurities are either retained by the chromatography material or penetrate the chromatography material at a different rate than the multispecific antibody.

[0145] In some embodiments, the loading density of a solution containing a multispecific antibody or antibody arm exceeds one of the following affinity chromatography material concentrations: approximately 10 g / L, approximately 20 g / L, approximately 30 g / L, approximately 40 g / L, approximately 50 g / L, approximately 60 g / L, approximately 70 g / L, approximately 80 g / L, approximately 90 g / L, approximately 100 g / L, approximately 110 g / L, approximately 120 g / L, approximately 130 g / L, approximately 140 g / L, or approximately 150 g / L. In some embodiments, the loading density of the solution containing the multispecific antibody or antibody arm is approximately 10 g / L to approximately 20 g / L, approximately 20 g / L to approximately 30 g / L, approximately 30 g / L to approximately 40 g / L, approximately 40 g / L to approximately 50 g / L, approximately 50 g / L to approximately 60 g / L, approximately 60 g / L to approximately 70 g / L, approximately 70 g / L to approximately 80 g / L, approximately 80 g / L to approximately 90 g / L, and approximately 90 g / L to approximately 100 g / L of the capture chromatography material (affinity chromatography material, e.g., protein A chromatography material, protein G chromatography material, protein A / G chromatography material, or protein L chromatography material).

[0146] In some embodiments of the methods described herein, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material (e.g., Q Sepharose® Fastflow (QSFF)). In some embodiments of the methods described herein, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material at a loading density of multispecific antibodies in the anion chromatography material (e.g., Q Sepharose® Fastflow (QSFF)) exceeding any of the following: about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L. In some embodiments, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material (e.g., Q Sepharose® Fast Flow (QSFF)) at a loading density of multispecific antibodies in the anion exchange chromatography material, which can be found to be approximately 10 g / L to 20 g / L, 20 g / L to 30 g / L, 30 g / L to 40 g / L, 40 g / L to 50 g / L, 50 g / L to 60 g / L, 60 g / L to 70 g / L, 70 g / L to 80 g / L, 80 g / L to 90 g / L, or 90 g / L to 100 g / L.

[0147] In some embodiments of the methods described herein, the eluate obtained after capture chromatography (optionally, after capture chromatography and one or more additional chromatographic steps including any of the chromatographic operations described herein) is loaded onto the first mixed-mode chromatography material (e.g., Capto® Adhere chromatography material or Capto® MMC chromatography material) at a loading density of multispecific antibodies exceeding any of the following: about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L. In some embodiments, the eluate obtained after capture-mode chromatography is loaded onto the first mixed-mode chromatography material (e.g., Capto® Adhere chromatography material or Capto® MMC chromatography material) at a loading density of multispecific antibodies of approximately 10 g / L to approximately 20 g / L, approximately 20 g / L to approximately 30 g / L, approximately 30 g / L to approximately 40 g / L, approximately 40 g / L to approximately 50 g / L, approximately 50 g / L to approximately 60 g / L, approximately 60 g / L to approximately 70 g / L, approximately 70 g / L to approximately 80 g / L, approximately 80 g / L to approximately 90 g / L, or approximately 90 g / L to approximately 100 g / L.

[0148] In some embodiments of the methods described herein, the eluate obtained after the first mixed-mode chromatography is loaded onto a second mixed-mode chromatography material (e.g., Capto® Adhere chromatography material or Capto® MMC chromatography material) at a loading density of multispecific antibodies exceeding any of the following: about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L. In some embodiments, the eluate obtained after the first mixed-mode chromatography is loaded onto a second mixed-mode chromatography material at a multispecific antibody loading density of approximately 10 g / L to 20 g / L, 20 g / L to 30 g / L, 30 g / L to 40 g / L, 40 g / L to 50 g / L, 50 g / L to 60 g / L, 60 g / L to 70 g / L, 70 g / L to 80 g / L, 80 g / L to 90 g / L, or 90 g / L to 100 g / L for the mixed-mode chromatography material (e.g., Capto® Adhere chromatography material or Capto® MMC chromatography material).

[0149] In some embodiments of the methods described herein, the eluate obtained after the second mixed-mode chromatography is loaded onto sequential chromatography materials (such as hydrophobic interaction (HIC) chromatography materials, anion exchange chromatography materials, cation exchange chromatography materials, size exclusion chromatography materials, affinity chromatography materials, or additional mixed-mode chromatography materials) at a loading density of multispecific antibodies exceeding any of the following: about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L. In some embodiments, the eluate obtained after the second mixed-mode chromatography is loaded onto sequential chromatography materials (such as hydrophobic interaction (HIC) chromatography materials, anion exchange chromatography materials, cation exchange chromatography materials, size exclusion chromatography materials, affinity chromatography materials, or additional mixed-mode chromatography materials) at a loading density of multispecific antibodies in one of the following sequential chromatography materials: approximately 10 g / L to approximately 20 g / L, approximately 20 g / L to approximately 30 g / L, approximately 30 g / L to approximately 40 g / L, approximately 40 g / L to approximately 50 g / L, approximately 50 g / L to approximately 60 g / L, approximately 60 g / L to approximately 70 g / L, approximately 70 g / L to approximately 80 g / L, approximately 80 g / L to approximately 90 g / L, or approximately 90 g / L to approximately 100 g / L.

[0150] Elution, as used herein, is the removal of a product, such as a multispecific antibody or an arm of an antibody, from the chromatographic material. Elution buffer is a buffer used to elute a multispecific antibody or other product of interest from the chromatographic material. Often, elution buffers have different physical characteristics from the loading buffer. For example, elution buffers may have different conductivity or pH than the loading buffer. In some embodiments, elution buffers have lower conductivity than the loading buffer. In some embodiments, elution buffers have higher conductivity than the loading buffer. In some embodiments, elution buffers have lower pH than the loading buffer. In some embodiments, elution buffers have higher pH than the loading buffer. In some embodiments, elution buffers have different conductivity and pH than the loading buffer. Elution buffers may have any combination of higher or lower conductivity and higher or lower pH.

[0151] In certain embodiments, the elution of a multispecific antibody from a chromatographic material is optimized for product production with minimal impurities and minimum elution volume or pool volume. For example, a composition containing a multispecific antibody (e.g., a bispecific antibody) or antibody arm may be loaded into a chromatographic material, e.g., a chromatographic column, in a loading buffer. Once loading is complete, the multispecific antibody or antibody arm is eluted in buffers of several different pH values ​​while maintaining a constant conductivity of the elution buffer. Alternatively, the multispecific antibody or antibody arm may be eluted from the chromatographic material in elution buffers of several different conductivity values ​​while maintaining a constant pH of the elution buffer. Once the elution of the multispecific antibody (e.g., a bispecific antibody) or antibody arm from the chromatographic material is complete, the amount of impurities in the pool fraction provides information about the separation of the multispecific antibody or antibody arm from impurities for a given pH or conductivity. Elution of a multispecific antibody or antibody arm in multiple column volumes (e.g., 8 column volumes) exhibits "tailing" of the elution profile. In some embodiments, elution tailing is minimized.

[0152] For example, various buffers may be used depending on the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the protein of interest, the chromatographic material, and the purification process (e.g., “binding and elution” or “flow-through” mode). In some embodiments of the methods described herein, the method includes the use of at least one buffer. The buffer may be a loading buffer, an equilibration buffer, an elution buffer, or a wash buffer. In some embodiments, one or more of the loading buffer, equilibration buffer, elution buffer, and / or wash buffer (such as loading buffers, equilibration buffers, and / or wash buffers used for any additional chromatography such as capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) are the same. In some embodiments, the loading buffer, equilibration buffer, and / or wash buffer (used for any additional chromatography such as capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) differs. In some embodiments of the methods described herein, the buffer contains a salt. The loading buffer (used for any additional chromatography such as capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) may contain sodium chloride, sodium acetate, Tris, arginine, phosphate, MOPS, MES, CHES, Bistris, ammonium sulfate, sodium phosphate, citrate, succinate, or mixtures thereof.In certain embodiments, the buffer is a sodium chloride buffer. In some embodiments, the buffer is a sodium acetate buffer. In certain embodiments, the buffer is a Tris, arginine, phosphate, MES, CHES, or MOPS buffer. In some embodiments, the buffer contains Tris. In some embodiments, the buffer contains arginine.

[0153] In some embodiments of the methods described herein, the loading buffer (such as the loading buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is approximately 1.0 mS / It has a conductivity greater than any of the following: cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, or approximately 20 mS / cm. The conductivity can be any of the following: approximately 1 mS / cm to approximately 20 mS / cm, approximately 4 mS / cm to approximately 10 mS / cm, approximately 4 mS / cm to approximately 7 mS / cm, approximately 5 mS / cm to approximately 17 mS / cm, approximately 5 mS / cm to approximately 10 mS / cm, or approximately 5 mS / cm to approximately 7 mS / cm. In some embodiments, the conductivity is one of approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, or approximately 20 mS / cm. In one embodiment, the conductivity is the conductivity of the loading buffer, equilibration buffer, and / or washing buffer.In some embodiments, the conductivity of one or more of the loading buffer, equilibration buffer, and / or wash buffer (such as loading buffer, equilibration buffer, and / or wash buffer used for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is the same. In some embodiments, the conductivity of the loading buffer is different from that of the wash buffer and / or equilibration buffer.

[0154] In some embodiments, the elution buffer (such as the elution buffer for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a conductivity less than that of the loaded buffer. In some embodiments of the methods described herein, the elution buffer (such as the elution buffer for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a conductivity less than any of the following: about 0 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, or about 7.0 mS / cm. The conductivity can be any of the following: approximately 0 mS / cm to approximately 7 mS / cm, approximately 1 mS / cm to approximately 7 mS / cm, approximately 2 mS / cm to approximately 7 mS / cm, approximately 3 mS / cm to approximately 7 mS / cm, or approximately 4 mS / cm to approximately 7 mS / cm, approximately 0 mS / cm to approximately 5.0 mS / cm, approximately 1 mS / cm to approximately 5 mS / cm, approximately 2 mS / cm to approximately 5 mS / cm, approximately 3 mS / cm to approximately 5 mS / cm, or approximately 4 mS / cm to approximately 5 mS / cm.In some embodiments, the conductivity of the elution buffer (such as the elution buffer for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is one of approximately 0 mS / cm, approximately 0.5 mS / cm, approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, or approximately 7.0 mS / cm.

[0155] In some embodiments, the elution buffer has a conductivity exceeding that of the loaded buffer. In some embodiments of the methods described herein, the elution buffer (e.g., elution buffer for any additional chromatography such as capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has conductivity of approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 It has a conductivity greater than any of the following: mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, approximately 11 mS / cm, approximately 12 mS / cm, approximately 13 mS / cm, approximately 14 mS / cm, approximately 15 mS / cm, approximately 16 mS / cm, approximately 17.0 mS / cm, approximately 18.0 mS / cm, approximately 19.0 mS / cm, approximately 20.0 mS / cm, approximately 21.0 mS / cm, approximately 22.0 mS / cm, approximately 23.0 mS / cm, approximately 24.0 mS / cm, approximately 25.0 mS / cm, approximately 26.0 mS / cm, approximately 27.0 mS / cm, approximately 28.0 mS / cm, approximately 29.0 mS / cm, or approximately 30.0 mS / cm. The conductivity can be any of the following: approximately 5.5 mS / cm to approximately 30 mS / cm, approximately 6.0 mS / cm to approximately 30 mS / cm, approximately 7 mS / cm to approximately 30 mS / cm, approximately 8 mS / cm to approximately 30 mS / cm, approximately 9 mS / cm to approximately 30 mS / cm, or approximately 10 mS / cm to approximately 30 mS / cm.In some embodiments, the conductivity of the elution buffer is approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, approximately 11 mS / cm, approximately 12 mS / cm, approximately 13 mS / cm, approximately 14 mS / cm, approximately 15 mS / cm, and approximately 16 mS The conductivity is one of the following: 1 / cm, approximately 17.0 mS / cm, approximately 18.0 mS / cm, approximately 19.0 mS / cm, approximately 20.0 mS / cm, approximately 21.0 mS / cm, approximately 22.0 mS / cm, approximately 23.0 mS / cm, approximately 24.0 mS / cm, approximately 25.0 mS / cm, approximately 26.0 mS / cm, approximately 27.0 mS / cm, approximately 28.0 mS / cm, approximately 29.0 mS / cm, or approximately 30.0 mS / cm. In some aspects of any of the embodiments described above, the conductivity of the elution buffer (e.g., elution buffer for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is changed from the charge and / or wash buffer by a stepwise or linear gradient.

[0156] In some embodiments, a solution containing a multispecific antibody is loaded onto a first mixed-mode chromatography material in a loading buffer having a conductivity of about <6.5 mS / cm, and the polypeptide is eluted from the first mixed-mode chromatography material in an elution buffer having a conductivity of about 1.5 mS / cm. In some embodiments, the loading buffer has a conductivity of about 6.5 mS / cm and the elution buffer has a conductivity of about 3 mS / cm. In some embodiments, the loading buffer has a conductivity of about 5.5 mS / cm and the elution buffer has a conductivity of about 2 mS / cm. In some embodiments, the loading buffer has a conductivity of about 5.5 mS / cm and the elution buffer has a conductivity of about 1 mS / cm. In further embodiments of the above embodiments, the first mixed-mode chromatography material is Capto® Adhere resin. In further embodiments of the above embodiments, the first mixed-mode chromatography material is Capto® MMC resin.

[0157] In some of the embodiments described above, the conductivity of the elution buffer is altered from the load and / or wash buffer by a stepwise or linear gradient. In some embodiments, a composition containing a multispecific antibody is loaded into a first mixed-mode chromatography (e.g., Capto® Adhere chromatography or Capto® MMC chromatography) at <6.5 mS / cm, and the multispecific antibody is eluted from the first mixed-mode chromatography by a stepwise conductivity gradient down to about 1.5 mS / cm.

[0158] In some embodiments, a solution containing a multispecific antibody is loaded onto a second mixed-mode chromatography material in a loading buffer having a conductivity of about <6.5 mS / cm, and the polypeptide is eluted from the second mixed-mode chromatography material in an elution buffer having a conductivity of about 1.5 mS / cm. In some embodiments, the loading buffer has a conductivity of about 6.5 mS / cm and the elution buffer has a conductivity of about 3 mS / cm. In some embodiments, the loading buffer has a conductivity of about 5.5 mS / cm and the elution buffer has a conductivity of about 2 mS / cm. In some embodiments, the loading buffer has a conductivity of about 5.5 mS / cm and the elution buffer has a conductivity of about 1 mS / cm. In further embodiments of the above embodiments, the second mixed-mode chromatography material is Capto® Adhere resin. In further embodiments of the above embodiments, the second mixed-mode chromatography material is Capto® MMC resin.

[0159] In some of the embodiments described above, the conductivity of the elution buffer is altered from the load and / or wash buffer by a stepwise or linear gradient. In some embodiments, a composition containing a multispecific antibody is loaded into a second mixed-mode chromatography (e.g., Capto® Adhere chromatography or Capto® MMC chromatography) at <6.5 mS / cm, and the multispecific antibody is eluted from the second mixed-mode chromatography by a stepwise conductivity gradient down to about 1.5 mS / cm.

[0160] In some embodiments, a composition containing a multispecific antibody is loaded into an anion exchange chromatography (e.g., QSFF chromatography) at <2.5 mS / cm, and the multispecific antibody is eluted from the anion exchange chromatography by a stepwise conduction gradient up to approximately 8.6 mS / cm.

[0161] In some embodiments, a composition containing a multispecific antibody is loaded into a cation exchange chromatograph (e.g., POROS50HS chromatography) at approximately 5.0 mS / cm, and the multispecific antibody is eluted from the cation exchange chromatograph by a stepwise conduction gradient up to approximately 27.5 mS / cm.

[0162] In some embodiments of the methods described herein, the loading buffer (such as a loading buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH less than any of about 10, about 9, about 8, about 7, about 6, or about 5, and includes any range between these values. In some embodiments of the methods described herein, the loading buffer (such as a loading buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH greater than any of about 4, about 5, about 6, about 7, about 8, or about 9, and includes any range between these values. The loading buffer (such as the loading buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH of approximately 4 to approximately 9, approximately 4 to approximately 8, approximately 4 to approximately 7, approximately 5 to approximately 9, approximately 5 to approximately 8, approximately 5 to approximately 7, and approximately 5 to approximately 6, and includes any range between these values.In some embodiments, the pH of the loading buffer (such as the loading buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is one of the following pH values: about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8, and includes any range between these values. The pH may be the pH of the loading buffer, equilibration buffer, or wash buffer (such as the loading buffer, equilibration buffer, and / or wash buffer used for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.). In some embodiments, the pH of one or more of the loading buffer, equilibration buffer, and / or wash buffer is the same. In some embodiments, the pH of the loading buffer is different from the pH of the equilibration buffer and / or wash buffer.

[0163] In some embodiments, the elution buffer (e.g., elution buffer for any additional chromatography such as capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH less than the pH of the loaded buffer. In some embodiments of the methods described herein, the elution buffer has a pH less than any of about 8, about 7, about 6, about 5, about 4, about 3, or about 2, and includes any range between these values. The pH of the elution buffer may be any of about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 9, about 6 to about 8, or about 6 to about 7, and includes any range between these values. In some embodiments, the pH of the elution buffer (such as the elution buffer for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is one of approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, or approximately 9.0, and includes any range between these values.

[0164] In some embodiments, the elution buffer (such as the elution buffer for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH greater than the pH of the loaded buffer. In some embodiments of the methods described herein, the elution buffer (such as the elution buffer for first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) has a pH greater than any of about 5, about 6, about 7, about 8, or about 9, and includes any range between these values. In some embodiments of the methods described herein, the elution buffer (such as the elution buffer for capture chromatography) has a pH greater than any of about 2, about 4, or about 4, and includes any range between these values. The pH of the elution buffer (such as the elution buffer for any additional chromatography, including capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) may be any of the following: about 2 to about 9, about 3 to about 9, about 4 to about 9, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 2 to about 7, about 3 to about 7, about 4 to about 7, about 2 to about 6, about 3 to about 6, and about 4 to about 6, and include any range between these values. In some embodiments, the pH of the elution buffer is any of the following: about 2.0, about 2.5, about 3.0, about 3.5, and about 4.0, and includes any range between these values.

[0165] In some embodiments, a solution containing a multispecific antibody or antibody arm is loaded into an affinity chromatograph (e.g., protein A chromatography) at approximately pH 7, and the multispecific antibody or antibody arm is eluted from the affinity chromatograph by a step gradient down to pH approximately 2.9.

[0166] In some of the embodiments described above, the pH of the elution buffer (such as the elution buffer for capture chromatography, first mixed-mode chromatography, second mixed-mode chromatography, and / or any additional chromatography such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, additional mixed-mode chromatography, etc.) is changed from the charge and / or wash buffer by a stepwise or linear gradient.

[0167] In some embodiments of the methods described herein, the flow rate is less than one of about 50 CV / hour, about 40 CV / hour, or about 30 CV / hour. The flow rate may be one of the following: about 5 CV / hour to about 50 CV / hour, about 10 CV / hour to about 40 CV / hour, or about 18 CV / hour to about 36 CV / hour. In some embodiments, the flow rate is one of the following: about 9 CV / hour, about 18 CV / hour, about 25 CV / hour, about 30 CV / hour, about 36 CV / hour, or about 40 CV / hour. In some embodiments of the methods described herein, the flow rate is less than one of about 100 cm / hour, about 75 cm / hour, or about 50 cm / hour. The flow rate may be one of the following: about 25 cm / hour to about 150 cm / hour, about 25 cm / hour to about 100 cm / hour, about 50 cm / hour to about 100 cm / hour, or about 65 cm / hour to about 85 cm / hour.

[0168] The floor height is the height of the chromatography material used. In some embodiments of the methods described herein, the floor height exceeds one of the following: about 5 cm, about 10 cm, about 15 cm, about 20 cm, about 25 cm, about 30 cm, about 35 cm, about 40 cm, about 45 cm, or about 50 cm. In some embodiments, the floor height is between about 5 cm and about 50 cm. In some embodiments, the floor height is determined based on the amount of polypeptide or impurities in the load.

[0169] In some embodiments, chromatography is performed in approximately 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, and 600 mL. The contents are in a column or container with a volume of approximately 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 25 L, 50 L, 100 L, 200 L, 300 L, 400 L, 500 L, 600 L, 700 L, 800 L, 900 L, or more than 1000 L.

[0170] In some embodiments, the fractions are collected from chromatography. In some embodiments, the collected fractions exceed about 0.01 CV, about 0.02 CV, about 0.03 CV, about 0.04 CV, about 0.05 CV, about 0.06 CV, about 0.07 CV, about 0.08 CV, about 0.09 CV, about 0.1 CV, about 0.2 CV, about 0.3 CV, about 0.4 CV, about 0.5 CV, about 0.6 CV, about 0.7 CV, about 0.8 CV, about 0.9 CV, about 1.0 CV, about 2.0 CV, about 3.0 CV, about 4.0 CV, about 5.0 CV, about 6.0 CV, about 7.0 CV, about 8.0 CV, about 9.0 CV, or about 10.0 CV.

[0171] In certain embodiments, fractions containing purified or partially purified products, such as multispecific antibodies (bispecific antibodies or bivalent F(ab')2) or antibody arms or Fabs, are pooled. The amount of polypeptide in the fraction can be determined by those skilled in the art, for example, by ultraviolet spectroscopy. In certain embodiments, the fraction is OD 280 However, if it exceeds any of the following: about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0, it is collected. In a particular embodiment, the fraction is OD 280 However, fractions are collected if they are in one of the following ranges: approximately 0.5 to approximately 1.0, approximately 0.6 to approximately 1.0, approximately 0.7 to approximately 1.0, approximately 0.8 to approximately 1.0, or approximately 0.9 to approximately 1.0. In certain embodiments, fractions containing multispecific antibodies (e.g., bispecific antibodies) or arms of antibodies are pooled.

[0172] In certain embodiments of any of the methods described herein, the impurities are product-specific impurities. Examples of product-specific impurities include, but are not limited to, non-paired hemiantibodies, non-paired antibody light chains, non-paired heavy chains, antibody fragments, homodimers (e.g., paired hemidimers of bispecific antibodies containing the same heavy and light chains), aggregates, high molecular weight species (MHWS) (very high molecular weight species (vHMWS)), multispecific antibodies with mispaired disulfides, light chain dimers, heavy chain dimers, low molecular weight species (LMWS), and charged variants (such as acidic and basic variants of antibodies).

[0173] In certain embodiments, the methods provided herein remove or reduce the level of non-reciprocal antibodies from a composition containing multispecific antibodies (e.g., bispecific antibodies) and non-reciprocal antibodies. Methods for determining the presence or level of non-reciprocal antibodies in a composition are known in the art and include, for example, mass spectrometry (e.g., liquid chromatography-mass spectrometry), CE-SDS, reverse-phase HPLC, and HIC HPLC. In certain embodiments of any of the methods described herein, the amount of non-reciprocal antibodies in the composition recovered from one or more purification steps (e.g., a chromatographic fraction) is reduced to more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and include any range between these values. In a particular embodiment, the amount of non-reciprocal antibody (such as a chromatographic fraction) in the composition recovered from one or more purification steps is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%. In some embodiments, the amount of non-reciprocal antibodies in the composition (e.g., chromatographic fraction) is reduced to one of the following percentages: approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In certain embodiments, the presence or reduction in the level of non-reciprocal antibodies is measured by comparing the amount of non-reciprocal antibodies in the composition recovered from the purification step(s) (e.g., chromatographic fraction) with the amount of non-reciprocal antibodies in the composition before the purification step(s).

[0174] In certain embodiments, the methods provided herein remove and reduce the levels of homodimers from a composition containing a multispecific antibody (e.g., a bispecific antibody) and homodimers. Methods for determining the presence or level of homodimers in a composition are known in the art and include, for example, mass spectrometry (e.g., liquid chromatography-mass spectrometry), reverse-phase HPLC, and HIC HPLC. In certain embodiments of any of the methods described herein, the amount of homodimers in the composition recovered from one or more purification steps (e.g., a chromatographic fraction) is reduced to more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and include any range between these values. In a particular embodiment, the amount of homodimers (such as a chromatographic fraction) in the composition recovered from one or more purification steps is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%. In some embodiments, the amount of homodimers in the composition (e.g., a chromatographic fraction) is reduced to one of the following: about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In certain embodiments, the presence or reduction in the level of homodimers is measured by comparing the amount of homodimers in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of homodimers in the composition before the purification step(s).

[0175] In certain embodiments, the methods provided herein remove or reduce the level of high molecular weight species (HMWS) proteins from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and HMWS proteins. HMWS proteins may include, for example, aggregated polypeptides (e.g., aggregated multispecific antibodies, aggregated half-antibodies, aggregated homodimers, etc.). In certain embodiments, aggregated polypeptides include heavy chain polymers, light chain polymers, and / or polymers of multispecific antibodies. HMWS proteins may include 2, 3, 4, 5, 6, 7, or 8 or more monomers of heavy or light chains, or 2, 3, 4, 5, 6, 7, or 8 or more aggregated multispecific antibodies. Methods for measuring aggregated proteins (e.g., HMWS proteins) are known in the art and are described, for example, in WO2011 / 150110. Such methods include, for example, size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), and liquid chromatography-mass spectrometry (LC-MS). In certain embodiments of any of the methods described herein, the amount of HMWS protein in the composition recovered from one or more purification steps (e.g., the chromatographic fraction) is reduced to more than one of the following: about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and includes any range between these values. In a particular embodiment, the amount of HMWS protein in the composition recovered from one or more purification steps (such as a chromatographic fraction) is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%.In some embodiments, the amount of HMWS protein in the composition (e.g., a chromatographic fraction) is reduced to one of the following percentages: approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In certain embodiments, the presence or reduction in the level of HMWS protein is measured by comparing the amount of HMWS protein in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of HMWS protein in the composition before the purification step(s).

[0176] In certain embodiments, the methods provided herein remove or reduce the level of low molecular weight species (LMWS) proteins from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and an LMWS protein. LMWS proteins may include fragmented polypeptides. In certain embodiments, the fragmented polypeptide is a fragment of a multispecific antibody, a fragment of an antibody arm, a heavy chain fragment, or a light chain fragment. Examples of LMWS proteins, but not limited to these, include Fab (i.e., fragment antigen-binding), Fc (fragment, crystallizable), a region, or a combination of both, or any randomly fragmented portion of the multispecific antibody, heavy chain, or light chain of interest, or a 1 / 2 antibody (containing a light / heavy chain pair of one antibody) or a 3 / 4 antibody (also indicated herein as HHL, containing heterodimers or homodimers of antibody heavy and antibody light chains). Methods for measuring fragmented proteins (e.g., LMWS proteins) are known in the art and are described, for example, in WO2011 / 150110. Such methods include, for example, size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), and liquid chromatography-mass spectrometry (LC-MS). In certain embodiments of any of the methods described herein, the amount of LMWS protein in the composition recovered from one or more purification steps (e.g., the chromatographic fraction) is reduced to more than one of the following: about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and includes any range between these values.In a particular embodiment, the amount of LMWS protein in the composition recovered from one or more purification steps (such as a chromatographic fraction) is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%. In some embodiments, the amount of LMWS protein in the composition (e.g., a chromatographic fraction) is reduced to one of the following percentages: approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In certain embodiments, the presence or reduction in the level of LMWS protein is measured by comparing the amount of LMWS protein in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of LMWS protein in the composition before the purification step(s).

[0177] In certain embodiments, methods provided herein remove or reduce the levels of acidic and / or basic variants from multispecific antibodies (e.g., bispecific antibodies) and compositions containing acidic and / or basic variants. Acidic variants of antibodies (multispecific antibodies, e.g., bispecific antibodies) are variants in which the pI of the antibody is less than the pI of the naturally intact antibody. Basic variants of antibodies (multispecific antibodies, e.g., bispecific antibodies) are variants in which the pI of the antibody is greater than the pI of the naturally intact antibody. Such charged variants (e.g., acidic and basic variants) may be the result of natural processes such as oxidation, deamidation, C-terminal treatment of lysine residues, N-terminal pyroglutamate formation, and glycosylation of antibodies. Methods for measuring charged variants are known in the art and include, for example, electrophoresis (iCIEF) such as image capillaries. In certain embodiments of any of the methods described herein, the amount of charged variants in the composition recovered from one or more purification steps (such as a chromatographic fraction) is reduced to more than one of the following: about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and includes any range between these values. In a particular embodiment, the amount of charged variants in the composition recovered from one or more purification steps (such as a chromatographic fraction) is reduced to one of the following: about 10-95%, about 10-99%, about 20-95%, about 20-99%, about 30-95%, about 30-99%, about 40-95%, about 40-99%, about 50-95%, about 50-99%, about 60-95%, about 60-99%, about 70-95%, about 70-99%, about 80-95%, about 80-99%, about 90-95%, or about 90-99%. In some embodiments, the amount of charged variants in the composition (such as chromatographic fractions) is reduced to one of the following: about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.In a particular embodiment, the presence or reduction in the level of charged variants is measured by comparing the amount of charged variants in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of charged variants in the composition before the purification step(s).

[0178] In certain embodiments of any of the methods described herein, the impurities are process-specific impurities. For example, process-specific impurities may include one or more of the following: leached protein A, host cell material, nucleic acids, other polypeptides, endotoxins, viral contaminants, cell culture medium components, carboxypeptidase B, gentamicin, etc. In certain embodiments, process-specific impurities may be, for example, prokaryotic cells, bacterial cells (such as E. coli cells), insect cells, eukaryotic cells, fungal cells, yeast cells, avian cells, or mammalian cells, such as host cell proteins (HCPs) from CHO cells.

[0179] In certain embodiments, the methods provided herein remove and reduce the level of leached protein A from a composition containing a multispecific antibody (e.g., a bispecific antibody) and leached protein A. Leached protein A is protein A that has been separated or washed from the solid phase to which it is bound. For example, leached protein A may be leached from a protein A chromatography column. The amount of protein A may be measured, for example, by ELISA, as described in WO2011 / 150110. In certain embodiments, the presence or reduction in the level of leached protein A is measured by comparing the amount of leached protein A in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of leached protein A in the composition before the purification step(s).

[0180] In certain embodiments, the methods provided herein remove and reduce levels of host cell proteins (HCPs) from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and HCPs. HCPs are proteins from host cells that have produced multispecific antibodies (e.g., bispecific antibodies). In certain embodiments, the HCP protein is a protein from a prokaryotic cell. In certain embodiments, the HCP is a protein from an E. coli cell (i.e., E. coli protein or ECP). Examples of prokaryotic HCPs (e.g., ECPs) include, but are not limited to, prokaryotic chaperones such as FkpA, DsbA, and DsbC. In certain embodiments, the HCP is a protein from a eukaryotic host cell, such as those described elsewhere herein. In certain embodiments, the HCP is a protein from a mammalian cell, such as a CHO cell protein (i.e., Chinese hamster ovary protein or CHOP). In certain embodiments, the amount of HCP (e.g., ECP, FkpA, DsbA, or DsbC, or, for example, CHOP) is measured by enzyme-linked immunosorbent assay (ELISA). For example, antibodies can be produced against ultra-high purity compositions of FkpA, DsbA, or DsbC. In certain embodiments, the amounts of FkpA, DsbA, and / or DsbC are measured by mass spectrometry. In some embodiments of the methods described herein, the amount of HCP (e.g., ECP, FkpA, DsbA, or DsbC, or, for example, CHOP). In certain embodiments of any of the methods described herein, the amount of HCP (e.g., ECP, FkpA, DsbA, or DsbC, or e.g., CHOP) in the composition (e.g., a chromatographic fraction) recovered from one or more purification steps is reduced to about 100 ppm, about 75 ppm, about 50 ppm, about 25 ppm, about 20 ppm, about 10 ppm, about 5 ppm, about 2 ppm, or less than about 1 ppm, and includes any range between these values.In some embodiments, the amount of HCP (e.g., ECP, FkpA, DsbA, or DsbC, or e.g., CHOP) in the composition (e.g., a chromatographic fraction) is reduced to less than about 100 ppm, less than about 75 ppm, less than about 50 ppm, less than about 25 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, less than about 2 ppm, or less than about 1 ppm, and includes any range between these values. In certain embodiments, the presence or reduction in the level of HCP (e.g., ECP, FkpA, DsbA, or DsbC, or e.g., CHOP) is measured by comparing the amount of HCP in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of HCP in the composition before the purification step(s).

[0181] In certain embodiments, the methods provided herein remove or reduce the level of nucleic acids (such as host cell DNA and / or RNA) from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and nucleic acids. Methods for measuring nucleic acids (host cell DNA and / or RNA) are known in the art and are described, for example, in WO2011 / 150110. Such methods include, for example, PCR of host cell DNA or RNA. In certain embodiments of any of the methods described herein, the amount of nucleic acids in the composition recovered from one or more purification steps (e.g., a chromatographic fraction) is reduced to more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and include any range between these values. In a particular embodiment, the amount of nucleic acid (such as a chromatographic fraction) in the composition recovered from one or more purification steps is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%. In some embodiments, the amount of nucleic acid in the composition (e.g., a chromatographic fraction) is reduced to one of the following percentages: approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95%. In certain embodiments, the presence or reduction in the level of nucleic acid is measured by comparing the amount of nucleic acid in the composition recovered from the purification step(s) (e.g., a chromatographic fraction) with the amount of nucleic acid in the composition before the purification step(s).

[0182] In certain embodiments, the method provided herein removes or reduces the levels of cell culture medium components from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and the cell culture medium components. “Cell culture medium components” refers to components present in the cell culture medium. In certain embodiments, “cell culture medium” refers to the cell culture medium at the time when the host cell(s) expressing the multispecific antibody (e.g., a bispecific antibody) or its arm is collected. In certain embodiments, the cell culture medium component is insulin or tetracycline. In certain embodiments, the amount of insulin or tetracycline is measured by ELISA. In certain embodiments of any of the methods described herein, the amount of cell culture medium components (such as a chromatographic fraction) in the composition recovered from one or more purification steps is reduced to more than one of the following: about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%, and includes any range between these values.

[0183] In a particular embodiment, the amount of cell culture medium components (such as a chromatographic fraction) in the composition recovered from one or more purification steps is reduced to one of the following percentages: approximately 10-95%, approximately 10-99%, approximately 20-95%, approximately 20-99%, approximately 30-95%, approximately 30-99%, approximately 40-95%, approximately 40-99%, approximately 50-95%, approximately 50-99%, approximately 60-95%, approximately 60-99%, approximately 70-95%, approximately 70-99%, approximately 80-95%, approximately 80-99%, approximately 90-95%, or approximately 90-99%. In some embodiments, the amount of cell culture medium components in the composition (e.g., chromatographic fraction) is reduced to one of the following: about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In certain embodiments, the presence or reduction in the level of cell culture medium components is measured by comparing the amount of cell culture medium components in the composition recovered from the purification step(s) (e.g., chromatographic fraction) with the amount of cell culture medium components in the composition before the purification step(s).

[0184] In a particular embodiment, a method for purifying a bispecific antibody comprising a first arm and a second arm is provided herein, wherein the first and second arms are generated separately, and the method involves subjecting the first and second arms to capture chromatography operated in binding and elution mode (such as one or a combination of any of the capture chromatography steps described elsewhere herein) to generate first and second capture elutes; forming a mixture comprising the first and second capture elutes under conditions sufficient to generate a composition comprising the multispecific antibody; and eluting the composition comprising the multispecific antibody to anion exchange chromatography in binding and elution mode (e.g., Q Sepharose® Fast Flow (QSFF)). The method comprises: subjecting the mixture to anion exchange eluate (which is gradient eluted) by anion exchange mixed-mode chromatography (e.g., Capto® Adhere chromatography) in binding and elution modes to produce a first mixed-mode eluate (which is gradient eluted); and subjecting the first mixed-mode eluate to cation exchange mixed-mode chromatography (e.g., Capto® MMC chromatography) in binding and elution modes to produce a second mixed-mode eluate (which is gradient eluted) and collecting a fraction containing a bispecific antibody, wherein the method reduces the amount of impurities in the fraction relative to the mixture containing the first and second arms.

[0185] In a particular embodiment, a method for purifying a bispecific antibody comprising a first arm and a second arm is provided herein, wherein the first and second arms are generated separately, and the method comprises: subjecting the first and second arms to capture chromatography in binding and elution mode (such as one or a combination of any of the steps of capture chromatography described elsewhere herein) to produce first and second capture elutes; forming a mixture comprising the first and second capture elutes under conditions sufficient to produce a composition comprising the bispecific antibody; subjecting the composition comprising the bispecific antibody to cation exchange mixed-mode chromatography (e.g., Capto® MMC chromatography) in binding and elution mode to produce a first mixed-mode elute, wherein the elution is a stepwise elution of pH and salt, and the method further comprises: subjecting the first mixed-mode elute to anion exchange mixed-mode chromatography (e.g., Capto® Adhere chromatography) in flow-through mode to produce a second mixed-mode elute; and collecting a fraction comprising the bispecific antibody, wherein the method reduces the amount of impurities in the fraction relative to the mixture comprising the first and second arms.

[0186] In a particular embodiment, a method for purifying a bispecific antibody comprising a first arm and a second arm is provided herein, wherein the first and second arms are generated separately, and the method involves subjecting the first and second arms to capture chromatography in binding and elution mode (such as one or a combination of any of the capture chromatography steps described elsewhere herein) to generate first and second capture elutes; forming a mixture comprising the first and second capture elutes under conditions sufficient to generate a composition comprising the multispecific antibody; and eluting the composition comprising the multispecific antibody by anion exchange mixed-mode chromatography in binding and elution mode (e.g., where elution is stepwise elution). The method comprises: subjecting the mixture to Capto® Adhere chromatography to produce a first mixed-mode eluate; subjecting the first mixed-mode eluate to cation exchange mixed-mode chromatography (e.g., Capto® MMC chromatography) in binding and elution modes to produce a second mixed-mode eluate in which elution is stepwise; subjecting the second mixed-mode eluate to hydrophobic interaction chromatography (e.g., hexyl-650C chromatography) in flow-through mode to produce a hydrophobic interaction eluate; and collecting a fraction containing a bispecific antibody, wherein the method reduces the amount of impurities in the fraction relative to the mixture containing the first and second arms.

[0187] In a particular embodiment, a method for purifying a bispecific antibody (such as bispecific F(ab')2) comprising a first arm and a second arm is provided herein, wherein the first and second arms are generated separately, and the method involves subjecting the first arm to capture chromatography operated in binding and elution mode (such as one or a combination of any of the capture chromatography steps described elsewhere herein) to produce a first capture-elute; subjecting the first capture-elute to cation exchange mixed-mode chromatography (e.g., Capto® MMC chromatography) in binding and elution mode to produce a first mixed-mode elute; and subjecting the second arm to capture chromatography operated in binding and elution mode (such as one or a combination of any of the capture chromatography steps described elsewhere herein) to produce a second capture-elute. The method comprises: generating a product; forming a mixture containing a first mixed-mode eluate and a second capture eluate under conditions sufficient to generate a composition containing a multispecific antibody; subjecting the composition containing the multispecific antibody to anion exchange mixed-mode chromatography (e.g., Capto® Adhere chromatography) to generate a second mixed-mode eluate; subjecting the second mixed-mode eluate to cation exchange chromatography (e.g., POROS® 50HS chromatography) in binding and elution mode to generate a cation exchange eluate; subjecting the cation exchange eluate to sequential cation exchange mixed-mode chromatography in binding and elution mode to generate a third mixed-mode eluate; and collecting a fraction containing a bispecific antibody, wherein the method reduces the amount of impurities in the fraction relative to the mixture containing the first and second arms.

[0188] In a particular embodiment, a method for purifying a bispecific antibody (such as bispecific F(ab')2) comprising a first arm and a second arm is provided herein, wherein the first and second arms are generated separately, and the method involves subjecting the first arm to capture chromatography in binding and elution mode (such as one or a combination of any of the capture chromatography steps described elsewhere herein) to produce a first capture eluate; subjecting the first capture eluate to cation exchange mixed-mode chromatography (e.g., Capto® MMC chromatography) in binding and elution mode to produce a first mixed-mode eluate; and subjecting the second arm to capture chromatography operated in binding and elution mode (such as the capture chromatography steps described elsewhere herein). The method comprises: producing a second capture-elute by subjecting the mixture to any one or a combination of the first and second arms; forming a mixture containing the first mixed-mode elute and the second capture-elute under conditions sufficient to produce a composition containing a multispecific antibody; subjecting the composition containing the multispecific antibody to anion exchange mixed-mode chromatography (e.g., Capto® Adhere chromatography) to produce a second mixed-mode elute; and subjecting the second mixed-mode elute to cation exchange chromatography (e.g., Capto® MMC chromatography) in binding and elution modes to produce a third mixed-mode elute; and collecting a fraction containing a bispecific antibody, wherein the method reduces the amount of impurities in the fraction relative to the mixture containing the first and second arms.

[0189] In some embodiments, multispecific antibodies (such as bispecific antibodies) are further purified by viral filtration. Viral filtration involves removing viral contaminants from the polypeptide purification feedstream. Examples of viral filtration include, for example, ultrafiltration and microfiltration. In some embodiments, polypeptides are purified using a parvovirus filter.

[0190] In some embodiments, the multispecific antibody is concentrated after chromatography (e.g., after a second mixed-mode chromatography, or after one or more chromatographic steps performed after the second mixed-mode chromatography). Examples of concentration methods are known in the art and are not limited to these, but include, for example, ultrafiltration and diafiltration (UFDF). In some embodiments, the multispecific antibody is concentrated by a first microfiltration, diafiltration, and a second ultrafiltration. In some embodiments, the ultrafiltration and / or diafiltration uses filters with cleavage less than about 5 kDal, about 10 kDal, about 15 kDal, about 20 kDal, or about 25 kDal or about 30 kDal. In some embodiments, the retained material from the first ultrafiltration is diafiltration to a pharmaceutical formulation.

[0191] In some embodiments, the concentration of the concentrated multispecific antibody is one of the following: approximately 10 mg / mL, approximately 20 mg / mL, approximately 30 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 110 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, approximately 140 mg / mL, approximately 150 mg / mL, approximately 160 mg / mL, approximately 170 mg / mL, approximately 180 mg / mL, approximately 190 mg / mL, approximately 200 mg / mL, or approximately 300 mg / mL. In some embodiments, the concentration of the multispecific antibody is approximately 10 mg / mL to 20 mg / mL, approximately 20 mg / mL to 30 mg / mL, approximately 30 mg / mL to 40 mg / mL, approximately 40 mg / mL to 50 mg / mL, approximately 50 mg / mL to 60 mg / mL, approximately 60 mg / mL to 70 mg / mL, approximately 70 mg / mL to 80 mg / mL, approximately 80 mg / mL to 90 mg / mL, approximately 90 mg / mL to 100 mg / mL, approximately 100 mg / mL to 110 mg / mL, and approximately 11 The dosage ranges are one of the following: 0 mg / mL to approximately 120 mg / mL, approximately 120 mg / mL to approximately 130 mg / mL, approximately 130 mg / mL to approximately 140 mg / mL, approximately 140 mg / mL to approximately 150 mg / mL, approximately 150 mg / mL to approximately 160 mg / mL, approximately 160 mg / mL to approximately 170 mg / mL, approximately 170 mg / mL to approximately 180 mg / mL, approximately 180 mg / mL to approximately 190 mg / mL, approximately 190 mg / mL to approximately 200 mg / mL, approximately 200 mg / mL, or approximately 300 mg / mL.

[0192] In some embodiments of the methods described herein, the method further comprises combining the purified polypeptide with a pharmaceutically acceptable carrier by a purification method. In some embodiments, the multispecific antibody is formulated into a pharmaceutically acceptable product by ultrafiltration / diafiltration.

[0193] In certain embodiments, the methods provided herein provide a composition comprising a multispecific antibody that is purer than any of the following: about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In certain embodiments, the multispecific antibody in the composition is purer than any of the following: about 96%, about 97%, about 98%, or about 99%.

[0194] In a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing an arm of non-paired antibodies in any of the following proportions: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing homodimers of any one of the following percentages: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 certain embodiments, the method provided herein generates a composition comprising a multispecific antibody containing aggregated proteins in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In certain embodiments, the method provided herein generates a composition comprising a multispecific antibody containing HMWS in any of the following amounts: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing LMWS in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing an acidic variant in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing a basic variant in any of the following proportions: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing leached protein A in any of the following amounts: about 0.1 ppm, about 0.2 ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, about 0.7 ppm, about 0.8 ppm, about 0.9 ppm, about 1 ppm, about 1.5 ppm, about 2 ppm, about 2.5 ppm, about 3 ppm, about 3.5 ppm, about 4 ppm, about 4.5 ppm, about 5 ppm, about 5.5 ppm, about 6 ppm, about 6.5 ppm, about 7 ppm, about 7.5 ppm, about 8 ppm, about 8.5 ppm, about 9 ppm, about 9.5 ppm, or about 10 ppm. In a particular embodiment, the method provided herein generates a composition comprising a multispecific antibody containing HCP in any of the following concentrations: about 0.1 ppm, about 0.2 ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, about 0.7 ppm, about 0.8 ppm, about 0.9 ppm, about 1 ppm, about 1.5 ppm, about 2 ppm, about 2.5 ppm, about 3 ppm, about 3.5 ppm, about 4 ppm, about 4.5 ppm, about 5 ppm, about 5.5 ppm, about 6 ppm, about 6.5 ppm, about 7 ppm, about 7.5 ppm, about 8 ppm, about 8.5 ppm, about 9 ppm, about 9.5 ppm, about 10 ppm, about 15 ppm, about 20 ppm, about 25 ppm, about 30 ppm, or about 35 ppm.In certain embodiments, the method provided herein generates a composition comprising a multispecific antibody containing nucleic acids in any of the following concentrations: about 2 ppm, about 2.5 ppm, about 3 ppm, about 3.5 ppm, about 4 ppm, about 4.5 ppm, about 5 ppm, about 5.5 ppm, about 6 ppm, about 6.5 ppm, about 7 ppm, about 7.5 ppm, about 8 ppm, about 8.5 ppm, about 9 ppm, about 9.5 ppm, or about 10 ppm. In certain embodiments, the composition comprising the multispecific antibody contains 0 ppm or less of nucleic acids. In certain embodiments, the nucleic acids in the composition comprising the multispecific antibody are below the detection level. In certain embodiments, the method provided herein generates a composition comprising a multispecific antibody containing any of the following concentrations of cell culture medium components: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%.

[0195] In a particular embodiment, a composition is provided comprising a multispecific antibody purified according to one of the methods described herein.

[0196] In certain embodiments, the multispecific antibody in the composition is purer than any of the following: about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In certain embodiments, the multispecific antibody in the composition is purer than any of the following: about 96%, about 97%, about 98%, or about 99%.

[0197] In a particular embodiment, the composition comprising the multispecific antibody includes an arm of non-paired antibodies in any of the following proportions: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 a particular embodiment, the composition comprising the multispecific antibody contains homodimers in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 certain embodiments, the composition containing the multispecific antibody contains aggregated protein in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In certain embodiments, the composition containing the multispecific antibody contains HMWS in any of the following amounts: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, the composition comprising the multispecific antibody contains LMWS in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 a particular embodiment, the composition comprising the multispecific antibody contains acidic variants in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In certain embodiments, the composition comprising the multispecific antibody contains a basic variant in any of the following amounts: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, the composition comprising a multispecific antibody contains leached protein A in any of the following amounts: about 0.1 ppm, about 0.2 ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, about 0.7 ppm, about 0.8 ppm, about 0.9 ppm, about 1 ppm, about 1.5 ppm, about 2 ppm, about 2.5 ppm, about 3 ppm, about 3.5 ppm, about 4 ppm, about 4.5 ppm, about 5 ppm, about 5.5 ppm, about 6 ppm, about 6.5 ppm, about 7 ppm, about 7.5 ppm, about 8 ppm, about 8.5 ppm, about 9 ppm, about 9.5 ppm, or about 10 ppm. In a particular embodiment, the composition comprising the multispecific antibody contains HCP in any of the following concentrations: approximately 0.1 ppm, approximately 0.2 ppm, approximately 0.3 ppm, approximately 0.4 ppm, approximately 0.5 ppm, approximately 0.6 ppm, approximately 0.7 ppm, approximately 0.8 ppm, approximately 0.9 ppm, approximately 1 ppm, approximately 1.5 ppm, approximately 2 ppm, approximately 2.5 ppm, approximately 3 ppm, approximately 3.5 ppm, approximately 4 ppm, approximately 4.5 ppm, approximately 5 ppm, approximately 5.5 ppm, approximately 6 ppm, approximately 6.5 ppm, approximately 7 ppm, approximately 7.5 ppm, approximately 8 ppm, approximately 8.5 ppm, approximately 9 ppm, approximately 9.5 ppm, approximately 10 ppm, approximately 15 ppm, approximately 20 ppm, approximately 25 ppm, approximately 30 ppm, or approximately 35 ppm.In a particular embodiment, the composition comprising the multispecific antibody contains nucleic acid in any of the following concentrations: approximately 0.1 ppm, approximately 0.2 ppm, approximately 0.3 ppm, approximately 0.4 ppm, approximately 0.5 ppm, approximately 0.6 ppm, approximately 0.7 ppm, approximately 0.8 ppm, approximately 0.9 ppm, approximately 1 ppm, approximately 1.5 ppm, approximately 2 ppm, approximately 2.5 ppm, approximately 3 ppm, approximately 3.5 ppm, approximately 4 ppm, approximately 4.5 ppm, approximately 5 ppm, approximately 5.5 ppm, approximately 6 ppm, approximately 6.5 ppm, approximately 7 ppm, approximately 7.5 ppm, approximately 8 ppm, approximately 8.5 ppm, approximately 9 ppm, approximately 9.5 ppm, approximately 10 ppm, approximately 15 ppm, approximately 20 ppm, approximately 25 ppm, approximately 30 ppm, or approximately 35 ppm. In certain embodiments, the composition containing the multispecific antibody comprises any of the following cell culture medium components: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%.

[0198] In some embodiments, compositions comprising a multispecific antibody are provided, the composition comprising a) at least about 95% to about 100% of the multispecific antibody, b) an arm of about 1% to less than 5% of the non-paired antibody, c) an antibody homodimer of about 1% to less than 5%, d) HMWS of about 1% or less or about 2%, e) LMWS of about 1% or less or about 2%, and / or f) 3 / 4 antibody of about 5% or less.

[0199] In certain embodiments, a composition is provided comprising a bispecific antibody purified according to one of the methods described herein. In certain embodiments, the bispecific antibody is a nob-in-hole (KiH) antibody, for example, a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody.

[0200] In certain embodiments, a composition is provided comprising a bispecific antibody that is purer than any of the following percentages: approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, and approximately 95%. In certain embodiments, the bispecific antibody in the composition is purer than any of the following percentages: approximately 96%, approximately 97%, approximately 98%, or approximately 99%. In certain embodiments, the bispecific antibody is a nob-in-hole (KiH) antibody, for example, a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody.

[0201] In a particular embodiment, a composition is provided comprising a bispecific antibody containing an arm of non-paired antibody in any of the following proportions: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 certain embodiments, a composition is provided comprising a bispecific antibody containing homodimers in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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 certain embodiments, a composition is provided comprising a bispecific antibody containing aggregated proteins in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In certain embodiments, a composition is provided comprising a bispecific antibody containing HMWS in any of the following amounts: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, a composition is provided comprising a bispecific antibody containing LMWS in an amount of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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% or less.In certain embodiments, a composition is provided comprising a bispecific antibody containing an acidic variant in any of the following amounts: about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 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%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In a particular embodiment, a composition is provided comprising a bispecific antibody containing a basic variant in any of the following amounts: about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%. In a particular embodiment, a composition is provided comprising a bispecific antibody containing leached protein A in an amount of approximately 0.1 ppm, approximately 0.2 ppm, approximately 0.3 ppm, approximately 0.4 ppm, approximately 0.5 ppm, approximately 0.6 ppm, approximately 0.7 ppm, approximately 0.8 ppm, approximately 0.9 ppm, approximately 1 ppm, approximately 1.5 ppm, approximately 2 ppm, approximately 2.5 ppm, approximately 3 ppm, approximately 3.5 ppm, approximately 4 ppm, approximately 4.5 ppm, approximately 5 ppm, approximately 5.5 ppm, approximately 6 ppm, approximately 6.5 ppm, approximately 7 ppm, approximately 7.5 ppm, approximately 8 ppm, approximately 8.5 ppm, approximately 9 ppm, approximately 9.5 ppm, or approximately 10 ppm or less. In a particular embodiment, a composition is provided comprising a bispecific antibody containing HCP in any of the following concentrations: approximately 0.1 ppm, approximately 0.2 ppm, approximately 0.3 ppm, approximately 0.4 ppm, approximately 0.5 ppm, approximately 0.6 ppm, approximately 0.7 ppm, approximately 0.8 ppm, approximately 0.9 ppm, approximately 1 ppm, approximately 1.5 ppm, approximately 2 ppm, approximately 2.5 ppm, approximately 3 ppm, approximately 3.5 ppm, approximately 4 ppm, approximately 4.5 ppm, approximately 5 ppm, approximately 5.5 ppm, approximately 6 ppm, approximately 6.5 ppm, approximately 7 ppm, approximately 7.5 ppm, approximately 8 ppm, approximately 8.5 ppm, approximately 9 ppm, approximately 9.5 ppm, approximately 10 ppm, approximately 15 ppm, approximately 20 ppm, approximately 25 ppm, approximately 30 ppm, or approximately 35 ppm.In certain embodiments, a composition is provided comprising a bispecific antibody containing nucleic acids in any of the following concentrations: approximately 2 ppm, approximately 2.5 ppm, approximately 3 ppm, approximately 3.5 ppm, approximately 4 ppm, approximately 4.5 ppm, approximately 5 ppm, approximately 5.5 ppm, approximately 6 ppm, approximately 6.5 ppm, approximately 7 ppm, approximately 7.5 ppm, approximately 8 ppm, approximately 8.5 ppm, approximately 9 ppm, approximately 9.5 ppm, or approximately 10 ppm. In certain embodiments, the composition comprising the bispecific antibody contains nucleic acids in 0 ppm or less. In certain embodiments, the nucleic acids in the composition comprising the bispecific antibody are below the detection level. In certain embodiments, a composition is provided comprising a bispecific antibody containing any of the following cell culture medium components: approximately 0.1%, approximately 0.5%, approximately 1%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, or approximately 35%. In certain embodiments, the bispecific antibody is a knob-in-hole (KiH) antibody, for example, a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody.

[0202] In some embodiments, compositions comprising a bispecific antibody are provided, the composition comprising a) at least about 95% to about 100% of the bispecific antibody, b) an arm of about 1% to less than 5% of the non-paired antibody, c) about 1% to less than 5% of the antibody homodimer, d) about 1% or less of HMWS, e) about 1% or less of LMWS, and / or f) about 5% or less of 3 / 4 antibody. In certain embodiments, the bispecific antibody is a nob-in-hole (KiH) antibody, e.g., a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody.

[0203] One embodiment reported herein is a multi-step chromatographic method for purifying an Fc region-containing heterodimer protein / polypeptide, the method comprising an affinity chromatography step followed by two different multimodal ion exchange chromatography steps, thereby purifying the Fc region-containing heterodimer polypeptide.

[0204] In certain embodiments, the method comprises i. an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step, or ii. an affinity chromatography step, followed by a multimodal cation exchange chromatography step, followed by a multimodal anion exchange chromatography step.

[0205] One embodiment reported herein is a method for producing an Fc-domain-containing heterodimer protein / polypeptide, comprising the steps of: i. culturing cells containing nucleic acids encoding an Fc-domain-containing heterodimer protein / polypeptide; ii. recovering the Fc-domain-containing heterodimer protein / polypeptide from the cells or culture medium; and iii. purifying the Fc-domain-containing heterodimer protein / polypeptide by the method reported herein, thereby producing an Fc-domain-containing heterodimer protein. The performance of the antibody purification process has been found to depend on the sequence of chromatographic steps used. By selecting a particular sequence / order of chromatographic steps, an improved process may be obtained.

[0206] The methods provided herein are based, at least in part, on the finding that the ultrafiltration / dialysis filtration step can be omitted by performing a multimodal anion exchange chromatography step immediately after the (initial) affinity chromatography step and before the multimodal cation exchange chromatography step. This step is required when the multimodal cation exchange chromatography step is performed before the multimodal anion exchange chromatography step.

[0207] In a particular embodiment, the multi-step chromatography method includes an affinity chromatography step, followed by a multimodal anion exchange chromatography step, and then a multimodal cation exchange chromatography step.

[0208] It has been found that good purity and yield can be achieved in only three chromatographic steps using the method described herein.

[0209] In a particular embodiment, the multi-step chromatography method includes exactly three chromatography steps.

[0210] It has been found that the removal of host cell proteins can be improved when the multimodal anion exchange chromatography method / step is performed in flow-through mode. In certain embodiments, the multimodal anion exchange chromatography method / step is performed in flow-through mode.

[0211] It has been found that the pH of the loading in the multimodal anion exchange chromatography step affects HCP by product and DNA removal. In one preferred embodiment of all embodiments, the multimodal anion exchange chromatography step is performed at a pH of about 7.0. Table 1 JPEG0007881651000001.jpg67170

[0212] The conductivity of a solution can have an effect on different parameters during the purification process. Here, it has been found that a low conductivity value in the loading of a multimodal anion exchange chromatography step (i.e., a solution containing Fc region-containing heterodimer polypeptides applied to the chromatography material) results in improved removal of HCP and DNA. Table 2 JPEG0007881651000002.jpg35170

[0213] In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value of less than 7 mS / cm. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value of less than 6 mS / cm. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value in the range of about 6 mS / cm to about 2 mS / cm. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value in the range of about 5 mS / cm to about 4 mS / cm. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity value of about 4.5 mS / cm.

[0214] In a particular embodiment, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied to a solution having a conductivity of about 4.5 mS / cm and a pH of about 7.

[0215] The methods provided herein are based, at least in part, on the finding that the protein loading in the multimodal anion exchange chromatography step also affects the performance of the purification process. When the loading is within a defined range, the overall purification process is improved, for example, in the removal of DNA contamination. Table 3: Starting dose of DNA: 80 pg / mg JPEG0007881651000003.jpg35170

[0216] In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied in an amount ranging from about 100 g to about 300 g per liter of chromatography material, i.e., the load is in the range of about 100 g / L to about 300 g / L. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied in an amount ranging from about 120 g to about 240 g per liter of chromatography material. In certain embodiments, during the multimodal anion exchange chromatography step, the Fc region-containing heterodimer polypeptide is applied in an amount ranging from about 160 g to about 200 g per liter of chromatography material.

[0217] Certain multimodal resin materials have been found to be particularly useful when applied in the methods reported herein.

[0218] In certain embodiments, the multimodal anion exchange chromatography material is a multimodal strong anion exchange chromatography material. In certain embodiments, the multimodal anion exchange chromatography material has a high-flow agarose matrix, multimodal strong anion exchange as a ligand, an average particle size of 36-44 μm, and an ion capacity of 0.08-0.11 mmol Cl- / mL medium. In certain embodiments, the multimodal anion exchange chromatography material is "Capto adhere ImpRes".

[0219] In certain embodiments, the multimodal cation exchange chromatography medium is a multimodal weak cation exchange chromatography medium. In certain embodiments, the multimodal cation exchange chromatography medium has a high-flow agarose matrix, multimodal weak cation exchange as ligand, an average particle size of 36–44 μm, and an ion capacity of 25–39 μmol / mL. In certain embodiments, the multimodal cation exchange chromatography medium is "Capto MMC ImpRes".

[0220] In certain embodiments, the multimodal cation exchange chromatography method / step is performed in binding and elution modes.

[0221] In certain embodiments, the affinity chromatography step is a protein A chromatography step, or a protein G affinity chromatography step, or a single-stranded Fv ligand affinity chromatography step, or a chromatography step using KappaSelect chromatography material, or a chromatography step using CaptureSelect chromatography material, or a chromatography step using CaptureSelect FcXL chromatography material. In certain embodiments, the affinity chromatography step is a protein A chromatography step. In certain embodiments, the affinity chromatography step is a CaptureSelect™ chromatography step. In certain embodiments, the affinity chromatography step is a protein A chromatography step.

[0222] In certain embodiments, the Fc-domain-containing heterodimer protein / polypeptide is an antibody, a bispecific antibody, or an Fc-fusion protein. In certain embodiments, the Fc-domain-containing heterodimer protein / polypeptide is a bispecific antibody. In certain embodiments, the Fc-domain-containing heterodimer protein / polypeptide is CrossMab. In certain embodiments, the Fc-domain-containing heterodimer protein / polypeptide is an Fc-fusion protein. In certain embodiments, the Fc-domain-containing heterodimer protein / polypeptide comprises a) a heavy and light chain of a first full-length antibody that specifically binds a first antigen, and b) a modified heavy and modified light chain of a full-length antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are substituted for each other.

[0223] In certain embodiments, the bispecific antibody is a bispecific antibody that binds ANG2 and VEGF. In certain embodiments, the Fc region-containing heterodimer protein / polypeptide is CrossMab that binds ANG2 and VEGF. In certain embodiments, the bispecific antibody is vanucizumab.

[0224] In certain embodiments, the bispecific antibody includes a first antigen-binding site comprising SEQ ID NO: 1 as the heavy chain variable domain (VH) and SEQ ID NO: 2 as the light chain variable domain (VL), and a second antigen-binding site comprising SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL). In certain embodiments, the bispecific antibody includes a first heavy chain having the amino acid sequence of SEQ ID NO: 9 and a second heavy chain having the amino acid sequence of SEQ ID NO: 10, and a first light chain having the amino acid sequence of SEQ ID NO: 11 and a second light chain having the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the bispecific antibody includes a first antigen-binding site comprising SEQ ID NO: 5 as the heavy chain variable domain (VH) and SEQ ID NO: 6 as the light chain variable domain (VL), and a second antigen-binding site comprising SEQ ID NO: 7 as the heavy chain variable domain (VH) and SEQ ID NO: 8 as the light chain variable domain (VL). In a particular embodiment, the bispecific antibody comprises a first heavy chain having the amino acid sequence of SEQ ID NO: 13, a second heavy chain having the amino acid sequence of SEQ ID NO: 14, a first light chain having the amino acid sequence of SEQ ID NO: 15, and a second light chain having the amino acid sequence of SEQ ID NO: 16. The amino acid sequences of SEQ ID NOs: 1 to 16 are provided in Table 4 below. Table 4 TIFF0007881651000004.tif255170TIFF0007881651000005.tif255170TIFF0007881651000006.tif23170

[0225] In certain embodiments, the purified Fc region-containing heterodimer polypeptide contains approximately 5% or less of 3 / 4 antibody. In certain embodiments, the purified Fc region-containing heterodimer polypeptide contains approximately 4% or less of 3 / 4 antibody. In certain embodiments, the purified Fc region-containing heterodimer polypeptide contains approximately 3% or less of 3 / 4 antibody. In certain embodiments, the purified Fc region-containing heterodimer polypeptide contains approximately 2% or less of 3 / 4 antibody. In certain embodiments, the purified Fc region-containing heterodimer polypeptide contains approximately 1% or less of 3 / 4 antibody.

[0226] One embodiment reported herein is a method for purifying a bispecific antibody conjugating ANG2 and VEGF by a multi-step chromatography method, the method comprising an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step, thereby purifying a bispecific antibody conjugating ANG2 and VEGF, wherein the bispecific antibody conjugating ANG2 and VEGF comprises a first antigen-binding site including SEQ ID NO: 1 as the heavy chain variable domain (VH) and SEQ ID NO: 2 as the light chain variable domain (VL), and a second antigen-binding site including SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL), or a first antigen-binding site including SEQ ID NO: 5 as the heavy chain variable domain (VH) and SEQ ID NO: 6 as the light chain variable domain (VL), and a second antigen-binding site including SEQ ID NO: 7 as the heavy chain variable domain (VH) and SEQ ID NO: 8 as the light chain variable domain (VL).

[0227] In one embodiment, a bispecific antibody binding ANG2 and VEGF comprises a) a heavy and light chain of a first full-length antibody containing a first antigen-binding site, and b) a modified heavy and modified light chain of a full-length antibody containing a second antigen-binding site, wherein the constant domains CL and CH1 are substituted for each other.

[0228] One aspect reported herein is the use of the method reported herein for the purification of Fc-containing heterodimer polypeptides.

[0229] One aspect reported herein is the use of the method reported herein for reducing Fc-containing heterodimer polypeptide-related impurities.

[0230] One embodiment reported herein is an Fc-containing heterodimer polypeptide obtained by the method reported herein for the manufacture of a pharmaceutical product for the treatment of cancer or eye disease.

[0231] One embodiment reported herein is an Fc-containing heterodimer polypeptide obtained by the method reported herein for use in the treatment of cancer or eye disease.

[0232] Polypeptide Monoclonal antibodies In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical and / or bind to the same epitope, but any expected variants that occur during the production of the monoclonal antibody are excluded, and such variants are generally present in small amounts. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is not a mixture of individual or polyclonal antibodies.

[0233] For example, monoclonal antibodies may be produced by the hybridoma method, first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (U.S. Patent No. 4,816,567).

[0234] In the hybridoma method, a mouse or other suitable host animal is immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable flux such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0235] The hybridoma cells thus prepared are grown in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of seeded, unfused parent myeloma cells. For example, if parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0236] In some embodiments, myeloma cells efficiently fuse, support stable high levels of antibody production by selected antibody-producing cells, and exhibit sensitivity to media such as HAT medium. Among these, in some embodiments, the myeloma cell lines are mouse myeloma lines such as MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, California, USA, and those derived from SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for human monoclonal antibody production (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0237] The culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0238] The binding affinity of a monoclonal antibody can be measured, for example, by scatchard analysis as described in Munson et al., Anal. Biochem. 107:220 (1980).

[0239] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0240] Monoclonal antibodies secreted by subclones can be suitably separated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as polypeptide A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, or ion exchange chromatography.

[0241] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which can then be transfected into host cells, such as E. coli cells, monkey COS cells, human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin polypeptides, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review papers on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0242] In further embodiments, antibodies or antibody fragments may be isolated from antibody phage libraries prepared using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol. Biol. 222:581-597 (1991) describe the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent publications describe chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)) as a strategy for constructing very large phage libraries, as well as the production of high-affinity (nM range) human antibodies by combination infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques represent a viable alternative to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0243] DNA may also be modified, for example, by substituting the coding sequence with the constant domains of the human heavy and light chains instead of homologous mouse sequences (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

[0244] Typically, such non-immunoglobulin polypeptides are substituted in place of the constant domain of an antibody, or they are substituted in place of the variable domain of one antigen-binding site of the antibody, to create a chimeric bivalent antibody containing one antigen-binding site specific to a particular antigen and another antigen-binding site specific to a different antigen.

[0245] In some embodiments of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

[0246] antibody fragment In some embodiments, the antibody is an antibody fragment. Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, the Fab'-SH fragment can be directly recovered from E. coli and chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, the F(ab')2 fragment can be directly isolated from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the optimal antibody is a single-stranded Fv fragment (scFv). See WO93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458. The antibody fragment may also be a “linear antibody,” as described, for example, U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0247] In some embodiments, antibody fragments described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, scFv, Fv, and diabodies.

[0248] Polypeptide variants and modifications In certain embodiments, amino acid sequence variants of proteins as described herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of a protein. Amino acid sequence variants of proteins can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the protein, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the protein, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct has the desired properties.

[0249] A "polypeptide variant" means a polypeptide as defined herein, such as an active polypeptide, that has at least about 80% amino acid sequence identity with the full-length native sequence of the polypeptide, a polypeptide sequence lacking a signal peptide, or the extracellular domain of a polypeptide with or without a signal peptide. Examples of such polypeptide variants include polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the full-length native amino acid sequence. Typically, polypeptide variants have at least about 80% amino acid sequence identity with the full-length native polypeptide sequence, a polypeptide sequence lacking a signal peptide, or the extracellular domain of a polypeptide with or without a signal peptide, or at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity. Optionally, a variant polypeptide may have one or fewer conserved amino acid substitutions compared to the native polypeptide sequence, or any of the following conserved amino acid substitutions with respect to the native polypeptide sequence: about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0250] Mutant polypeptides may be cleaved at the N-terminus or C-terminus, or may lack internal residues compared to, for example, the full-length natural polypeptide. Certain mutant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These mutant polypeptides having cleavage, deletion, and insertion may be prepared by any of several conventional techniques. The desired mutant polypeptide may be chemically synthesized. Another preferred technique involves isolating and amplifying the nucleic acid fragment encoding the desired mutant polypeptide by polymerase chain reaction (PCR). Oligonucleotides defining the desired ends of the nucleic acid fragment are used in PCR as 5' and 3' primers. Preferably, the mutant polypeptide shares at least one biological and / or immunological activity with the natural polypeptide disclosed herein.

[0251] Amino acid sequence insertions include fusions of amino-terminuses and / or carboxyl-terminuses having a polypeptide length ranging from one residue to more than 100 residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue, or antibodies fused to cytotoxic polypeptides. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme or polypeptide that increases the serum half-life of the antibody.

[0252] For example, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide. Amino acid sequence variants of polypeptides are prepared by introducing appropriate nucleotide changes into antibody nucleic acids or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of a polypeptide, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions is made to arrive at a final construct, provided that the final construct has the desired characteristics. Amino acid changes can also modify the post-translational processes of polypeptides (e.g., antibodies), for example, by changing the number or location of glycosylation sites.

[0253] Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the polypeptide sequence with that of known homologous polypeptide molecules and minimizing the number of amino acid sequence changes in highly homologous regions.

[0254] A useful method for identifying specific residues or regions of a polypeptide (e.g., an antibody) that are favorable sites for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction between the amino acid and the antigen. The amino acid position demonstrating functional sensitivity to the substitution is then refined by introducing further or other variants at or in place of the substitution site. Thus, while the site for introducing amino acid sequence mutations is predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for desired activity.

[0255] Another type of mutant is the amino acid substitution mutant. These mutants have at least one amino acid residue in the antibody molecule that has been replaced with a different residue. The most interesting sites for substitution mutagenesis are the hypervariable regions, but FR modifications are also considered. If such substitutions result in changes in biological activity, they are indicated as “exemplary substitutions” in Table 5, or more substantial changes are introduced as further described below with reference to amino acid classes, and the product may be screened. Table 5 JPEG0007881651000007.jpg168170

[0256] Substantial modification of the biological properties of a polypeptide is achieved by selecting substitutions that vary widely in their effects on (a) the structure of the polypeptide backbone in the substitution region, such as sheet or helical conformations, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, Biochemistry second ed., pp. 73-75, Worth Publishers, New York (1975)).

[0257] (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

[0258] (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

[0259] (3) Acidic: Asp (D), Glu (E)

[0260] (4) Basic: Lys (K), Arg (R), His (H)

[0261] Alternatively, naturally occurring residues can be grouped based on common side chain properties.

[0262] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile

[0263] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln

[0264] (3) Acidic: Asp, Glu

[0265] [[ID=3就5]] (4) Basic: His, Lys, Arg

[0266] (5) Residues affecting chain orientation: Gly, Pro

[0267] (6) Aromatic: Trp, Tyr, Phe

[0268] Non-conservative substitutions involve the exchange of a member of one of these classes with a member of another class.

[0269] Any cysteine ​​residues that do not contribute to maintaining the proper conformation of the antibody can generally be substituted with serine, improving the oxidative stability of the molecule and preventing abnormal crosslinking. Conversely, cysteine ​​bonds(s) can be added to a polypeptide to improve its stability (specifically, when the antibody is an antibody fragment such as an Fv fragment).

[0270] An example of a substitution variant involves the substitution of one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody). Generally, the variant(s) selected and obtained for further development have improved biological properties compared to the parent antibody from which they are produced. A favorable method for generating such substitution variants involves affinity maturation using phage display. Briefly, 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 in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13, packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, 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. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such mutants are generated, a panel of mutants may be screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0271] Another type of amino acid variant in a polypeptide alters the original glycosylation pattern of the antibody. Polypeptides may contain non-amino acid moieties. For example, polypeptides can be glycosylated. Such glycosylation may occur spontaneously during polypeptide expression in host cells or host organisms, or it may be a planned modification resulting from human intervention. Modification means the deletion of one or more carbohydrate moieties found in the polypeptide, and / or the addition of one or more glycosylation sites that were not present in the polypeptide.

[0272] Polypeptide glycosylation is typically either N-linked or O-linked. N-linking refers to the binding 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 binding of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences within a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0273] The addition of a glycosylation site to a polypeptide is conveniently achieved by modifying the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for the N-linked glycosylation site). The modification may also be carried out by the addition or substitution of one or more serine or threonine residues to the original antibody sequence (for the O-linked glycosylation site).

[0274] The removal of the carbohydrate moiety on a polypeptide can be achieved chemically, enzymatically, or by mutational substitution of codons encoding amino acid residues that function as targets for glycosylation. Enzymatic cleavage of the carbohydrate moiety on a polypeptide can be achieved using various endo- and exo-glycosidases.

[0275] Other modifications include deamidation of glutaminyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of γ-amino groups of lysine, arginine, and histidine side chains; acetylation of N-terminal amines; and amidation of any C-terminal carboxyl group.

[0276] Chimeric polypeptide Polypeptides described herein may be modified by methods for forming chimeric molecules, including a polypeptide fused to another heterologous polypeptide or an amino acid sequence. In some embodiments, the chimeric molecule involves the fusion of a polypeptide with a tagged polypeptide that provides an epitope to which an anti-tagged antibody can selectively bind. The epitope tag is generally located at the amino-terminus or carboxyl-terminus of the polypeptide. The presence of a polypeptide in such an epitope-tagged form can be detected using an antibody against the tagged polypeptide. The provision of an epitope tag also makes it possible to readily purify the polypeptide by affinity purification using an anti-tagged antibody or another type of affinity matrix that binds to the epitope tag.

[0277] multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to c-met and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of c-met. Bispecific antibodies can also be used to localize cytotoxic drugs to cells expressing c-met. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0278] Techniques for making multispecific antibodies include recombinant co-expression of two immunoglobulin heavy-chain / light-chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knobs-in-holes" maneuver (see, e.g., U.S. Patent No. 5,731,168), among others. Multispecific antibodies can also be made by manipulating the electrostatic steering effect to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), making bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), as well as preparing trispecific antibodies (which can be made, e.g., by Tutt et al. J. Immunol. 147:60 (1991).

[0279] Antibodies or fragments herein also include multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112,193, WO2010 / 115,589, WO2010 / 136,172, WO2010 / 145,792, and WO2010 / 145,793.

[0280] Modified antibodies having three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, US2006 / 0025576A1).

[0281] The antibodies or fragments described herein also include “dual-acting Fab” or “dual-acting Fab” (DAF) which include antigen-binding sites that bind to a first epitope (e.g., on a first antigen) and another different epitope (e.g., on the first antigen or a second different antigen) (see, e.g., US2008 / 0069820, Bostrom et al. (2009) Science, 5921, 1610-1614).

[0282] Methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, each having two heavy chains with different specificities (Milstein and Cuello, Nature 305:537 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is somewhat cumbersome and results in low product yields. Similar procedures are disclosed in WO93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655 (1991).

[0283] According to a different, more preferred approach, an antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. This fusion is preferably with an immunoglobulin heavy chain constant domain that includes at least a portion of the hinge, CH2, and CH3 regions. Preferably, at least one of the fusions has a first heavy chain constant region (CH1) that includes a site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. This provides excellent flexibility in adjusting the relative ratios of the three polypeptide chains in embodiments where an unequal ratio of the three polypeptide chains used in construction yields the optimal yield. However, if a high yield is obtained by expressing at least two polypeptide chains in equal ratios, or if their ratios are not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0284] In a preferred embodiment of this approach, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from undesirable immunoglobulin chain combinations, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy method of separation. This approach is disclosed in WO94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0285] Nobu in Hole Alternatively, the interface between a pair of antibody molecules can be manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. A preferred interface includes at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains originating from the interface of the first antibody molecule are replaced (knobs or bumps) with larger side chains (e.g., tyrosine or tryptophan). Compensatory "caves" (holes) of the same or similar size as the larger side chains are created on the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing heterodimer yields over other undesirable end products, such as homodimers. Knobs and holes are further described herein.

[0286] The use of knobs into holes as a method for producing multispecific antibodies and / or monoarm antibodies and / or immunoadhesins is well known in the art. See U.S. Patent No. 5,731,168, assigned to Genentech and approved on March 24, 1998; PCT Publication WO2009089004, assigned to Amgen and published on July 16, 2009; and U.S. Patent Publication 2009 / 0182127, assigned to Novo Nordisk A / S and published on July 16, 2009. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9. A brief discussion is provided herein.

[0287] A “bump” refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide and is therefore capable of being positioned within a compensatory cavity at the adjacent interface (i.e., the interface of the second polypeptide), thereby stabilizing the heteromultimer and making heteromultimerization more favorable than homomultimerization. Bumps can be present at the original interface or can be introduced synthetically (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is modified to encode a bump. To achieve this, the nucleic acid encoding at least one “original” amino acid residue at the interface of the first polypeptide is replaced with a nucleic acid encoding at least one “imported” amino acid residue having a larger side chain volume than the original amino acid residue. It will be understood that there may be two or more original residues and corresponding imported residues. The upper limit on the number of original residues to be replaced is the total number of residues at the interface of the first polypeptide. The side chain volumes of various amino residues are shown in the table below. Table 6: Characteristics of amino acids TIFF0007881651000008.tif159170 a The molecular weight of an amino acid minus the molecular weight of water. Value from Handbook of Chemistry and Physics, 43rd ed., Cleveland, Chemical Rubber Publishing Co., 1961. b Value from AAZamyatnin, Prog. Biophys. Mol. Biol., 24:107-123, 1972. c Values ​​from C. Chothia, J. Mol. Biol. 105:1-14, 1975. The writeable surface area is defined in Figures 6-20 of this reference.

[0288] Preferred translocation residues for ridge formation are generally naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In one embodiment, the original residue for ridge formation has a small side-chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain for ridge formation include, but are not limited to, the T366W substitution.

[0289] A “cavity” refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide and thus accommodates a corresponding bulge on the adjacent interface of the first polypeptide. Cavities can be present at the original interface or can be introduced synthetically (e.g., by modifying the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is modified to encode a cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue at the interface of the second polypeptide is replaced with DNA encoding at least one “imported” amino acid residue having a smaller side chain volume than the original amino acid residue. It will be understood that there may be two or more original residues and corresponding imported residues. The upper limit of the number of original residues to be replaced is the total number of residues at the interface of the second polypeptide. Side chain volumes of various amino residues are shown in Table 2 above. Preferred imported residues for cavity formation are usually naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Serine, alanine, or threonine are most preferred. In one embodiment, the original residue for forming the cavity has a large side-chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain for generating the cavity include, but are not limited to, the T366S substitution, L368A substitution, and Y407A substitution.

[0290] The “original” amino acid residue is replaced by an “introduced” residue that may have a smaller or larger side-chain volume than the original residue. The introduced amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, but is preferably a naturally occurring amino acid residue. A “naturally occurring” amino acid residue is one that is encoded by the genetic code and is listed in Table 2 above. A “non-naturally occurring” amino acid residue is one that is not encoded by the genetic code but can covalently bond to an adjacent amino acid residue(s) in the polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). The procedures of Noren et al., Science 244:182 (1989) and Ellman et al. (see above) may be used to generate such non-naturally occurring amino acid residues. In short, this involves the chemical activation of a suppressor tRNA having amino acid residues not naturally occurring, followed by in vitro transcription and translation of the RNA. The method provided herein involves the substitution of at least one original amino acid residue, but two or more original residues may be substituted. Typically, the number of original amino acid residues to be substituted does not exceed the total number of residues at the interface of the first or second polypeptide. Typically, the original residues to be substituted are “filled.” “Filled” means that the residue is essentially inaccessible to the solvent. Generally, the replacement residue is not cysteine ​​to prevent possible oxidation or mispairing of the disulfide bond.

[0291] The ridges are "situable" within the cavity, meaning that the spatial position of the ridge and cavity at the interface of the first and second polypeptides, respectively, and the size of the ridge and cavity, are such that the ridge can be positioned within the cavity without significantly disrupting the normal association of the first and second polypeptides at the interface. Since ridges such as Tyr, Phe, and Trp typically do not extend perpendicularly from the interface axis and do not have a favorable conformation, the alignment of the ridge and the corresponding cavity depends on modeling the ridge / cavity pair based on its three-dimensional structure, such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques widely accepted in the art.

[0292] The term "original or template nucleic acid" refers to the nucleic acid encoding the target polypeptide that can be "modified" (i.e., genetically engineered or mutated) to encode a bulge or cavity. The original or initiating nucleic acid may be a naturally occurring nucleic acid or may include a pre-modified nucleic acid (e.g., a humanized antibody fragment). To "modify" a nucleic acid means that the original nucleic acid is mutated by the insertion, deletion, or substitution of at least one codon encoding the target amino acid residue. Typically, the codon encoding the original residue is replaced by a codon encoding the replacement residue. Techniques for genetically modifying DNA in this manner are outlined in Mutagenesis: A Practical Approach, MJ McPherson, Ed., IRL Press, Oxford, UK (1991), and include, for example, site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis. By mutating the original / template nucleic acid, the original / template polypeptide encoded by the original / template nucleic acid is modified accordingly.

[0293] Raised bumps or cavities may be “introduced” to the interface of a first or second polypeptide by synthetic means, for example, by recombinant techniques, in vitro peptide synthesis, the aforementioned techniques for introducing amino acid residues that do not exist in nature, enzymatic or chemical coupling of peptides, or some combination of these techniques. Thus, “introduced” bumps or cavities are “not naturally occurring” or “unnatural,” meaning they do not exist in natural or original polypeptides (e.g., humanized monoclonal antibodies).

[0294] Generally, the transplanted amino acid residues that form ridges have a relatively small number of "rotational isomers" (e.g., about 3 to 6). A "rotational isomer" is an energetically favorable conformation of the amino acid side chain. The number of rotational isomers of various amino acid residues is outlined in Ponders and Richards, J.Mol.Biol.193:775-791 (1987).

[0295] In one embodiment, a first Fc polypeptide and a second Fc polypeptide contact / interact at an interface. In some embodiments where the first Fc polypeptide and the second Fc polypeptide contact at an interface, the interface (arrangement) of the second Fc polypeptide includes a situable bump (also referred to as a "knob") within a cavity (also referred to as a "hole") of the interface (arrangement) of the first Fc polypeptide. In one embodiment, the first Fc polypeptide is modified from the template / original polypeptide to encode a cavity, or the second Fc polypeptide is modified from the template / original polypeptide to encode a bump, or both. In one embodiment, the first Fc polypeptide is modified from the template / original polypeptide to encode a cavity, and the second Fc polypeptide is modified from the template / original polypeptide to encode a bump. In one embodiment, the interface of the second Fc polypeptide includes a ridge that can be positioned within the cavity of the interface of the first Fc polypeptide, and the cavity, the ridge, or both are introduced at the interface of the first Fc polypeptide and the second Fc polypeptide, respectively. In some embodiments where the first Fc polypeptide and the second Fc polypeptide are in contact at the interface, the interface of the first Fc polypeptide (arrangement) includes a ridge that can be positioned within the cavity of the interface (arrangement) of the second Fc polypeptide. In one embodiment, the second Fc polypeptide is modified from the template / original polypeptide to encode a cavity, or the first Fc polypeptide is modified from the template / original polypeptide to encode a ridge, or both. In one embodiment, the second Fc polypeptide is modified from the template / original polypeptide to encode a cavity, and the first Fc polypeptide is modified from the template / original polypeptide to encode a ridge. In one embodiment, the interface of the first Fc polypeptide includes a ridge that can be positioned within the cavity of the interface of the second Fc polypeptide, and the ridge, the cavity, or both are introduced at the interfaces of the first Fc polypeptide and the second Fc polypeptide, respectively.

[0296] In one embodiment, the ridges and cavities each contain naturally occurring amino acid residues. In one embodiment, the Fc polypeptide containing ridges is produced by replacing an original residue from the interface of the template / original polypeptide with an import residue having a larger side-chain volume than the original residue. In one embodiment, the Fc polypeptide containing ridges is produced by a method comprising the step of replacing a polynucleotide encoding an original residue from the interface of the polypeptide with a polynucleotide encoding an import residue having a larger side-chain volume than the original residue. In one embodiment, the original residue is threonine. In one embodiment, the original residue is T366. In one embodiment, the import residue is arginine (R). In one embodiment, the import residue is phenylalanine (F). In one embodiment, the import residue is tyrosine (Y). In one embodiment, the import residue is tryptophan (W). In one embodiment, the import residue is R, F, Y, or W. In one embodiment, the ridges are produced by replacing two or more residues of the template / original polypeptide. In one embodiment, the Fc polypeptide containing the ridge includes the substitution of threonine at position 366 with tryptophan (amino acid numbering according to the EU numbering scheme of Kabat et al. (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, MD))).

[0297] In some embodiments, cavity-containing Fc polypeptides are produced by replacing original residues at the interface of a template / original polypeptide with import residues having smaller side-chain volumes than the original residues. For example, cavity-containing Fc polypeptides may be produced by a method comprising the step of replacing a polynucleotide encoding an original residue derived from the polypeptide interface with a polynucleotide encoding an import residue having smaller side-chain volumes than the original residue. In one embodiment, the original residue is threonine. In one embodiment, the original residue is leucine. In one embodiment, the original residue is tyrosine. In one embodiment, the import residue is not cysteine ​​(C). In one embodiment, the import residue is alanine (A). In one embodiment, the import residue is serine (S). In one embodiment, the import residue is threonine (T). In one embodiment, the import residue is valine (V). Cavities may be produced by replacing one or more original residues of the template / original polypeptide. For example, in one embodiment, the cavity-containing Fc polypeptide includes the substitution of two or more original amino acids selected from the group consisting of threonine, leucine, and tyrosine. In one embodiment, the cavity-containing Fc polypeptide includes two or more transfer residues selected from the group consisting of alanine, serine, threonine, and valine. In some embodiments, the cavity-containing Fc polypeptide includes the substitution of two or more original amino acids selected from the group consisting of threonine, leucine, and tyrosine, where the original amino acids are replaced by transfer residues selected from the group consisting of alanine, serine, threonine, and valine. In some embodiments, the original amino acids to be replaced are T366, L368, and / or Y407. In one embodiment, the cavity-containing Fc polypeptide includes the substitution of threonine at position 366 with serine (amino acid numbering according to the EU numbering scheme of Kabat et al. (see above)). In one embodiment, the Fc polypeptide containing a cavity includes the substitution of leucine at position 368 with alanine (amino acid numbering according to the EU numbering scheme of Kabat et al. (see above)).In one embodiment, the Fc polypeptide containing a cavity includes a substitution of valine for tyrosine at position 407 (amino acid numbering according to the EU numbering scheme of Kabat et al. (see above)). In one embodiment, the Fc polypeptide containing a cavity includes two or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V (amino acid numbering according to the EU numbering scheme of Kabat et al. (see above)). In some embodiments of these antibody fragments, the Fc polypeptide containing a ridge includes a substitution of tryptophan for threonine at position 366 (amino acid numbering according to the EU numbering scheme of Kabat et al. (see above)).

[0298] In one embodiment, the antibody contains Fc mutations that constitute a “knob” and a “hole” as described in WO2005 / 063816. For example, the hole mutation may be one or more of T366A, L368A, and / or Y407V in the Fc polypeptide, and the knob mutation may be T366W in the IgG1 or IgG4 backbone. Those skilled in the art can produce equivalent mutations in other immunoglobulin isotypes. Furthermore, those skilled in the art will readily understand that it is preferable that the two half-antibodies used for bispecificity are of the same isotype.

[0299] CrossMab technology Schaefer et al. (Roche Diagnostics GmbH) describe a method for expressing two heavy chains and two light chains derived from two existing antibodies as a human bivalent bispecific IgG antibody without using an artificial linker (PNAS (2011) 108(27):11187-11192 and US2009 / 0232811). The method involves exchanging one or more heavy chain and light chain domains within the antigen-binding fragment (Fab) of half of the bispecific antibody (CrossMab). Correct association of the light chains and their homogeneous heavy chains is achieved by the exchange of heavy chain and light chain domains within the antigen-binding fragment (Fab) of half of the bispecific antibody. This "crossover" preserves antigen-binding affinity, but because the two arms are different, mispairing of the light chains can no longer occur. See WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, and WO2010 / 145793, each incorporated herein by reference in its entirety. These recent advantages, for example, resulting from the development of methodologies such as the "Knobs into holes" (KiH) or "CrossMab" technology expression of multispecific antibodies, can still lead to the undesirable formation of product-specific impurities specifically associated with their generation. These product-specific impurities may include, for example, a 1 / 2 antibody (containing a single heavy / light chain pair), a 3 / 4 antibody (containing a complete antibody lacking a single light chain), or a product 5 / 4 antibody (containing an additional heavy or light chain variable domain).

[0300] BiTE technology Another form used for bispecific T cell engager (BiTE) molecules (e.g., Wolf et al. (2005) Drug Discovery Today 10:1237-1244) is based on a single-strand variable fragment (scFv) module. The scFv consists of light and heavy chain variable regions of an antibody fused via a flexible linker, which, generally, can fold appropriately so that the regions can bind to alloantigens. BiTEs link the different specificities of two scFv in tandem on a single strand. This configuration eliminates the generation of molecules with two copies of the same heavy chain variable region. In addition, the linker configuration is designed to ensure correct pairing of the respective light and heavy chains.

[0301] Other bispecific antibody forms Strop et al. (Rinat-Pfizer Inc.) describe a method for generating a stable bimodality antibody by separately expressing and purifying two target antibodies and then mixing them together under specific redox conditions (J.Mol.Biol.(2012)420:204-19).

[0302] Other heterodimerizing domains that prefer heterodimer formation over homodimer formation may be incorporated into this multispecific antigen-binding protein. Examples, though not limited to these, include, for example, WO2007 / 147901 (Kjaergaard et al. - Novo Nordisk (explaining ionic interactions)), WO2009089004 (Kannan et al. - Amgen (explaining electrostatic steering effects)), and WO2010 / 034605 (Christensen et al. - Genentech (explaining coiled-coils)). See also, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992) (explaining leucine zippers), or Pack et al., Bio / Technology 11, 1271-1277 (1993) (explaining helix-turn-helix motifs). The terms “heteromultimerization domain” and “heterodimerization domain” are used synonymously herein. In certain embodiments, the multispecific antigen-binding protein comprises one or more heterodimerization domains.

[0303] Zhu et al. (Genentech) generated heterodimeric diabodies by manipulating mutations at the VL / VH interface of diabodies constructed from variant domain antibody fragments completely lacking the constant domain (Protein Science (1997) 6:781-788). Similarly, Igawa et al. (Chugai) also manipulated mutations at the VL / VH interface of single-stranded diabodies to promote selective expression and inhibit structural isomerization of the diabodies (Protein Engineering, Design & Selection (2010) 23:667-677).

[0304] U.S. Patent Publication 2009 / 0182127 (Novo Nordisk, Inc.) describes the generation of a bispecific antibody by modifying amino acid residues at the Fc interface and CH1:CL interface of a light-heavy chain pair to reduce the ability of one pair of light chains to interact with the other pair of heavy chains.

[0305] Examples of bispecific antibodies include crosslinked or "heteroconjugated" antibodies. For example, in a heteroconjugated antibody, one antibody may be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells against undesirable cells (U.S. Patent No. 4,676,980) and to treat HIV infection (WO91 / 00360, WO92 / 200373, and EP03089). Heteroconjugated antibodies can be produced using any simple crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with several crosslinking techniques.

[0306] Techniques for generating bispecific antibodies from antibody fragments are also described in this literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science 229:81 (1985) describe a procedure in which an intact antibody is proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize the adjacent dithiol and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of another Fab'-TNB derivative to form a bispecific antibody. The produced bispecific antibody can be used as an agent for selective enzyme immobilization.

[0307] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J.Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' portion of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, and then reoxidized to form an antibody heterodimer. This method can also be used for the production of antibody homodimers. The “diabody” technique described by Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) provides an alternative mechanism for producing bispecific antibody fragments. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between two domains on the same chain. Therefore, the VH and VL domains of one fragment are paired with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single-chain Fv(scFv) dimers has also been reported. See Gruber et al., J.Immunol. 152:5368 (1994).

[0308] An overview of various bispecific and multispecific antibodies is provided in Klein et al., (2012) mAbs4:6,653-663, Spiess et al. (2015) “Alternative molecular formats andrapeutic applications for bispecific antibodies.” Mol.Immunol. Published online January 27, 2015, doi:10.1016 / j.molimm.2015.01.003, and Kontermann et al. (2015) Drug Discovery Today 20,838-847.

[0309] Polynucleotides, vectors, host cells, and recombination methods The multispecific antibodies used in the purification methods described herein may be obtained using methods known in the art, including recombinant methods. The following sections provide guidance on these methods.

[0310] Polynucleotides As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA.

[0311] Polynucleotides encoding polypeptides can be obtained from any source, including, but not limited to, cDNA libraries prepared from tissues thought to possess polypeptide mRNA and express it at detectable levels. Therefore, polypeptide-encoding polynucleotides can be readily obtained from cDNA libraries prepared from human tissues. Polypeptide-coding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0312] For example, a polynucleotide can encode an entire immunoglobulin molecular chain, such as a light chain or a heavy chain. A complete heavy chain has a heavy chain variable region (V H ) as well as the heavy chain steady region (C H ) also includes, which typically consists of three constant domains: C H 1, C H 2, and C H 3; and includes the "hinge" region. In some cases, the presence of a steady-state region is desirable.

[0313] Other polypeptides that can be encoded by polynucleotides include antigen-binding antibody fragments, e.g., single-domain antibodies ("dAb"), Fv, scFv, Fab', and F(ab')2, as well as "mini-bodies." Mini-bodies are (typically) C H 1 and C K or C LThese are bivalent antibody fragments from which the domain has been excised. Because the minibodies are smaller than conventional antibodies, they should achieve good tissue penetration for clinical / diagnostic applications, but they are bivalent and must maintain higher binding affinity than monovalent antibody fragments such as dAb. Therefore, unless otherwise specified in the context, the term “antibody,” as used herein, encompasses not only all antibody molecules but also antigen-binding antibody fragments of the types considered above. Preferably, each framework region present in the encoded polypeptide contains at least one amino acid substitution relative to the corresponding human receptor framework. Thus, for example, a framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the receptor framework region.

[0314] Preferably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0315] The term “isolated polynucleotide” is intended to indicate that the molecule has been removed or separated from its normal or natural environment, or has been produced in a manner not present in its normal or natural environment. In some embodiments, the polynucleotide is a purified polynucleotide. The term “purified” is intended to indicate that at least some contaminating molecules or substrates have been removed.

[0316] Preferably, the polynucleotides are substantially purified such that the relevant polynucleotides constitute the dominant (i.e., most abundant) polynucleotides present in the composition.

[0317] Polynucleotide expression The following description primarily concerns the production of polypeptides by culturing cells converted or transfected with a polypeptide-coding polynucleotide vector. Naturally, alternative methods well known in the art may be used to prepare polypeptides. For example, a suitable amino acid sequence or portion thereof can be produced by direct peptide synthesis using solid-phase techniques (see Stewart et al., Solid-Phase Peptide Synthesis WH Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitro protein synthesis can be performed using manual or automated techniques. Automated synthesis can be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) following the manufacturer's instructions. Various parts of a polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired polypeptide.

[0318] The polynucleotides described herein are inserted into an expression vector(s) for polypeptide production. The term “regulatory sequence” refers to a DNA sequence required for the expression of a manipulably linked coding sequence in a particular host organism. Regulatory sequences include, but are not limited to, promoters (e.g., naturally occurring or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.

[0319] A polynucleotide is "operably linked" when it is placed in a functional relationship with another polynucleotide sequence. For example, a pre-sequence or secretion leader nucleic acid is operably linked to the polypeptide nucleic acid when expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked nucleic acid sequences are contiguous, and in the case of a secretion leader, they are contiguous and in the leading phase. Enhancers, however, do not have to be contiguous. Linking is achieved by ligation at a convenient restriction site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional practice.

[0320] For antibodies, the light and heavy chains can be cloned in the same or different expression vectors. The nucleic acid segment encoding the immunoglobulin chain is manipulably ligated to a control sequence in the expression vector(s) that ensures the expression of the immunoglobulin polypeptide.

[0321] CrossMab, containing four different polypeptide chains, utilizes four expression titer sets. These can be cloned in two or four different expression vectors. Each nucleic acid segment encoding an immunoglobulin chain is operably ligated to a control sequence in the expression vector(s) that ensures the expression of the immunoglobulin polypeptide. When two or more expression titer sets are present on the same expression vector, they can be organized unidirectionally or bidirectionally.

[0322] Vectors containing polynucleotide sequences (e.g., variable heavy and / or variable light chains encoding sequences and any expression control sequences) can be transferred to host cells by known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, microparticle guns, or viral-based transfection may be used for other cell hosts. (See Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989) for general information.) Other methods used to transfect mammalian cells include polyblens, protoplast fusion, liposomes, electroporation, and microinjection. For the production of transgenic animals, the transgene may be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells and the nuclei of such cells transferred to enucleated oocytes.

[0323] vector The term "vector" includes expression vectors, as well as transformation vectors and shuttle vectors.

[0324] The term "expression vector" refers to a construct that can be expressed in vivo or in vitro.

[0325] The term "transformation vector" refers to a construct that can be transferred from one host to another, which may be of the same species or a different species. When a construct can be transferred from one host to another, such as from an Escherichia coli plasmid to a bacterium such as a Bacillus, then the transformation vector is sometimes called a "shuttle vector." It may also be a construct that can be transferred from an E. coli plasmid to Agrobacterium, a plant.

[0326] The vector can be converted into a suitable host cell and provide polypeptide expression, as described herein. A variety of vectors are publicly available. The vector may be, for example, a plasmid, cosmid, viral particle, or phage. A suitable nucleic acid sequence can be inserted into the vector by various procedures. Generally, the DNA is inserted into a suitable restriction endonuclease site(s) using techniques known in the art. A suitable vector construct containing one or more of these components is prepared using standard ligation techniques known to those skilled in the art.

[0327] The vector may be, for example, a plasmid, virus, or phage vector provided as an origin of replication, optionally a promoter for polynucleotide expression, and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes known in the art.

[0328] These expression vectors are typically replicable in the host organism as either episomes or as an integral part of host chromosomal DNA.

[0329] For the production of multispecific antibodies, the nucleic acids (or multiple nucleic acids) encoding the multispecific antibody (or the arms of the multispecific antibody, i.e., heavy / light chain pairs) are typically isolated and inserted into a replicable vector for further cloning, amplification, and / or expression. The antibody-encoding DNA is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector selection is partially dependent on the host cell used. Constant regions of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used for this purpose, and it is understood that such constant regions can be obtained from any human or animal species.

[0330] host cell The host cell may be, for example, a bacterial cell, a yeast cell, another fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0331] Genetically engineered transgenic multicellular host organisms can be used to generate polypeptides. These organisms may, for example, be transgenic mammalian organisms (e.g., transgenic goat or mouse strains).

[0332] Suitable prokaryotes include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC31,446), E. coli X1776 (ATCC31,537), E. coli strain W3110 (ATCC27,325), and K5 772 (ATCC53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative and not limiting. Strain W3110 is one particularly preferred host or parental host because it is a common host strain for the fermentation of recombinant polynucleotide products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes.For example, strain W3110 can be modified to affect gene mutations in genes encoding endogenous polypeptides in the host, and examples of such hosts include E. coli W3110 strain 1A2 with complete genotype tonA, E. coli W3110 strain 9E4 with complete genotype tonA ptr3, E. coli W3110 strain 27C7 (ATCC55,244) with complete genotype tonA ptr3phoA E15(argF-lac)169degP ompT kan', E. coli W3110 strain 37D6 with complete genotype tonA ptr3phoA E15(argF-lac)169degP ompT rbs7ilvG kan', E. coli W3110 strain 40B4 which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation, and E. coli strains with mutant periplasmic proteases. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are preferred. In some embodiments, a prokaryotic host cell (e.g., E. coli host cell) expresses one or more chaperones to facilitate antibody folding and assembly. In some embodiments, the chaperone is one or more of FkpA, DsbA, or DsbC. In some embodiments, the chaperone is expressed from an endogenous chaperone gene. In some embodiments, the chaperone is expressed from an exogenous chaperone gene. In some embodiments, the chaperone gene is an E. coli chaperone gene (e.g., E. coli FkpA gene, E. coli DsbA gene, and / or E. coli DsbC gene).

[0333] In these prokaryotic hosts, one can be used to construct an expression vector, which typically contains an expression regulatory sequence compatible with the host cell (e.g., an origin of replication). In addition, there will be any number of different well-known promoters, such as lactose promoter systems, tryptophan (TRP) promoter systems, beta-lactamase promoter systems, or lambda phage promoter systems. Promoters typically control expression and optionally have operator sequences, as well as ribosome-binding site sequences for the initiation and completion of transcription and translation.

[0334] Eukaryotic microorganisms can be used for expression. Eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for polypeptide encoding vectors. Saccharomyces cerevisiae is a commonly used lower-order eukaryotic host microorganism. Other examples include Schizosaccharomyces pombe; for example, K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis (ATCC12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC36,906), K. thermolerans, and K. marxianus, which are Kluyveromyces hosts; yarrowia (EP402,226); Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces Examples of suitable hosts include Schwanniomyces such as occidentalis; filamentous fungi such as Neurospora, Penicillium, and Tolypocladium; and Aspergillus hosts such as A. nidulans and A. niger. Methylotrope yeasts, preferred and not limited to those herein, include methanol-grown yeasts selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces is a preferred yeast host having a suitable vector with expression regulatory sequences (e.g., promoters), origins of replication, termination sequences, etc., as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among many, alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0335] In addition to microorganisms, mammalian tissue cell cultures may also be used to express and produce polypeptides as described herein, and are preferred in some examples (see Winnacker, From Genes to Clones VCH Publishers, NY, NY (1987)). For some embodiments, several suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art, and eukaryotic cells may be preferred, as they include CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines, or transformed B- cells or hybridomas. In some embodiments, the mammalian host cell is a CHO cell.

[0336] In some embodiments, the host cells are vertebrate host cells. Examples of useful mammalian host cell lines include: monkey kidney CV1 cell line converted by SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO or CHO-DP-12 cell line); mouse Sertoli cells; monkey kidney cells (CV1ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor cells (MMT060562, ATCC These include CCL51 cells, TRI cells, MRC5 cells, FS4 cells, and human liver cancer cell line (Hep G2).

[0337] Generation of multispecific antibodies using prokaryotic host cells Vector construction Polynucleotide sequences encoding polypeptide components of multispecific antibodies purified according to the methods provided herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequence encoding the polypeptide (two or more heavy chains and / or two or more light chains) is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells (such as E. coli cells). Many vectors available and known in the art can be used for the purposes of the methods and compositions provided herein. The selection of a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, origins of replication, selection marker genes, promoters, ribosome-binding sites (RBS), signal sequences, heterologous nucleic acid insertions, and transcription termination sequences.

[0338] Generally, plasmid vectors containing species-derived replicons and regulatory sequences compatible with host cells are used in relation to these hosts. These vectors typically have replication sites and marking sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically converted using pBR322, a plasmid derived from the E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing a convenient means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by microorganisms for the expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0339] In addition, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, bacteriophages such as GEM(trademark)-11 can be used to create recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0340] An expression vector may contain two or more promoter-cistron pairs that encode each of the polypeptide components. The promoter is a non-translating regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inductive promoters and constitutive promoters. Inductive promoters are promoters that initiate transcription of cistrons at levels increased under their control in response to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature.

[0341] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be manipulatively bound to cistron DNA encoding a light or heavy chain by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into a vector. Both native promoter sequences and many heterologous promoters can be used to induce amplification and / or expression of a target gene. In some embodiments, heterologous promoters are utilized because they generally result in greater transcription and higher yield of the expressed target gene compared to native target polypeptide promoters.

[0342] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the α-lactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters, such as the tac or trc promoter. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences are publicly available, allowing those skilled in the art to ligate them operably to cistrons encoding target light and heavy chains using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0343] The translation initiation region (TIR) ​​is a major determinant of the overall translation level of a protein. The TIR contains a polynucleotide encoding a signal sequence and extends from just upstream of the Shine-Dalgano sequence to approximately 20 nucleotides downstream of the start codon. Generally, this vector contains the TIR, and both the TIR and variant TIRs are known in the art, as are methods for generating the TIR. A range of nucleic acid sequence variants can be produced at a range of translation intensities, thereby providing a convenient means for tuning this factor for optimal secretion of many different polypeptides. The use of a reporter gene fused to these variants, such as PhoA, provides a method for quantifying the relative translation intensities of different translation initiation regions. Variant or mutant TIRs are provided as a background in a plasmid vector, thereby providing a set of plasmids into which a gene intended to establish an optimal range of translation intensities for the maximal expression of a mature polypeptide is inserted, and its expression is measured. Variant TIRs are disclosed in USP8,241,901.

[0344] In one embodiment, each cistron in the recombinant vector contains a secretory signal sequence component that induces transposition of the expressed polypeptide across the membrane. Generally, the signal sequence may be a component of the vector or part of the target polypeptide DNA inserted into the vector. The selected signal sequence should be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of a prokaryotic host cell that does not recognize and process a signal sequence specific to the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence. Such sequences are well known in the art. In addition, the vector may contain a signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Lpp, or a heat-stable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP.

[0345] In one embodiment, one or more polynucleotides (e.g., an expression vector) collectively encode an antibody. In one embodiment, a single polynucleotide encodes the light chain of the antibody, and a separate polynucleotide encodes the heavy chain of the antibody. In one embodiment, a single polynucleotide encodes both the light and heavy chains of the antibody. In several embodiments, one or more polynucleotides (e.g., an expression vector) collectively encode a one-arm antibody. In one embodiment, a single polynucleotide encodes (a) the light and heavy chains of the one-arm antibody, and (b) the Fc polypeptide. In one embodiment, a single polynucleotide encodes both the light and heavy chains of the one-arm antibody, and a separate polynucleotide encodes the Fc polypeptide. In one embodiment, separate polynucleotides encode the light chain component, the heavy chain component, and the Fc polypeptide of the one-arm antibody, respectively. For the production of one-arm antibodies, see, for example, WO2005063816.

[0346] Suitable prokaryotic host cells for antibody expression include Archaeobacteria and Eubacteria, such as Gram-negative or Gram-positive bacteria. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as host cells. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219, ATCC deposit numbers 27,325) and its derivatives, including strain 33D3 having the genotype W3110ΔfhuA(ΔtonA)ptr3lacIq lacL8ΔompTΔ(nmpc-fepE)degP41kanR (US Patent No. 5,639,635), as well as strains 63C1 and 64B4. In some embodiments, the E. coli strain is a W3110 derivative named 62A7 (ΔfhuA(ΔtonA)ptr3,lacIq,lacL8,ompTΔ(nmpc-fepE)ΔdegP ilvG repair type). Other strains and their derivatives, such as E. coli 294 (ATCC31,446), E. coli B, E. coli λ1776 (ATCC31,537), and E. coli RV308 (ATCC31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing any derivative of any of the above-mentioned bacteria having the defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990).In general, it is necessary to select an appropriate bacterium considering the replication potential of the replicon in bacterial cells. For example, when providing a replicon using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410, E. coli, Serratia, or Salmonella species can be suitably used as hosts. Typically, the host cell should secrete only minimal amounts of protease, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0347] Bacterial cells can be modified to improve the yield and quality of polypeptide production in bacterial cultures. For example, to improve the proper assembly and folding of secreted antibody polypeptides, bacterial host cells may contain additional vectors expressing chaperone proteins such as FkpA and Dsb proteins (DsbB, DsbC, DsbD, and / or DsbG), which can be used to co-transform host prokaryotic cells. It has been demonstrated that chaperone proteins facilitate the proper folding and lysis of heterologous proteins produced in bacterial host cells.

[0348] Multispecific antibody production Host cells are transformed with the expression vector described above and cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selectio...

Claims

1. Culturing cells containing nucleic acids encoding bispecific antibodies in a culture medium, and Purifying the bispecific antibody from the culture medium, A method for producing a bispecific antibody, including A bispecific antibody comprises multiple arms, each arm comprising a VH / VL unit, and each arm of the bispecific antibody is generated separately. The bispecific antibodies are as follows: a) Each arm of the bispecific antibody is subjected to capture chromatography to generate the capture eluate of each arm of the bispecific antibody. b) Forming a mixture containing the capture elutions of each arm of the bispecific antibody under conditions sufficient to produce a composition containing the bispecific antibody, c) A composition containing a bispecific antibody is subjected to first mixed-mode chromatography to produce a first mixed-mode eluate. d) Subjecting the first mixed-mode eluate to a second mixed-mode chromatography to produce a second mixed-mode eluate, and e) Collect fractions containing bispecific antibodies. Purified from the culture medium by a method that sequentially includes the following: The method reduces the amount of product-specific impurities in the composition, wherein the product-specific impurities are one or more of the following: non-counter-antibody arms, antibody homodimers, aggregates, high molecular weight species (HMWS), low molecular weight species (LMWS), acidic variants, and basic variants; the first mixed-mode chromatography is a mixed-mode anion exchange chromatography including a quaternary amine and a hydrophobic moiety; and the second mixed-mode chromatography is a mixed-mode cation exchange chromatography including a carboxyl group. The bispecific antibody is either a knob-in-hole (KiH) bispecific antibody or a CrossMab bispecific antibody. method.

2. The method according to claim 1, wherein the product-specific impurity is one or more of the non-counter antibody arm and antibody homodimer.

3. The method according to claim 1 or 2, wherein the bispecific antibody is a full-length antibody.

4. The method according to any one of claims 1 to 3, wherein the culture medium containing the bispecific antibody is subjected to ion exchange chromatography before the first mixed-mode chromatography, and optionally the ion exchange chromatography contains a quaternary amine, or the culture medium containing the bispecific antibody is subjected to hydrophobic interaction (HIC) chromatography before the first mixed-mode chromatography.

5. The method according to any one of claims 1 to 4, wherein the capture chromatography is protein L chromatography, protein A chromatography, protein G chromatography, or protein A and protein G chromatography.

6. The method according to any one of claims 1 to 5, wherein the first mixed-mode chromatography and the second mixed-mode chromatography are continuous.

7. (a) The first mixed-mode chromatography is performed in binding and elution mode or flow-through mode, (b) The second mixed-mode chromatography is performed in binding and elution mode or flow-through mode. The method according to any one of claims 1 to 6.

8. (a) The first mixed-mode chromatography is performed in flow-through mode, (b) A second mixed-mode chromatography is performed in binding and elution modes. The method according to claim 7.

9. The method according to claim 7, wherein the first mixed-mode chromatography is performed in binding and elution modes, and the elution is gradient elution.

10. The method according to any one of claims 7 to 9, wherein the second mixed-mode chromatography is performed in binding and elution modes, and the elution is gradient elution.

11. The capture chromatography is protein A chromatography, and the protein A chromatography uses one or more of protein A equilibration buffer, protein A loading buffer, and protein A washing buffer. The equilibration buffer, loading buffer, and / or washing buffer have a pH of approximately 7 to 8. The method according to any one of claims 5 to 10.

12. The method according to claim 11, wherein the protein A equilibration buffer comprises about 25 mM Tris and about 25 mM NaCl.

13. The method according to any one of claims 1 to 12, wherein the fraction comprises at least about 95% bispecific antibodies.

14. The fraction is (a) Arm with 5% non-counterantibody, (b) 5% antibody homodimer, (c) 2% aggregates or high molecular weight species (HMWS), (d) 2% low molecular weight species (LMWS), (e) 50% acidic variant, (f) 35% basic variant, and (g) 5% 3 / 4 antibody, The method according to any one of claims 1 to 13, comprising one or more of the above.

15. The fraction is (a) Bispecific antibodies with a concentration of at least 95% to 100%, (b) Arms with approximately 1% to 5% or less of non-counter antibodies, (c) Antibody homodimers containing approximately 1% to 5% or less (d) HMWS of approximately 1% or 2% or less, (e) LMWS of approximately 1% or 2% or less, and (f) Contains 3 / 4 antibodies of approximately 5% or less, The method according to any one of claims 1 to 13.

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