MULTI-SPECIFIC ANTIBODY PURIFICATION

Abstract

This paper provides methods for purifying multispecific antibodies. The methods comprise the sequential steps of performing a capture chromatography, a first mixed-mode chromatography, and a second mixed-mode chromatography. In some respects, it provides multispecific antibody compositions, wherein these compositions have reduced levels of one or more product-specific and / or process-specific impurities.

Description

PRESENTATION OF THE LIST OF SEQUENCES IN TEXT FILE ASCII The contents of the following ASCII text file presentation are incorporated herein in their entirety by this reference: A Computer Readable Form (CRF) of the List of Sequences (file name: 146392036341SEQLIST.TXT, registration date: July 9, 2017, size: 32 KB). FIELD OF INVENTION Methods are provided for purifying multispecific antibodies from a composition comprising the multispecific antibody and at least one impurity, including at least one specific impurity of the product. In some embodiments, the specific impurity of the product is, for example, a precursor, an aggregate and / or a variant of the multispecific antibody. Also provided are multispecific antibodies purified according to the methods and compositions, and formulations comprising said multispecific antibodies. BACKGROUND OF THE INVENTION For recombinant biopharmaceutical proteins to be acceptable for administration to human patients, it is important that residual impurities resulting from the manufacturing and purification processes are removed from the final biological product. These process components include culture media proteins, immunoglobulin affinity ligands, viruses, endotoxins, DNA, and host cell proteins (HCPs). The development of new antibody formats, such as multispecific antibodies, presents new challenges as conventional manufacturing and purification processes are inadequate to sufficiently remove specific impurities from the product, including mismatched antibody arms and antibodies. poorly assembled. Compared to antibody purification Standard, purification of multispecific antibodies from production media presents unique challenges. While a bivalent antibody Standard monospecific antibody is the result of the dimerization of identical heavy chain / light chain subunits, the production of a multispecific antibody requires the dimerization of at least two different heavy chain / light chain subunits, where each comprises a different heavy chain, as well as a different light chain. Production and purification of the final correct and complete multispecific antibody, with minimal amounts of mismatched, misassembled or incomplete molecules presents different challenges. Chain mismatches (e.g., homodimerization of identical heavy chain peptides or inappropriate heavy chain / light chain associations) are commonly observed, as well as incomplete protein assembly due to unbalanced host cell expression of different proteins. antibody chains. Commonly observed product-specific impurities include half (¾) antibodies (comprising a single heavy chain / light chain pair), three-quarters (¾) antibodies (comprising a full antibody lacking a single light chain) and homodimers. Additional product-specific impurities may be observed depending on the multispecific format used. For example, when a multispecific antibody variable domain is constructed as a single-chain Fab (scFab), a 5 / 4 antibody byproduct (comprising an additional heavy or light chain variable domain) can be observed. These corresponding product-specific impurities would not appear in standard antibody production. Conventional purification techniques designed to remove process-related impurities such as HCP, DNA, endotoxins, and other materials that have characteristics and properties very different from antibodies may be inadequate when implemented to remove impurities that are more similar to antibodies. multispecies antibodies. Thus, it is necessary to develop manufacturing and purification schemes that effectively remove specific impurities from the product and produce a sufficient quantity of the correct and complete multispecific antibody. All references mentioned herein, including publications and patent applications, are incorporated in their entirety by this reference. DIGEST OF THE INVENTION As described and exemplified herein, applicants have discovered that the use of at least two mixed-mode chromatography steps (also referred to herein as multi-modal or multimodal), following an initial capture chromatography step , produces greater removal of product-specific impurities and an improved process for purifying multispecific antibodies. Therefore, in certain embodiments, a method is provided for purifying a multispecific antibody from a composition comprising the multispecific antibody and an impurity, wherein the multispecific antibody comprises multiple arms, each arm comprising a VH / VL unit, wherein The method comprises the sequential steps of: 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 generate a first mixed mode eluate; 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 specific impurity of the product of the composition. In some embodiments according to (or applied to) any of the above embodiments, 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 (or applied to) any of the above embodiments, the second mixed mode eluate is subjected to ion exchange chromatography (e.g. e.g., anion exchange) or hydrophobic interaction chromatography. In certain embodiments, a method is provided for purifying a multispecific antibody from a composition comprising the multispecific antibody and an impurity, wherein the multispecific antibody comprises multiple arms, each arm comprising a VH / VL unit, wherein each arm of the antibody multispecific antibody is produced separately, where the method comprises the sequential steps of a) subjecting each arm of the multispecific antibody to capture chromatography to produce capture eluates for each arm of the multispecific antibody, b) forming a mixture comprising 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 generate a first mixed mode eluate and d) subjecting the first eluate of mixed mode to a second mixed mode chromatography to generate a second mixed mode eluate; and e) collecting a fraction comprising the multispecific antibody, wherein the method reduces the amount of a specific impurity of the product of the composition. In some embodiments according to (or applied to) any of the above embodiments, 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 (or applied to) any of the above embodiments, the second mixed mode eluate is subjected to ion exchange chromatography (e.g., anion exchange) or hydrophobic interaction chromatography. In some embodiments according to (or applied to) any of the above embodiments, capture chromatography is affinity chromatography. In some embodiments according to (or applied to) any of the above embodiments, affinity chromatography is protein L chromatography, protein A chromatography, protein G chromatography, protein A and protein G chromatography. With (or applied to) any of the above modalities, affinity chromatography is protein A chromatography. In some embodiments according to (or applied to) any of the above embodiments, capture chromatography is performed in binding and elution mode. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography and the second mixed mode chromatography are contiguous. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography is a mixed mode anion exchange chromatography. In some embodiments according to (or applied to) any of the above embodiments, the second mixed mode chromatography is a mixed mode cation exchange chromatography. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography is a mixed mode cation exchange chromatography. In some embodiments according to (or applied to) any of the above embodiments, the second mixed mode chromatography is a mixed mode anion exchange chromatography. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography is performed in junction mode and φ elution or in continuous flow mode. In some embodiments according to (or applied to) any of the embodiments in which the first mixed mode chromatography is performed in binding and elution mode, the elution is a gradient elution. 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 in continuous flow 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, the elution is a gradient elution. In some embodiments according to (or applied to) any of the above embodiments, the method further comprises the step of subjecting the second mixed mode eluate to ultrafiltration. In some embodiments according to (or applied to) any of the above embodiments, the ultrafiltration sequentially comprises a first ultrafiltration, a diafiltration and a second ultrafiltration. In some embodiments according to (or applied to) any of the above embodiments, protein A chromatography comprises protein A bound to agarose. In some embodiments according to (or applied to) any of the above embodiments, protein A chromatography is protein A chromatography. MAbSelect™, MAbSelect™ SuRe and MAbSelect™ SuRe LX, ProsepVA, Prosep-VA Ultra Plus, Fast Flow Protein A Sepharose, or Toyopearl. In some embodiments according to (or applied to) any of the above embodiments, protein A chromatography utilizes one or more of a protein A equilibration buffer, a protein A loading buffer, or a protein A wash buffer. , wherein the equilibration buffer, a load buffer and / or the wash buffer have between about pH 7 and about pH 8. In some embodiments according to (or applied to) any of the above embodiments, the equilibration buffer of protein A has approximately pH 7.7. In some embodiments according to (or applied to) any of the above embodiments, the protein A equilibration buffer comprises approximately 25 mM Tris and approximately 25 mM NaCl. In some embodiments in accordance with (or applied a) In any of the above embodiments, protein A chromatography is washed with equilibration buffer after loading. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from protein A by applying low pH protein A elution buffer to protein A chromatography. With (or applied to) any of the above embodiments, the protein A elution buffer comprises approximately 150 mM acetic acid and approximately pH 2.9. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from protein A chromatography by pH gradient. In some embodiments according to (or applied to) any of the above embodiments, anion exchange mixed mode chromatography comprises a quaternary amine and a hydrophobic moiety. In some embodiments according to (or applied to) any of the above embodiments, anion exchange mixed mode chromatography comprises a quaternary amine and a hydrophobic moiety linked to highly cross-linked agarose. In some embodiments according to (or applied to) any of the above embodiments, the mixed mode chromatography is a Capto™ Adhere chromatography or a Capto™ Adhere ImpRes chromatography. In some embodiments according to (or applied to) any of the above embodiments, cationic mixed mode chromatography comprises N-benzyl-n-methyl ethanolamine exchange. In some embodiments according to (or applied to) any of the above embodiments, the mixed mode chromatography is a Capto™ MMC chromatography or a Capto™ MMC ImpRes chromatography. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography uses one or more of a mixed mode pre-equilibration buffer, a mixed mode equilibration buffer, a loading buffer mixed mode or a mixed mode wash damper, wherein the mixed mode prebalance damper, the mixed mode balance damper, the mixed mode load damper and / or the mixed mode wash damper have between approximately pH 6 and about pH 7. In some embodiments, an anion mixed mode equilibration buffer has between about pH 6.5 and about pH 8. In some embodiments according to (or applied to) any of the above embodiments, the second mixed-mode chromatography utilizes one or more of a mixed moto pre-balancing damper, a mixed-mode balancing damper, an e mixed-mode charge buffer or a mixed-mode wash buffer, wherein the mixed-mode pre-equilibration buffer, the mixed-mode equilibration buffer and / or the mixed-mode wash buffer has between about pH 5 and about pH 8, optionally between about pH 5 and about pH 7 or between about pH 5 and about pH 6 or between about pH 6 and about pH 7. In some embodiments according to (or applied to) any of the above embodiments, the mixed-mode pre-equilibration buffer, the mixed-mode equilibration buffer, and / or the mixed-mode wash buffer has approximately pH 5.5. . In some embodiments according to (or applied to) any of the above embodiments, the mixed mode pre-equilibration buffer comprises approximately 500 mM acetate. In some embodiments according to (or applied to) any of the above embodiments, the mixed mode equilibration buffer comprises approximately 50 mM acetate. In some embodiments according to (or applied to) any of the above embodiments, the first mixed mode chromatography is washed with wash buffer after loading. In some embodiments according to (or applied to) any of the above embodiments, the second mixed mode chromatography is washed with wash buffer after loading. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from the first mixed mode chromatography by salt gradient and / or pH gradient or stepwise pH elution. In some embodiments in accordance with (or applied a) In any of the above embodiments, the multispecific antibody is eluted from the first mixed mode chromatography by applying a low pH mixed mode elution buffer to the mixed mode exchange chromatography. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from the second mixed mode chromatography by salt gradient and / or pH gradient or stepwise pH elution. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from the second mixed mode chromatography by applying a low pH mixed mode elution buffer to the mode exchange chromatography. mixed. In some embodiments according to (or applied to) any of the above embodiments, the mixed mode elution buffer comprises about 25 mM acetate, about pH 5.5. In some embodiments according to (or applied to) any of the above embodiments, anion exchange chromatography comprises quaternary amine. In some embodiments according to (or applied to) any of the above embodiments, anion exchange chromatography comprises quaternary amine linked to cross-linked agarose. In some embodiments according to (or applied to) any of the above embodiments, anion exchange chromatography is QSFF chromatography. In some embodiments according to (or applied to) any of the above embodiments, anion exchange chromatography utilizes one or more of an anion exchange pre-equilibration buffer, an anion exchange equilibration buffer, or an anion exchange loading buffer. anion exchange, wherein the anion exchange pre-equilibration buffer, the anion exchange equilibration buffer and / or the anion exchange loading buffer has between about pH 8 and about pH 9. In some embodiments according to (or applied a) any of the above embodiments, the anion exchange pre-equilibration buffer, the anion exchange equilibration buffer and / or the anion exchange loading buffer has approximately pH 8.5. In some embodiments according to (or applied to) any of the above embodiments, the anion exchange pre-equilibration buffer comprises approximately 50 mM Tris, 500 mM sodium acetate. In some embodiments according to (or applied to) any of the above embodiments, the anion exchange equilibration buffer comprises approximately 50 mM Tris. In some embodiments according to (or applied to) any of the above embodiments, the anion exchange chromatography is washed with anion exchange equilibration buffer after loading. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from the anion exchange chromatography by salt gradient. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is eluted from the anion exchange chromatography by applying an anion exchange elution buffer with an increasing salt concentration to the anion exchange chromatography. anion exchange. In some embodiments according to (or applied to) any of the above embodiments, the anion exchange elution buffer comprises approximately 50 mM Tris, 100 mM sodium acetate at approximately pH 8.5. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody arms are produced in a cell. In some embodiments according to (or applied to) any of the above embodiments, the cell is a prokaryotic cell. In some embodiments according to (or applied to) any of the above embodiments, the prokaryotic cell is an E. coli cell. In some embodiments according to (or applied to) any of the above embodiments, the cell is modified to express one or more chaperones. In some embodiments according to (or applied to) any of the above embodiments, the chaperone is one or more of FkpA, DsbA or DsbC. In some embodiments according to (or applied to) any of the above embodiments, the chaperone is an E. coli chaperone. In some embodiments according to (or applied to) the above embodiments, the cell is a eukaryotic cell. In some embodiments according to (or applied to) the above embodiments, the eukaryotic cell is a yeast cell, an insect cell, or a mammalian cell. In some embodiments according to (or applied to) the above embodiments, the eukaryotic cell is a CHO cell. In some embodiments according to (or applied to) any of the above embodiments, cells are lysed to generate a cell lysate comprising the multispecific antibody or an arm of the multispecific antibody prior to capture chromatography. In some embodiments according to (or applied to) any of the above embodiments, cells are lysed using a microfluidizer. In some embodiments according to (or applied to) any of the above embodiments, polyethyleneimine (PEI) is added to the cell lysate prior to chromatography. In some embodiments according to (or applied to) any of the above embodiments, PEI is added to the lysate at a final concentration of about 0.4%. In some embodiments according to (or applied to) any of the above embodiments, the cell lysate is clarified by centrifugation. In some embodiments according to (or applied to) any of the above embodiments, the cell lysate of a mammalian cell, e.g. For example, a CHO cell is subjected to one or more of the following treatments: heat inactivation, low pH inactivation, viral inactivation by addition of detergent. In some embodiments in accordance with (or applied to) and In any of the above embodiments, the method reduces the amount of a process-specific impurity, such as any host cell protein (HCP), leached protein A, nucleic acid, cell culture media components or viral impurities in the composition. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody is a bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g., a KiH bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a bispecific antibody of CrossMab. In some embodiments according to (or applied to) any of the above methods, a fraction is collected after the last chromatography step, comprising at least about 95%, at least about 96%, at least about 97%. %, at least about 98%, at least about 99% or about 100% multispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, a fraction is collected after the last chromatography step, comprising a reduced amount of a product-specific impurity, wherein the product-specific impurity is a or more of: unpaired antibody arms, antibody homodimers, high molecular weight species (HMWS), low molecular weight species (LMWS), or % antibodies. In some embodiments according to (or applied to) any of the above embodiments, the fraction contains less than about 5%, less than about 4%, less than about 3%, less than about 2% or less of approximately 1% unpaired antibody arms. In some embodiments according to (or applied to) any of the above embodiments, the fraction contains less than about 5%, less than about 4%, less than about 3%, less than about 2% or less of approximately 1% homodimers of the antibody. In some embodiments according to (or applied to) any of the above embodiments, the fraction contains no more than about 1% or no more than about 2% HMWS. In some embodiments according to (or applied to) any of the above embodiments, the fraction contains no more than about 2% or no more than about 1% LMWS. In some embodiments in accordance with (or applied Yo a) any of the above embodiments, the fraction contains no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2% or no more than about 1% of % antibodies. In some embodiments according to (or applied to) any of the above embodiments, the fraction contains a) at least about 95% - 100% multispecific antibody; b) less than approximately 1%-5% unpaired antibody arms; c) less than about 1%-5% homodimers of the antibody; d) not more than about 1% or 2% HMWS; e) no more than approximately 1% or 2% of LMWS; and / or f) no more than approximately 5% ¾ antibodies. In certain embodiments, a composition is provided comprising a multispecific antibody purified through the method of any of the methods described above. In some embodiments according to (or applied to) any of the above methods, a composition is provided comprising a multispecific antibody, wherein the composition comprises at least about %, at least about 96%, at least about 97% %, at least about 98%, at least about 99% or about 100% multispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, a composition is provided comprising a multispecific antibody, wherein the composition comprises a reduced amount of a specific impurity of the product, wherein the specific impurity of the product is one or more of: unpaired antibody arms, antibody homodimers, high molecular weight species (HMWS), low molecular weight species (LMWS), or ¾ antibodies. In some embodiments according to (or applied to) any of the above embodiments, the composition contains less than about 5%, less than about 4%, less than about 3%, less than about 2% or less of approximately 1% unpaired antibody arms. In some embodiments according to (or applied to) any of the above embodiments, the composition contains less than about 5%, less than about 4%, less than about 3%, less than about 2% or less of approximately 1% homodimers of the antibody. In some embodiments according to (or applied to) any of the above embodiments, the composition contains no more than about 1% or no more than about 2% HMWS. In some embodiments according to (or applied to) any of the above embodiments, the composition contains no more than about 2% or no more than about % LMWS. In some embodiments according to (or applied to) any of the above embodiments, the composition contains no more than about 5%, no more than about 4%, no more than about 2%, no more than about 2%. or no more than approximately 1% of ¾ antibodies. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody of the composition is a bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g., a KiH bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a bispecific antibody of CrossMab. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody binds to ANG-2 and VEGF. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody that binds ANG-2 and VEGF comprises a) the heavy chain and the light chain of a first full-length antibody comprising the first antigen binding site; and b) the modified heavy chain and the modified light chain of a full-length antibody comprising the second antigen binding site, wherein the CL and CHl constant domains replace each other. In some embodiments according to (or applied to) any of the above embodiments, the composition contains: a) at least about 95% - 100% multispecific antibody; b) less than approximately %-5% unpaired antibody arms; c) less than about 1%-5% homodimers of the antibody; d) no more than approximately 1% or 2% of HMWS; e) no more than approximately 1% or 2% of LMWS; and / or f) no more than approximately 5% ¾ antibodies. In some embodiments according to (or applied to) any of the above embodiments, the multispecific antibody of the composition is a bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g., a KiH bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody is a CrossMab bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody binds to ANG-2 and VEGF. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody that binds ANG-2 and VEGF comprises a) the heavy chain and the light chain of a first full-length antibody comprising the first antigen binding site; and b) the modified heavy chain and the modified light chain of a full-length antibody comprising the second antigen binding site, wherein the CL and CHl constant domains replace each other. In some embodiments according to (or applied to) any of the above embodiments, a composition is provided comprising a multispecific or bispecific antibody (such as a bispecific antibody that binds ANG2 and VEGF) purified through any of the methods. described above for the treatment of cancer or eye disease. In some embodiments according to (or applied to) any of the above embodiments, there is provided the use of a composition comprising a multispecific or bispecific antibody (such as a bispecific antibody that binds ANG2 and VEGF) purified through either of the methods described above for the manufacture of a medicament for the treatment of cancer or eye disease. In some embodiments according to (or applied to) any of the above embodiments, the methods provided herein are used for the purification of a heterodimeric polypeptide containing Faith. In some embodiments according to (or applied to) any of the above embodiments, use of any of the methods provided herein is provided for the reduction of impurities related to Fe-containing heterodimeric polypeptides in a composition. In certain embodiments, a method is provided for purifying a region-containing heterodimeric polypeptide. Fe with a multi-step chromatography method, wherein the method comprises an affinity chromatography step followed by two different multimodal ion exchange chromatography steps, and thereby purifying the Fe region-containing heterodimeric polypeptide. In some embodiments according to (or applied to) any of the above embodiments, the multi-stage 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. In some embodiments according to (or applied to) any of the above embodiments, the multi-stage chromatography method comprises an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by an affinity chromatography step. multimodal cation exchange; In some embodiments according to (or applied to) any of the above embodiments, the multi-stage chromatography method comprises exactly three chromatography steps. In some embodiments according to (or applied to) any of the above embodiments, the multimodal anion exchange chromatography step is performed in continuous flow mode. In some embodiments according to (or applied to) any of the above embodiments, in the multimodal <---I anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with a lower conductivity value at 7 mS / cm. In some embodiments according to (or applied to) any of the above embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with a conductivity value in the range of about mS / cm and approximately 2 mS / cm. In some embodiments according to (or applied to) any of the above embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with a conductivity value of about 4.5 mS / cm. In some embodiments according to (or applied to) any of the above embodiments, the multimodal anion exchange chromatography step is performed at a pH of about 7. In some | embodiments according to (or applied to) any of the above embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with 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, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in the range of between about 100 g and about 300 g per liter of chromatography material. In some embodiments according to (or applied to) any of the above embodiments, the multimodal anion exchange chromatography material is a multimodal strong anion exchange chromatography material. In some embodiments according to (or applied to) any of the above embodiments, the multimodal anion exchange chromatography material has a high flux agarose matrix, a multimodal strong anion exchanger as a ligand, an average particle size of 36 -44 pm and an ionic capacity of between 0.08 and 0.11 mmol in Cl- / mL medium. In some embodiments according to (or applied to) any of the above embodiments, the multimodal cation exchange chromatography medium is a multimodal weak cation exchange chromatography medium. In some embodiments according to (or applied to) any of the above embodiments, the multimodal cation exchange chromatography medium has a high flux agarose matrix, a multimodal weak cation exchanger as a ligand, an average particle size of 36 -44 pm and an ionic capacity of between 25 and 39 pmol / mL. In some embodiments according to (or applied to) any of the above embodiments, the multimodal anion exchange chromatography step is performed in binding and elution mode. In some embodiments according to (or applied to) any of the above embodiments, capture chromatography is carried out by affinity chromatography. In some embodiments, the affinity chromatography is a protein A affinity chromatography, a protein G affinity chromatography, a single chain Fv ligand affinity chromatography, a chromatography step with chromatography material CaptureSelect or a chromatography step with CaptureSelect FcXL chromatography material. In some embodiments according to (or applied to) any of the preceding embodiments, affinity chromatography is a protein A chromatography step. In some embodiments according to (or applied to) any of the preceding embodiments, the chromatography step Affinity is a chromatography step with chromatography material CaptureSelect™. In some embodiments according to (or applied to) any of the above embodiments, the Fe region-containing heterodimeric polypeptide is an antibody, a bispecific antibody or Fe fusion proteins. In some embodiments according to (or applied to) any Of the above embodiments, the Fe region-containing heterodimeric polypeptide is a bispecific antibody. In some embodiments according to (or applied to) any of the above embodiments, the Fe region-containing heterodimeric polypeptide is a CrossMab. In some embodiments in accordance with (or I applied a) any of the previous modalities, the If Fe region-containing heterodimeric polypeptide is a bispecific antibody comprising a) a heavy chain and a light chain of a full-length first antibody that specifically binds to a first antigen; and b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds to a second antigen, where the CL and CH1 constant domains replace each other. : In some embodiments in accordance with (or applied to) In any of the above embodiments, the bispecific antibody binds to ANG2 and VEGF. In some embodiments according to (or applied to) any of the above embodiments, CrossMab binds to ANG2 and VEGF. In some embodiments in accordance with (or applied to) | any of the above modalities, the bispecific antibody is vanucizumab. In some embodiments in accordance with (or applied to) any of the embodiments • above, the bispecific antibody comprises a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 1 and, as a light chain variable domain (VL), SEQ ID NO: 2 ; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 3 and, as a light chain variable domain (VL), SEQ ID NO: 4. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody comprises a first heavy chain with the amino acid sequence of SEQ ID NO: 9 and a second heavy chain with the amino acid sequence of the SEQ ID NO: 10 and a first light chain with the amino acid sequence of SEQ ID NO: 11 and a second light chain with the amino acid sequence of SEQ ID NO: 12. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody comprises a first antigen binding site comprising, as a heavy chain variable (VH) domain, SEQ ID NO: 5 and, as light chain variable domain (VL), SEQ ID NO: 6; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 7 and, as a light chain variable domain (VL), SEQ ID NO: 8. In some embodiments of in accordance with (or applied to) any of the • In the above embodiments, the bispecific antibody comprises a first heavy chain with the amino acid sequence of SEQ ID NO: 13 and a second heavy chain with the amino acid sequence of SEQ ID NO: 14 and a first light chain with the amino acid sequence of SEQ ID NO: 15 and a second light chain with the amino acid sequence of SEQ ID NO: 16. In some embodiments according to (or applied to) any of the above embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about 5% ¾ antibodies. In certain embodiments, a method is provided for purifying a bispecific antibody that binds to ANG2 and VEGF with a multi-step chromatography method, wherein the method comprises an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step, and thereby purifying the bispecific antibody that binds to ANG2 and VEGF, where the bispecific antibody that binds ANG2 and VEGF comprise a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 1 and, as a light chain variable domain (VL), SEQ ID NO: 2; and a second binding site as an antigen chain variable domain comprising, heavy (VH), SEQ ID NO: 3 and, as a light chain variable domain (VL), SEQ ID NO: 4 or comprising a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 5 and, as a light chain variable domain (VL), SEQ ID NO: 6; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: and, as a variable light chain (VL) domain, the SEQ ID NO: 8. In some embodiments according to (or applied to) any of the above embodiments, the bispecific antibody that binds ANG2 and VEGF comprises a) the heavy chain and the light chain of a first full-length antibody comprising the first antigen binding site; and b) the modified heavy chain and the modified light chain of a full-length antibody comprising the second antigen binding site, wherein the CL and CH1 constant domains replace each other. In some embodiments, the use of any method according to (or applied to) any of the above embodiments is provided for the reduction of impurities related to the heterodimeric polypeptide containing Faith. In some embodiments, a polypeptide is provided Fe-containing heterodimeric obtained with the method according to (or applied to) any of the above modalities for the manufacture of a medicament for the treatment of cancer or ocular disease. In some embodiments, an Fe-containing heterodimeric polypeptide obtained from the method according to (or applied to) any of the above embodiments is provided for use in the treatment of cancer or ocular disease. In certain embodiments, a method is provided for producing an Fe-containing heterodimeric polypeptide comprising the following steps (i) culturing a cell comprising a nucleic acid encoding an Fe-containing heterodimeric polypeptide; (ii) recovering the Fe-containing heterodimeric protein from the cell or culture medium; (iii) purifying the Fe-containing heterodimeric polypeptide by using a method according to (or applied to) any of the above embodiments and thereby producing the Fe-containing heterodimeric polypeptide. In certain embodiments, a method is provided for producing a bispecific antibody that binds to ANG-2 and VEGF comprising the steps of: (i) culturing a cell comprising a nucleic acid encoding the bispecific antibody; (ii) recovering the bispecific antibody from the cell or culture medium; (iii) purifying the bispecific antibody by using a method according to (or applied to) any of the above embodiments, and thereby producing the bispecific antibody that binds ANG-2 and VEGF. BRIEF DESCRIPTION OF THE FIGURES Figure IA represents a first purification scheme used in Example 1. Figure IB represents a second purification scheme used in Example 1. Figure IC represents a third purification scheme used in Example 1. Figure 2 represents a purification scheme used in Example 2. Figure 3 A represents a first purification scheme used in Example 3. Figure 3 B represents a second purification scheme used in Example 3. Figure 4 represents a purification scheme used in Example 4. Figure 5A represents a first purification scheme used in Example 6. Figure 5B represents a second purification scheme used in Example 6. Figure 6A represents a first purification scheme used in Example 7. Figure 6B represents a second purification scheme used in Example 7. Figure 7A represents a first purification scheme described in Example 8. Figure 7B represents a second purification scheme described in Example 8. Figure 7C represents a third purification scheme described in Example 8. DETAILED DESCRIPTION OF THE INVENTION Provided herein are methods for purifying a multispecific antibody (such as a bispecific antibody or a divalent F(ab')2) comprising the sequential steps of subjecting a composition comprising the multispecific antibody to a) capture chromatography, b) a first mixed mode chromatography and c) a second mixed mode chromatography. In some aspects, methods are provided for purifying a multispecific antibody wherein each of the individual arms of the multispecific antibody is produced in separate cultures and purified separately by capture chromatography. The purified antibody arms are assembled to produce the multispecific antibody. The assembled multispecific antibody is subjected to a first mixed-mode anion exchange chromatography followed by a second mixed-mode chromatography. As described in more detail below, each of the capture chromatography, the first mixed mode chromatography and / or second mixed mode chromatography are preceded and / or followed by one or more additional chromatography steps. The terms mixed mode chromatography and multimodal chromatography are used interchangeably herein. In some aspects, compositions are provided that They comprise multispecific antibodies that have reduced levels of one or more product-specific and / or process-specific impurities, such as unpaired antibody arms, homodimers, aggregates, low molecular weight species, acidic and basic variants. In some aspects, compositions are provided comprising multispecific antibodies having reduced levels of one or more process-specific impurities, such as, e.g. e.g., prokaryotic host cell protein, eukaryotic host cell protein (such as CHO or CHOP proteins), nucleic acid and chaperones (such as prokaryotic chaperones, e.g., FkpA, DsbA and DsbC). In certain embodiments, the compositions provided herein are obtained by using a method provided herein. In certain embodiments, the compositions provided herein have reduced levels of one or more product-specific and / or process-specific impurities than compositions obtained using methods known in the art. In some aspects, uses of the methods as described herein are provided for the purification of an Fe-containing heterodimeric polypeptide and for the reduction of impurities related to Fe-containing heterodimeric polypeptide. A reduction is achieved improved removal of specific product impurities. In the case of CrossMab-specific impurities, a reduction of e.g. e.g., ¾ antibodies. Definitions The terms polypeptide and protein are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids and may be interrupted by non-amino acids. The terms also encompass a polymer of amino acids that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The terms polypeptide and protein, as used herein, specifically encompass antibodies. Purified polypeptide (e.g., antibody or immunoadhesin) means that the purity of the polypeptide has been increased so that it exists in a purer form than in its natural environment and / or when initially synthesized and / or amplified under conditions of laboratory. 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 degree of purity of a desired molecule (such as a multispecific antibody, e.g., a bispecific antibody) from a composition. or sample comprising the desired molecule and one or more impurities. Typically, the degree of impurity of the desired molecule is increased by removing (completely or partially) at least one impurity from the composition. A multispecific antibody that binds to an antigen of interest is one that binds to the antigen, e.g. e.g., a protein, with sufficient affinity so that the multispecific antibody is useful as a diagnostic and / or therapeutic agent to target a protein, cell or tissue that expresses the protein, and does not exhibit considerable cross-reactivity with other proteins. In such embodiments, the extent of binding of the multispecific antibody to a non-target protein will be less than about 10% of the binding of the multispecific antibody to its particular target protein as determined, p. For example, fluorescence-activated cell sorting assay (FACS, radioimmunoprecipitation (RIA) or ELISA. Regarding the binding of a multispecific antibody to a molecule target, the term specific binding, binds specifically to or specific to a polypeptide or a particular epitope or a particular polypeptide target indicates a binding significantly different from a non-specific interaction (e.g., a non-specific interaction may be a binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule. similar to the target, for example, an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term specific binding, specifically binds to or is specific for a particular epitope or polypeptide on a particular polypeptide target as used herein may be displayed, for example, by a molecule with a Kd for the target of at least about 200 nM, alternatively to about 150 nM, alternatively to about 100 nM, alternatively to 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 higher affinity. In one embodiment, the term specific binding refers to binding in which a multispecific antigen binding protein is binds a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. Binding affinity generally refers to the sum total stress of 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 intrinsic binding affinity that reflects an interaction 1:1 between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule with respect to its partner Y can generally be represented by the dissociation constant (Kd). For example, the 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 nM or less, about 2 0 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 calculated using common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigen more quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art and any of them can be used for the purposes of the methods provided herein. In one embodiment, the Kd or Kd value according to the present invention is measured by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized target (e.g., antigen) CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with Netyl-N'-(3-dimethylaminopropyl)-carbodiimide 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 pg / ml (~0.2 μΜ) before injection at a flow rate of 5 μΙ / minute to achieve approximately 10 response units ( RU) coupled protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. To take kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) in PBS with 0.05% Tween 20 (PBST) were injected at 25 °C at a flow rate of approximately 25 μΙ / min. Association rates (kon) and dissociation rates (kOff) are calculated using a one-to-one Langmuir simple binding model (BIAcore evaluation software version 3.2) by simultaneous association and dissociation sensorogram fitting. The dissociation equilibrium constant (Kd) is calculated as the ratio kOff / kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 106 M1s_1according to the surface plasmon resonance assay mentioned above, it is possible to determine the association rate using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) at 25 °C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow spectrophotometer (Aviv Instruments) or an SLM-Aminco 8000 series spectrophotometer (ThermoSpectronic) with a stirring cuvette. Active or activity for the purposes herein refers to forms of a polypeptide (such as a multispecific polypeptide) that maintains a biological and / or immunological activity of the native or naturally occurring polypeptide, where the biological activity refers to a biological function. (whether inhibitory or stimulatory) caused by a native or naturally occurring polypeptide other than the ability to induce the production of an antibody against an antigenic epitope of a native or naturally occurring polypeptide and an immunological activity refers to the ability to induce the production of an antibody against an antigenic epitope of a native or naturally occurring polypeptide. Biologically active, biological activity and biological characteristics, with respect to a multispecific antigen-binding protein provided herein, such as an antibody, fragment or derivative thereof, means having the ability to bind to a biological molecule, except where specified otherwise. The term antibody is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments. , provided they exhibit the desired biological activity. The term immunoglobulin (Ig) is used interchangeably with antibody herein. Antibodies are naturally occurring immunoglobulin molecules that have various structures, all based on the immunoglobulin fold. For example, antibodies IgGs have two heavy chains and two light chains that bind to disifide to form a functional antibody. Each heavy and light chain comprises a constant region (C) and a variable region (V). The V regions determine the antigen-binding specificity of the antibody, while the C regions provide structural support and function in non-specific interactions of the antigen with immune effectors. The φ Antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a particular antigen. The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. The variability is not uniformly distributed over a 110 amino acid stretch of the variable domains. In contrast, V regions consist of relatively invariant expanses called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions,” each 9-12 amino acids in length. Each of the variable domains of natural heavy and light chains comprises four FR, which generally adopt a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, and in some cases form part, of the β-sheet structure. The hypervariable regions of each chain are held together in proximity to each other by the FRs and, with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibodies (see Kabat et al., Sequences of Proteins of Imiaunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC). Each V region typically comprises three complementarity-determining regions (CDRs, each containing a hypervariable loop) and four framework regions. An antibody binding site, the minimum structural unit necessary to bind with substantial affinity to a particular desired antigen, therefore typically includes the three CDRs and at least three, preferably four, framework regions interspersed between them to support and present the CDR in the proper conformation. Classical four-chain antibodies have antigen binding sites that are defined by the VH and VL domains. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites formed only by heavy chains. Immunoglobulins designed with a single domain, in which the binding sites are formed by heavy chains or light chains alone, can be prepared in the absence of cooperation between VH and VL. The term variable refers to the fact that certain parts of the variable domains differ widely in sequence between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed across the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The most widely conserved parts of variable domains are called flanking regions (FR). Each of the variable domains of natural heavy and light chains comprises four FRs, which generally adopt a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, and in some cases form part, of the structure of leaf β. The hypervariable regions of each chain are held together in proximity to each other by the FRs and, with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, M.D. (1991)). Constant domains are not directly involved in the binding of an antibody to an antigen, but have various effector functions, such as Antibody involvement in antibody-dependent cellular cytotoxicity (ADCC). When used herein, the term "hypervariable region" refers to amino acid residues of an antibody that are responsible for binding to antigen. The hypervariable region may comprise amino acid residues from a complementarity determining region or CDR (e.g., around approximately residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the VLy around approximately 31-35B (Hl), 50-65 (H2) and 95-102 (H3) in the VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or the residues of a hypervariable loop (e.g., residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in VLy 26-32 ( Hl), 52A-55 (H2) and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework region" or "FR" residues are those variable domain residues other than hypervariable region residues as defined herein. Hinge region in the context of an antibody or hemiantibody, is generally defined as an extension from Glu216 to Pro230 of human IgGl (Burton, Molec. φ Immunol.22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgGl sequence by placing the first and last cysteine ​​residues that form S-S bonds between heavy chains in the same positions. The lower hinge region of an Fe region is typically defined as the extension of residues immediately C terminal with respect to the hinge region, i.e., residues 233 to 239 of the Fe region. Prior to the present application, FcyR binding was generally attributed to amino acid residues in the lower hinge region of an Fe region of IgG. The "CH2 domain" of a human IgG Fe region generally extends from about residue 231 to about 340 of the IgG. The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains of an intact natural IgG molecule. It has been speculated that the carbohydrate may provide a substitute for interdomain pairing and help stabilize the CH2 domain. Burton, Molec. Immunol.22:161206 (1985). The CH3 domain comprises the extension of C-terminal residues of the CH2 domain into the Fe region (i.e., from approximately amino acid residue 341 to approximately amino acid residue 447 of an IgG). "Antibody fragments" comprise a part of an intact antibody, preferably comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., US Patent No. 5,641,870, Example 2; 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 the antibody fragments (e.g., including, but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv or (di ,tri)-scFv) in tandem; and bispecific T cell recruiters (BiTE). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fe" fragment, the name of which reflects the ability to crystallize easily. The Fab fragment consists of a complete L chain along with the variable region domain of the H chain (VH) and the first constant domain of a φ chain heavy (CHl). Pepsin treatment of an antibody produces a single large F(ab')2 fragment that approximately corresponds to two disulfide-linked Fab fragments that have divalent antigen-binding activity and can still cross-link the antigen. The fragments Fab' differ from Fab fragments because they have few additional residues at the carboxyl terminus of the domain. CHl that includes one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains carry a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. Fv is the minimal antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in strong, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific hypervariable regions) has the capacity to recognize and bind antigens, albeit at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the CH1 heavy chain domain that includes one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. Antibody light chains (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, kappa (k) and lambda (λ), based on the amino acid sequences of their constant domains. Based on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g. e.g., IgGl, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, Y and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. Single chain Fv or scFv antibody fragments comprise the VH and VL domains of the antibody, where these domains are present on a single polypeptide chain. In some embodiments, the Fv polypeptide additionally comprises a polypeptide bond between the domains VHy VLwhich allows the scFv to form the desired structure for antigen binding. To analyze the scFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term diabodies refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). ). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Nati. Academic Sci. USA, 90:6444-6448 (1993). The term half-antibody or hemimer, as used herein, refers to a monovalent antigen-binding polypeptide. In certain embodiments, a half-antibody or hemimer comprises a VH / VL unit and, optionally, at least a portion of an immunoglobulin constant domain. In certain embodiments, a half-antibody or hemimer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain or an antigen-binding fragment thereof. In certain embodiments, a half-antibody or hemimer is monospecific, that is, it binds to a single antigen or epitope. One skilled in the art will readily appreciate that a half-antibody may have an antigen-binding domain consisting of a single variable domain, e.g. e.g., which originates from a camelid. The term VH / VL unit refers to the antigen binding region of an antibody comprising at least one VH HVR and at least one VL HVR. In certain embodiments, the VH / VL unit comprises 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, the VH / VL unit further comprises at least a portion of a framework region (FR). In some embodiments, a VH / VL unit comprises three VH HVRs and three VL HVRs. In some of said embodiments, a VH / VL unit comprises at least one, at least two, at least three or all four FRs of VH and at least one, at least two, at least three or all four FRs of VL. The term multispecific antibody is used in the broadest sense and specifically encompasses an antibody that comprises an antigen-binding domain that has polyepitope specificity (i.e., is capable of specifically binding to two, or more, different epitopes on a biological molecule or is capable of specifically binding to epitopes on two, or more, different biological molecules). In some embodiments, an antigen binding domain of a multispecific antibody (such as a bispecific antibody or a divalent F(ab')2) comprises two units. VH / VL, wherein a first VH / VL unit specifically binds to a first epitope and a second unit VH / VL specifically binds to a second epitope, where each VH / VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). These multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments, such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and tribodies, antibody fragments. covalently or non-covalently linked. A unit VH / VL further comprising at least a portion of a heavy chain constant region and / or at least a portion of a 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 of said embodiments, a bispecific antibody that comprises two half-antibodies and binds two antigens comprises a first half-antibody that binds to the first antigen or first epitope but not to the second antigen or second epitope and a second half-antibody that binds to the second antigen or second epitope and 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 an affinity of between 5 M and 0.001 pM, between 3 M and 0.001 pM, between 1 M and 0.001 pM, between 0, 5 M and 0.001 pM or between 0.1 M and 0.001 pM. In some embodiments, a hemimer comprises a sufficient portion of a heavy chain variable region to allow intramolecular disulfide bonds to form with a second hemimer. In some embodiments, a hemimer comprises a button mutation or a buttonhole mutation, for example, to allow heterodimerization with a second hemimer or half-antibody comprising a complementary buttonhole mutation or button mutation. Button mutations and buttonhole mutations are further described below. A bispecific antibody is a multispecific antibody that comprises an antigen-binding domain that is capable of specifically binding to two different epitopes on one biological molecule or is capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody may also be referred to herein as having dual specificity or as being dual specific. Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody are listed in a bispecific antibody name is arbitrary. In some embodiments, a bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and, optionally, at least a portion of a heavy chain constant region, and a single light chain variable region and , optionally, at least a portion of a light chain constant region. In certain embodiments, a bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and comprises no more than one single heavy chain variable region and comprises no more than a single light chain variable region. In some embodiments, a bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to a first antigen and not to a second antigen and the second half-antibody binds to the second antigen and not to the first antigen. As used herein, the term buttonhole or KiH refers to the technology aimed at pairing two polypeptides with each other in vitro or in vivo by introducing a protuberance (button) in one polypeptide and a cavity (eyelet) in the other polypeptide. in an interface in which they interact. For example, KiHs have been introduced into the Fe:Fe junction interfaces, CL:CH1 interfaces, or VH / VL interfaces of antibodies (see, e.g., US 2011 / 0287009, US2007 / 0178552, WO 96 / 027011, WO 98 / 050431 and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, KiHs trigger the pairing of two different heavy chains during the manufacture of multispecific antibodies. For example, multispecific antibodies with KiH in their Fe regions may further comprise single variable domains linked to each Fe region or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KiH technology can also be used to pair two different receptor extracellular domains to any other polypeptide sequence comprising different target recognition sequences (e.g., including affibodies, peptibodies, and other Fe fusions). The term "knob mutation," as used herein, refers to a mutation that introduces a protuberance (knob) in a polypeptide at an interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a buttonhole mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, which are incorporated herein by this reference in their entirety). The term "eyelet mutation" as used herein refers to a mutation that introduces a pocket (eyelet) in a polypeptide at an interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a button mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, which are incorporated herein by this reference in their entirety). The term single domain antibodies (sdAb) or single variable domain antibodies (SVD) generally refer to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse shark) and those derived from recombinant human and mouse antibody methods (Nature (1989) 341:544 -546;Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; FEBS Lett (1994) 339:285-290; WO00 / 29004; WO 02 / 051870). The term monoclonal antibody, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for Possible variants that may arise during the production of the monoclonal antibody, such variants are generally present in minor quantities. Unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are not contaminated by other immunoglobulins. The monoclonal modifier indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods. (see, e.g., US patent φ η.° 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. Monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) wherein a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while that the rest of the chain or chains are identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, as long as they exhibit the desired biological activity ( U.S. Patent No. 4,816,567; Morrison et al., Proc. Nati. Academic Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g. old world monkey, such as baboon, rhesus or cynomolgus monkey) and region sequences. human constant (US patent No. 5,693,780). “Humanized” forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced with residues from a hypervariable region of a non-human species (donor antibody), such as mouse , rat, rabbit or non-human primate that has the desired specificity, affinity and capacity. In some cases, human immunoglobulin framework region (FR) residues are replaced with corresponding non-human residues. Likewise, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or almost all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or almost all of the FRs are those of a human immunoglobulin sequence, except for FR substitutions as indicated above. The humanized antibody will also optionally comprise at least a portion of a region immunoglobulin constant, usually a human immunoglobulin. For details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). For the purposes herein, an intact antibody is one that comprises heavy and light chain variable domains as well as an Fe region. The constant domains may be constant domains of a native sequence (for example, constant domains of a human native sequence ) or an amino acid sequence variant of these. Preferably, the intact antibody has one or more effector functions. Natural antibodies are generally heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide bridges spaced at regular intervals. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the light chain constant domain aligned with the first heavy chain constant domain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. A naked antibody is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel. As used herein, the term immunoadhesin indicates molecules that combine the binding specificity of a heterologous protein (an adhesin) with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with a desired binding specificity, wherein said amino acid sequence is distinct from the antigen recognition and binding site of an antibody (i.e., is heterologous compared to the antigen binding site). with a constant region of an antibody) and an immunoglobulin constant domain sequence (e.g., CH2 and / or CH3 sequence of an IgG). Examples of adhesin sequences include contiguous amino acid sequences that comprise a portion of a receptor or a ligand that binds to a protein of interest. Adhesin sequences can also be sequences that bind a protein of interest, but are not receptor or ligand sequences (e.g., adhesin sequences in peptibodies). Such polypeptide sequences can be selected or identified with various methods including phage display techniques and high-throughput sorting methods. The immunoglobulin constant domain sequence in immunoadhesin can be obtained from any immunoglobulin, such as subtypes IgG-1, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. In certain embodiments, the Fe region-containing heterodimeric polypeptide is an antibody, a bispecific antibody, or Fe fusion proteins. In certain embodiments, the Fe fusion protein produced according to a method provided herein is a targeted immunocytokine. In certain embodiments, the targeted immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the immunocytokine CEA-IL2v is RG7813. In certain embodiments, the targeted immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the immunocytokine FAP-IL2v is RG7461. In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced according to a method provided herein binds to CEA and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced according to a method provided herein binds cytokine and at least one additional target molecule. In certain embodiments, a multispecific antibody produced according to a method provided herein is fused to IL2v (i.e., an interleukin variant 2) and to at least one additional target molecule. In certain embodiments, a multispecific antibody produced according to a method provided herein is a T cell bispecific antibody (i.e., a bispecific T cell linker or BiTE). In some embodiments, antibody effector functions refer to biological activities that can be attributed to the Fe region (a natural sequence Fe region or an amino acid sequence variant Fe region) of an antibody and vary depending on the antibody isotype. . Examples of effector functions of antibodies include: Clq binding and complement-dependent cytotoxicity, Fe receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of surface receptors cell phone. 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 (Clq) to a molecule (e.g., polypeptide (e.g., an antibody)) in complex with a cognate antigen. To assess complement activation, a CDC assay can be performed, e.g. e.g. , as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody-dependent cell-mediated cytotoxicity and ADCC refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fe receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies. binding on a target cell and subsequently causing lysis of the target cell. The primary cells to mediate ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. The expression of FcR in 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 a molecule of interest, an in vitro ADCC assay can be performed, such as that described in US Patent No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, e.g. e.g. , in an animal model, such as that described in Clynes et al., Proc. Nati. Academic Sci. (USA) 95:652-656 (1998). Human effector cells are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcyRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. PBMC cells are preferred and NK. The term Fe receptor or FcR is used to describe a receptor that binds to the Fe region of an antibody. In some embodiments, the FcR is a naturally occurring human FcR. Furthermore, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII and Fcy RUI subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. The receivers FcyRII include FcyRIIA (an activating receptor) and FcyRIIB (an inhibitory receptor), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Daéron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:2534 (1994); and de Haas et al. , J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those that will be identified in the future, are encompassed by the term FcR herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The terms host cell, host cell line and host cell culture are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of said cells. Host cells include transforming cells and transformed cells that include the transformed primary cell and progeny derived from it regardless of the number of passages. The nucleic acid content of the progeny may not be completely identical to that of a parental cell, but may contain mutations. Included herein is mutant progeny with the same biological function or activity as tested or selected in the originally transformed cell. Impurities refer to materials that are different from the desired polypeptide product. The impurity may refer to product-specific polypeptides, such as single-arm antibodies and misassembled antibodies, antibody variants, including basic variants and acidic variants, and aggregates. Other impurities include process-specific impurities, including, but not limited to: host cell materials, such as host cell protein (HCP); leached protein A; nucleic acid; another polypeptide; endotoxin; viral contaminant; component of cell culture media, etc. In some examples, the impurity may be an HCP from, for example, but not limited to, a bacterial cell, such as an E cell. coli (ECP), an insect cell, a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an avian cell, a fungal cell. In some examples, the impurity may be an HCP from a mammalian cell, such as a CHO cell, i.e., a CHO cell protein (CHOP). The impurity may refer to accessory proteins used to facilitate the expression, binding or assembly of multispecific antibodies; for example, prokaryotic chaperones, such as FkpA, DsbA and DsbC. Complex or in complex, as used herein, refers to the association of two or more molecules that interact with each other through bonds and / or forces (e.g., van der Waals forces, hydrophilic, hydrophilic) that are not peptide bonds. In one embodiment, the complex is heteromultimeric. The term protein complex or polypeptide complex, as used herein, will be understood to include complexes that have a non-protein entity conjugated to a protein in the protein complex (e.g., including, but not limited to, molecules chemicals, such as a toxin or a detection agent). An isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that commonly contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location different from its natural chromosomal location. Percent (%) amino acid sequence identity, 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 aligning the sequences and introducing spaces, if necessary, to achieve the maximum percentage of sequence identity and without considering any conservative substitution as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in various ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. In certain embodiments, % amino acid sequence identity values ​​are generated by the ALIGN-2 sequence comparison software. The ALIGN-2 sequence comparison software program was created by Genentech, Inc. and the source code was submitted with user documentation to the Office of United States Copyright, Washington D.C., 20559, where it is registered under US Copyright Registration No. 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 on an operating system UNIX, including digital UNIX V4.0D. The ALIGN2 program establishes all the sequence comparison parameters and these do not change. In situations where ALIGN-2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a specific amino acid sequence B (which may be expressed as alternatively as a specific amino acid sequence A having or comprising a given % amino acid sequence identity to, with or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where amino acid in B. It will be noted that when 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 with respect to B will not be equal to the % identity of amino acid sequence of B with respect to A. Unless specifically indicated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the computer program ALIGN2. The terms variable region or variable domain refer to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a natural antibody generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). for its acronym in English). (See, p. e j. , Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., page 91 (2007).) A single VH domain or VL may be sufficient to confer antigen binding specificity. Additionally, antibodies that bind to a specific antigen can be isolated using a VH domain or VL of an antigen-binding antibody to screen a library of complementary VL or VH domains, respectively. See, p. e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). The term vector, as used herein, refers to a nucleic acid molecule that can propagate another nucleic acid to which it is bound. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it is introduced. Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as expression vectors. The term sequential, as used herein with respect to chromatography, refers to chromatography steps in a specific sequence; p. e.g. , a first chromatography stage followed by a second chromatography stage followed by a third chromatography stage, etc. Additional steps may be included among the sequential chromatography steps. The term continuous, as used herein with respect to chromatography, refers to having a first chromatography material and a second chromatography material directly connected or some other mechanism that allows continuous flow between the two chromatography materials. Charge density refers to the quantity, e.g. e.g., grams, of composition that is brought into contact with a volume of chromatography material, e.g. e.g., liters. In some examples, the charge density is expressed in g / L. A sample refers to a small portion of a larger amount of material. Generally, testing according to the methods described herein is performed on a sample. The sample is usually obtained from a recombinant polypeptide preparation obtained, for example, from cultured recombinant polypeptide-expressing cell lines, also referred to herein as product cell lines, or from cultured host cells. As used herein, host cells do not contain genes for the expression of recombinant polypeptides of interest or products. A sample may be obtained from, for example, but not limited to, collected cell culture fluid, from a processing pool at a given stage in a purification process, or from the final purified product. The sample may also include diluents, buffers, detergents and contaminating species, debris and the like that are mixed with the desired molecule (such as a multispecific antibody, e.g., a bispecific antibody). Reference to about a value or parameter herein includes (and describes) variations directed to that value or parameter itself. For example, the description that refers to about X includes the description of X. The aspects and embodiments of the invention described herein are understood to include, comprise, and consist essentially of aspects and embodiments. As used herein and in the appended claims, the singular forms a, an, or, the, and the include plural referents, unless the context clearly indicates otherwise. Aspects and variations of the invention described herein are understood to include consisting of and / or consisting essentially of aspects and variations. All references mentioned herein, including publications and patent applications, are incorporated herein in their entirety by this reference. Methods for purification of a multispecific antibody Methods for purifying a multispecific antibody are provided herein. In certain embodiments, the multispecific antibody is a bispecific antibody. In certain embodiments, the multispecific antibody is a divalent 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 dual antibody. specific, that is, an antibody that - has two antigen-binding arms that are identical in amino acid sequence and where each Fab arm is capable of recognizing two antigens (such as a Fab antibody of φ dual action). In some aspects, purification of the multispecific antibody comprises the sequential steps of capture chromatography, a first mixed mode chromatography, and a second mixed mode chromatography. In some In embodiments, the multispecific antibody is assembled prior to capture chromatography. In some embodiments, the multispecific antibody is assembled after capture chromatography. In some embodiments, the multispecific antibody (such as a bispecific antibody or a divalent F(ab')2) comprises two or more antibody arms, where different arms of the antibody bind to different epitopes. In certain embodiments, the different epitopes are found on the same antigen. In certain embodiments, each epitope is found on a different antigen. In certain embodiments, the antibody arms comprise VH / VL units. In certain embodiments, the antibody arms comprise hemimers, also known as half-antibodies. To facilitate assembly, in certain embodiments, the heavy chain of one arm of the antibody is modified to comprise a button and the heavy chain of the other arm of the antibody comprises an eyelet, such that the button of the first heavy chain fits into the eyelet of the second heavy chain. In certain embodiments, each arm of the multispecific antibody is produced in a separate cell culture. After expression of the antibody arm in the host cell, the entire cell broth is collected and homogenized, and the antibody arm is extracted. In certain embodiments, polyethyleneimine (PEI) is added to the cellular waste prior to chromatography. In some embodiments, the cell lysate is centrifuged prior to chromatography. Each arm of the multispecific antibody is purified by capture chromatography (such that each arm is purified on a separate membrane or chromatography column). In certain embodiments, capture chromatography is affinity chromatography. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the affinity chromatography is protein chromatography. G. In certain embodiments, 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; for example, by SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody are then combined and allowed to assemble, as described in detail elsewhere herein. In other embodiments, each arm of the multispecific antibody is produced in a separate cell culture. After expression of the antibody arm in the host cell, the entire cell broth is collected and homogenized. 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 prior to chromatography. In some embodiments, the cell lysate is centrifuged prior to chromatography. The combined arms of the multispecific antibody are then purified by affinity chromatography. In some embodiments, affinity chromatography is protein A chromatography. At that point, the purified antibody arms can be analyzed; for example, by SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody are then combined and allowed to assemble via the methods described herein. In other embodiments, each arm of the multispecific antibody is produced in the same cell culture. After expression of the antibody arm in the host cell, the entire cell broth is collected and homogenized and the antibody arms are extracted. In some embodiments, polyethyleneimine (PEI) is added to the Used cell before chromatography. In some embodiments, the cellular waste is centrifuged prior to chromatography. The multispecific antibody arms are then purified by affinity chromatography. In some embodiments, the affinity chromatography is protein A chromatography. At that point, the purified antibody arms can be analyzed; for example, by SDS-PAGE, SEC chromatography, mass spectrometry, etc. The purified arms of the multispecific antibody are then allowed to assemble via the methods described herein. In some embodiments, the final concentration of PEI in the cell lysate is at least about any of the 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0. In some embodiments, the final concentration of PEI in the cell lysate is approximately any of 0.1% to 5%, 0.1% to 1%, 0.1% to 0. .5%, between 0.5% and 5%, between 0.5% and 1% or between 1% and 5%. In some embodiments, the cell lysate that or comprises PEI is maintained for more than approximately any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 12 hr, 14 hr, 16 hr, 18 hr , 8 p.m. or 24 p.m. In some embodiments, cellular use comprising PEI is maintained for approximately any of between approximately 1 hour and 24 hours, between 1 hour and 6 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 18 hours and 24 hours. In some embodiments, the cellular waste comprising PEI is maintained for approximately any of between 10 h and h. In some embodiments, the cellular waste comprising PEI is maintained between about 4 °C and °C. In some embodiments, the cellular waste comprising PEI is maintained at approximately room temperature. In some embodiments, the cellular waste is clarified by centrifugation prior to chromatography. In some embodiments, the cell lysate is filtered prior to chromatography. In some embodiments, the cell lysate is filtered through a 0.22 μm filter prior to chromatography. Examples of affinity chromatography include, but are not limited to, e.g. e.g. , protein A chromatography, protein G chromatography, protein A / G chromatography or protein L chromatography. Examples of o material affinity chromatography include, but are not limited to, ProSep®vA, ProSep® Ultra Plus, Fast Flow Protein A Sepharose®, Toyopearl® AF-rProtein 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 binding and elution mode (alternatively referred to as the binding and elution process). Binding and elution mode refers to a technique of product separation in which a product (such as multispecific antibody) in the sample binds to the affinity chromatography material and is eluted subsequently of the affinity chromatography material. In some embodiments, the elution is a step elution, in which the composition of the mobile phase is changed gradually, on one or more occasions, during the elution process. In certain embodiments, the elution is gradient elution, in which the composition of the mobile phase is continuously changed 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 MAbSeiect chromatography. SuRe. In certain embodiments, chromatography of or affinity is CaptureSelect chromatography. In certain embodiments, affinity chromatography is chromatography CaptureSelect FcXL. In certain embodiments, the eluate from the affinity chromatography step is subsequently applied to a first mixed mode chromatography. In certain embodiments, the first mixed mode material comprises functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, pi-pi bonding interactions, hydrophilic interactions, thiophilic interactions, and hydrophobic interactions. In certain embodiments, the first mixed mode material comprises functional groups capable of anion exchange and hydrophobic interactions. In certain embodiments, the first mixed mode material comprises functional groups capable of cation exchange and hydrophobic interactions. In certain embodiments, the first mixed mode material contains N-benzyl-N-methyl ethanol amine, 4-mercapto-ethylpyridine, 2-benzamido-4-mercaptobutanoic acid, hexylamine or phenylpropylamine or cross-linked 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, φ membrane Nuvia™cPrime™. In some embodiments, the first mixed mode material is Capto™ Adhere resin. In certain embodiments, the first mixed mode material is resin Capto™ Adhere. In certain embodiments, the first mixed mode material is Capto™ MMC. In certain embodiments, the first mixed mode chromatography does not include ceramic hydroxyapatite chromatography. In certain embodiments, the first mixed mode chromatography is performed in binding and elution mode. In some embodiments, the elution is a stepwise elution. In certain embodiments, the elution is gradient elution. In certain embodiments, the first mixed mode chromatography is performed in continuous flow mode. In certain embodiments of the above, the first mixed mode material is in a column. In certain embodiments of the above, the first mixed mode material is in a membrane. In certain embodiments, the capture chromatography and the first mixed mode chromatography are continuous, e.g. e.g., wherein the capture chromatography material and the first mixed mode material are directly connected or connected through some other mechanism that allows continuous flow between the capture chromatography material and the first mixed mode material. In certain embodiments, capture chromatography and the first or Mixed mode chromatography are contiguous, where the first mixed mode chromatography is performed directly after capture chromatography. In certain embodiments, the capture chromatography eluate is subjected to one or more additional chromatography steps before being applied to the first mixed mode resin. For example, the capture chromatography eluate may be subjected to one or more of the following chromatography steps in any order and / or in any combination before being subjected to a first mixed mode chromatography: hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite chromatography (CHT), hydrophilic interaction liquid chromatography (HILIC), etc. Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to hydrophobicity. Examples of HIC chromatography materials include, but are not limited to, e.g. e.g. , Toyopearl® Hexyl-650, Toyopearl® Butyl-650, Toyopearl® Phenyl-650, Toyopearl® Ether-650, HiTrap® Sepharose, Octyl Sepharose®, Phenyl Sepharose® or Butyl Sepharose®. In some embodiments, the HIC chromatography material comprises φ phenyl sepharose. In certain embodiments, HIC chromatography is performed in binding and elution mode. In some embodiments, HIC chromatography is performed in continuous flow mode. In some embodiments of the above, the HIC chromatography material is in a column. In some embodiments of the above, the HIC chromatography material is contained in a membrane. The anion exchange chromatography material is a solid phase that is positively charged and has free anions for exchange with anions in an aqueous solution (such as a composition comprising a multispecific antibody and an impurity) that is passed over or through the phase. solid. In some embodiments of any of the methods described herein, the anion exchange material may be a membrane, a monolith or resin. In one embodiment, the anion exchange material may be a resin. In some embodiments, the anion exchange material may comprise a primary amine, a secondary amine, a tertiary amine, or a quaternary ammonium ionic functional group, a polyamine functional group, or a diethylaminoethyl functional group. Examples of anion exchange materials are known in the art and include, but are not limited to, Poros*8HQ 50, Poros” Pl 50, Poros” D, Mustang® Q, Q Sepharose” fast flow (QSFF), resin Accell™ Plus Quaternary Methyl Amine (QMA), Sartobind STIC and DEAE-Sepharose®. In some embodiments, chromatography Anion exchange is carried out in binding and elution mode. In some embodiments, anion exchange chromatography is performed in continuous flow mode. In some embodiments of the above, the anion exchange chromatography material is in a column. In some embodiments of the above, the exchange chromatography material anionic is a column. The cation exchange chromatography material is a negatively charged solid phase having free anions for exchange with cations in an aqueous solution (such as a composition comprising a multispecific antibody and an impurity) that is passed over or through the phase. solid. In some embodiments of any of the methods described herein, the cation exchange material may be a membrane, a monolith or resin. In some embodiments, the cation exchange material may be a resin. The cation exchange material may comprise a carboxylic acid functional group or a sulfonic acid functional group, such as, among others, sulfonate, carboxylic acid, carboxymethyl sulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, or orthophosphate. In some modalities of Yo Or sulfoxyethyl above, cation exchange chromatography material is a cation exchange chromatography column. In some embodiments of the above, the cation exchange chromatography material is a cation exchange chromatography membrane. Examples of cation exchange materials are known in the art and include, among others, Mustang® S, Sartobind® S, S03 Monolith (such as, e.g., CIM®, CIMmultus® and CIMac® S03), S Ceramic HyperD®, Poros* EMD Se Hicap, Fractogel® EMD SO3_o Fractogel® EMD COO. In some embodiments, cation exchange chromatography is performed in binding and elution mode. In some embodiments, cation exchange chromatography is performed in continuous flow mode. In some embodiments of the above, the cation exchange chromatography material is in a column. In some embodiments of the above, the cation exchange chromatography material is located on a membrane. The functional groups of hydroxyapatite chromatography material (Ca10(PO4)$ (OH)2) comprise positively charged pairs of crystalline calcium ions (C sites) and groups of six positively charged oxygen atoms associated with crystalline phosphate triplets (C sites). P). The C sites, P sites, and hydroxyls are distributed in a fixed pattern on the crystal surface. Proteins are normally adsorbed on hydroxyapatite in a low concentration (e.g., 10-25 mM) of phosphate buffer, although certain acidic proteins can be adsorbed if loaded in water, saline, or a non-phosphate buffer. Proteins are usually eluted by an increasing phosphate gradient, although Ca2+, Mg2+, or Cl” ion gradients can also be used, such as for the selective elution of basic proteins. In some embodiments of any 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 of the above, the hydroxyapatite chromatography material is a column. Examples of hydroxyapatite chromatography materials are known in the art and include, among others, CHT™ ceramic hydroxyapatite, CHT type I ceramic hydroxyapatite support, CHT type II ceramic hydroxyapatite support. In some In embodiments, hydroxyapatite chromatography is performed in binding and elution mode. In some embodiments, hydroxyapatite chromatography is performed in © mode. continuous flow. In one embodiment, the method as described herein may further comprise a method for separating a bispecific antibody comprising an Fe domain from a solution comprising said bispecific antibody, wherein said method comprises (a) contacting said solution with a hydroxyapatite chromatography medium, (b) adsorbing said bispecific antibodies in said hydroxyapatite chromatography medium and (c) eluting said bispecific antibody from said hydroxyapatite chromatography medium in the presence of chloride ions, wherein said solution further comprises one or more fragments of said bispecific antibody, which one or more fragments comprise an Fe domain; and / or wherein said solution further comprises one or more polypeptides having a molecular weight greater than the molecular weight of said bispecific antibody and comprises at least one of the two heavy chains of said bispecific antibody, which one or more polypeptides further comprise a domain Faith mentioned in WO2015024896. In certain embodiments, the capture chromatography eluate is subjected to anion exchange chromatography. In certain embodiments, the anion exchange chromatography material is Q Sepharose." In certain embodiments, the Or fast flow (QSFF). In anion exchange chromatography it is performed in binding and elution mode. In certain embodiments, an eluate collected after the first mixed mode chromatography is subsequently applied to a second mixed mode chromatography. In certain embodiments, the second mixed mode material comprises functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, pi-pi bonding interactions, hydrophilic interactions, thiophilic interactions, and hydrophobic interactions. In certain embodiments, the second mixed mode material comprises functional groups capable of anion exchange and hydrophobic interactions. In certain embodiments, the second mixed mode material comprises functional groups capable of cation exchange and hydrophobic interactions. In certain embodiments, the second mixed mode material contains N-benzyl-N-methyl ethanol amine, 4-mercapto-ethyl-pyridine, 2-benzamido-4-mercaptobutanoic acid, hexylamine or phenylpropylamine or cross-linked 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, ο membrane Nuvia™cPrime™. In some embodiments, the second mixed mode material is Capto™ Adhere resin. In certain embodiments, the second mixed mode material is resin Capto™ Adhere. In certain embodiments, the second mixed mode material is Capto™ MMC. In certain embodiments, the second mixed mode chromatography does not include ceramic hydroxyapatite chromatography. In certain embodiments, the second mixed mode chromatography is performed in binding and elution mode. In some embodiments, the elution is a stepwise elution. In certain embodiments, the elution is gradient elution. In certain embodiments, the first mixed mode chromatography is performed in continuous flow mode. In certain embodiments of the above, the second mixed mode material is located in a column. In certain embodiments of the above, the second mixed mode material is a column. In certain embodiments, the first mixed mode chromatography and the second mixed mode chromatography are continuous, e.g. e.g., wherein the capture chromatography material and the first mixed mode material are directly connected or connected through some other mechanism that allows 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 contiguous, wherein the second mixed mode chromatography is performed directly after the first mixed mode chromatography. In certain embodiments, the eluate from the first mixed mode chromatography is subjected to one or more additional chromatography operations before being applied to the second mixed mode resin. For example, the eluate from the first mixed mode chromatography may be subjected to one or more of the following chromatography steps in any order and / or in any combination before being subjected to a second mixed mode chromatography: hydrophobic interaction chromatography (HIC ), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite chromatography (CHT), hydrophilic interaction liquid chromatography (HILIC), etc. In certain embodiments of any of the methods described herein, the eluate from the second mixed mode chromatography is subjected to one or more additional chromatography steps. For example, the eluate from the second mixed mode chromatography can be subjected to one or more of the following chromatography steps in any or order and / or in any combination: hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite chromatography (CHT), hydrophilic interaction liquid chromatography ( HILIC), mixed mode chromatography, etc. In certain embodiments of any of the methods described herein, the methods comprise using a damper. Various buffers can be employed during the purification of the multispecific antibody depending on, for example, the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the multispecific antibody being purified, and the purification method. The damper can be a load damper, a balance damper or a wash damper. In certain embodiments, one or more of the load damper, the balance damper and / or the wash damper are the same. In certain embodiments, the load damper, the balancing damper and / or the wash damper are different. In certain embodiments of any of the methods described herein, the buffer comprises a salt. In certain embodiments, the buffer comprises chloride 100 or sodium, sodium acetate, Tris HCl, Tris acetate, sodium phosphate, potassium phosphate, MES, CHES, MOPS, BisTris, arginine, arginine HCI or a mixture of these. 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. Loading refers to the composition that is loaded into the chromatography material. The loading buffer is the buffer used to load the composition (e.g., a composition comprising a multispecific antibody and an impurity or a composition comprising an antibody arm and an impurity) into a chromatography material (such as any of the chromatography materials described herein). The chromatography material may be equilibrated with an equilibration buffer prior to loading of the composition to be purified. The wash buffer is used after loading the composition onto a chromatography material. An elution buffer is used to elute the polypeptide of interest from the solid phase. Loading a composition comprising the multispecific antibody (such as a composition comprising the 101 or multispecific antibody and an impurity) in any of the chromatography materials described herein can be optimized to separate the multispecific antibody from the impurity. In some embodiments, loading of the composition comprising the multispecific antibody (such as a composition comprising the multispecific antibody and an impurity) into the chromatography material is optimized to bind the multispecific antibody to the chromatography material when chromatography is performed. in the binding and elution mode (p. e.g., affinity chromatography, mixed mode chromatography, and ion exchange chromatography, as designated herein). Conductivity refers to the ability of an aqueous solution to carry an electrical current between two electrodes. In solution, current flows by transport of ions. Therefore, with an increase in the amount of ions present in the aqueous solution, the solution has a higher conductivity. The basic unit of measurement for conductivity is the Siemens (mS / cm) or ohms (mho) and can be measured using a conductivity meter, such as various models of conductivity meters. Orion. Because electrolytic conductivity is the ability of ions in a solution to carry electric current, the conductivity of a solution is 102 or can be altered by changing the concentration of ions in it. For example, the concentration of a buffering agent and / or the concentration of a salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be altered to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is varied to achieve the desired conductivity. For example, in certain embodiments, the composition comprising the multispecific antibody (such as a composition comprising the multispecific antibody and an impurity) is loaded into the chromatography material, e.g. e.g. , a chromatography column comprising any of the chromatography materials described herein, in a loading buffer at a series of different pH values ​​while the conductivity of the loading buffer is constant. Alternatively, the solution comprising the multispecific antibody can be loaded onto the chromatography material in a loading buffer at a range of different conductivities while the pH of the loading buffer is constant. Upon completion of loading the composition comprising the multispecific antibody (such as a composition comprising the multispecific antibody and an impurity) into the 103 or chromatography and the elution of the multispecific antibody from the chromatography material in a pool fraction, the amount of impurity remaining in the pool fraction provides information on the separation of the multispecific antibody from the impurity for a given pH or conductivity. Likewise, for chromatography where the multispecific antibody flows through the chromatography material, the loading buffer is optimized for pH and conductivity such that the multispecific antibody flows through the chromatography whereby the chromatography material retains the impurity. or flows through the chromatography material at a speed different from that of the multispecific antibody. In some embodiments, the charge density of the solution comprising the multispecific antibody or antibody arms is greater than about any of 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L or 150 g / L of the affinity chromatography material (e.g. e.g. , protein A chromatography material). In some embodiments, the charge density of the solution comprising the multispecific antibody or antibody arms is between approximately any of 10 g / L and 20 g / L, 20 g / L and 30 g / L, 30 g / L and 40 g / L, 40 g / L and 50 g / L, 50 g / L and 60 g / L, 60 g / L and 70 g / L, 70 g / L and 80 g / L, 80 g / L and 104 or g / L, 90 g / L and 100 g / L of the capture chromatography material (such as an affinity chromatography material, p. e.g., a protein A chromatography material, a protein G chromatography material, a protein A / G chromatography material or a protein L chromatography material). In some embodiments of any of the methods described herein, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material (e.g., Q Sepharose” fast flow (QSFF)). In some embodiments of any of the methods described herein, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material at a multispecific antibody charge density greater than about any of 30 g / L. , 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L , 140 g / L or 150 g / L of the anionic chromatography material (e.g., Q Sepharose” fast flow (QSFF)). In some embodiments, the eluate obtained after capture chromatography is loaded onto an anion exchange chromatography material at a loading density of the multispecific antibody between about any of 10 g / L and 20 g / L, 20 g / L and 30 g / L, 30 g / L and 40 g / L, 40 g / L and 50 g / L, 50 g / L and 60 g / L, 105 or g / L and 70 g / L, 70 g / L and 80 g / L, 80 g / L and 90 g / L, 90 g / L and 100 g / L of the anion exchange chromatography material (e.g. , Q Sepharose® Fast Flow (QSFF)). In some embodiments of any of the methods described herein, the eluate obtained after capture chromatography (optionally after capture chromatography and one or more additional chromatography steps comprising any of the chromatography operations described herein ) is loaded into a first mixed-mode chromatography material at a charge density of the multispecific antibody greater than approximately any of 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L , 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L or 150 g / L of the first mixed mode chromatography material (p (e.g., Capto™ Adhere chromatography material or a Capto™ MMC chromatography material). In some embodiments, the eluate obtained after capture chromatography is loaded into a first mixed-mode chromatography material at a loading density of the multispecific antibody between about any of 10 g / L and 20 g / L, 20 g / L and 30 g / L, 30 g / L and 40 g / L, 40 g / L and 50 g / L, 50 g / L and 60 g / L, 60 g / L and 70 g / L, 70 g / L and 80 g / L, 80 g / L and 90 g / L, 90 g / L and 100 g / L of the first mixed mode chromatography material (e.g., Capto™ Adhere chromatography material or a 106 methods or Capto™ MMC chromatography material). In some embodiments of any of the present invention, the eluate obtained after the first mixed-mode chromatography is loaded onto a second mixed-mode chromatography material at a multispecific antibody loading density greater than about 30 g / L. , 40 g / L, 50 g / L, g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L or 150 g / L of the second mixed-mode chromatography material (e.g., Capto™ Adhere chromatography material or a Capto™ MMC chromatography material). In some embodiments, the eluate obtained after mixed mode chromatography is loaded into the second mixed mode chromatography material at a loading density of the multispecific antibody between about any of 10 g / L and 20 g / L, 20 g / L. L and 30 g / L, 30 g / L and 40 g / L, 40 g / L and 50 g / L, 50 g / L and 60 g / L, 60 g / L and 70 g / L, 70 g / L L and 80 g / L, 80 g / L and 90 g / L, 90 g / L and 100 g / L of the mixed mode chromatography material (e.g. (e.g., Capto™ Adhere chromatography material or a Capto™ MMC chromatography material). In some embodiments of any of the methods described herein, the eluate obtained after the second mixed mode chromatography is loaded into a material 107 of subsequent chromatography (such as a hydrophobic interaction chromatography material (HIC), anion exchange chromatography material, cation exchange chromatography material, size exclusion chromatography material, affinity chromatography material or a chromatography material additional mixed mode) at a charge density of the multispecific antibody greater than about any of 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L p 150 g / L of the subsequent chromatography material. In some embodiments, the eluate obtained after the second mixed mode chromatography is loaded into the subsequent chromatography material (such as a hydrophobic interaction chromatography (HIC) material, anion exchange chromatography material, cation exchange chromatography material , size exclusion chromatography material, affinity chromatography material or an additional mixed mode chromatography material) at a loading density of the multispecific antibody between approximately any of 10 g / L and 20 g / L, 20 g / L L and 30 g / L, 30 g / L and 40 g / L, 40 g / L and 50 g / L, 50 g / L and 60 g / L, 60 g / L and 70 g / L, 70 g / L L and 80 g / L, 80 g / L and 90 g / L, 90 g / L and 100 g / L of the subsequent chromatography material. 108 present, is the φ Elution, as used in product disposal, e.g. e.g., the multispecific antibody or antibody arm, of the chromatography material. Elution buffer is the buffer used to elute the multispecific antibody or other product of interest from a chromatography material. In many cases, an elution buffer has a different physical characteristic than the loading buffer. For example, the elution buffer may have a different conductivity than the loading buffer or a different pH than the loading buffer. In some embodiments, the elution buffer has a lower conductivity than the loading buffer. In some embodiments, the elution buffer has a higher conductivity than the loading buffer. In some embodiments, the elution buffer has a lower pH than the loading buffer. In some embodiments, the elution buffer has a higher pH than the loading buffer. In some embodiments, the elution buffer has a different conductivity and a different pH than the loading buffer. The elution buffer may have any combination of higher or lower conductivity and higher or lower pH. In certain embodiments, the elution of the antibody 109ο Multispecific chromatography material is optimized to provide product with minimal impurity and at minimum elution volume or pool volume. For example, the composition containing the multispecific antibody (e.g., bispecific antibody) or the antibody arms can be loaded into the chromatography material, e.g. e.g., a chromatography column, in a loading buffer. Upon completion of loading, the multispecific antibody or antibody arm is eluted with buffers at a series of different pH values ​​while the conductivity of the elution buffer is constant. Alternatively, the multispecific antibody or antibody arm can be eluted from the chromatography material in an elution buffer at a range of different conductivities while the pH of the elution buffer is constant. Upon completion of elution of the multispecific antibody (e.g., bispecific antibody) or antibody arm from the chromatography material, the amount of an impurity in the pool fraction provides information about the separation of the multispecific antibody or antibody arm from the chromatography material. impurities for a given pH or conductivity. Elution of the multispecific antibody or antibody arm in a large number of column volumes (e.g., eight column volumes) indicates prolongation of the elution profile. In some 110 or modalities, elution prolongation is minimized. Various buffers can be used depending on, for example, the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the protein of interest, the chromatography material, and the purification process (e.g., binding and elution mode). or continuous flow). In some embodiments of any of the methods described herein, the methods comprise the use of at least one damper. The buffer may be a load buffer, an equilibration buffer, an elution buffer, or a wash buffer. In some embodiments, one or more of the loading buffer, the equilibration buffer, the elution buffer and / or the wash buffer (such as a load buffer, an equilibration buffer and / or a wash buffer used for the capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, a mixed mode chromatography additional, etc.) are the same. In some embodiments, the loading buffer, the equilibration buffer and / or the wash buffer (such as a loading buffer, an equilibration buffer and / or a wash buffer used for capture chromatography, the first chromatography mixed mode, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) are different . In some embodiments of any of the methods described herein, the buffer comprises a salt. The loading buffer (such as a loading buffer, an equilibration buffer and / or a 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, a further mixed mode chromatography, etc.) may comprise sodium chloride, sodium acetate, Tris, arginine, phosphate, MOPS, MES, CHES, BisTris, ammonium sulfate, sodium sulfate, citrate, succinate or mixtures of these. In certain embodiments, the buffer is a sodium chloride buffer. In some embodiments, the buffer is a sodium acetate buffer. In certain embodiments, the shock absorber is 112 2? Tris, arginine, phosphate, MES, CHES or MOPS buffer. In some embodiments, the damper comprises Tris. In some embodiments, the buffer comprises arginine. In some embodiments of any of the methods described herein, the loading buffer (such as a loading buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography , such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, exclusion chromatography by size, an additional mixed mode chromatography, etc. ) has a conductivity greater than approximately any of 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm , 4.0 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 mS / cm, 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm or 20 : mS / cm. The conductivity can be between approximately any of 1 mS / cm and 20 mS / cm, 4 mS / cm and mS / cm, 4 mS / cm and 7 mS / cm, 5 mS / cm and 17 mS / cm, 5 mS / cm and 10 mS / cm or 5 mS / cm and 7 mS / cm. In some embodiments, the conductivity is approximately any of mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, mS / cm, 4 mS / cm, 4.5 mS / cm, 5, 0 mS / cm, 5.5 mS / cm, mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 mS / cm, 113 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm or 20 mS / cm. In one aspect, the conductivity is the conductivity of the load buffer, the balance buffer and / or the wash buffer. In some embodiments, the conductivity of one or more of the loading buffer, the equilibration buffer, and the wash buffer (such as a loading buffer, an equilibration buffer, and / or a wash buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc. ) it's the same. In some embodiments, the conductivity of the load buffer is different from the conductivity of the wash buffer and / or the balance buffer. In some embodiments, the elution buffer (such as an elution buffer for capture chromatography, first mixed mode chromatography, second i mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, a 114 additional mixed mode, etc.) has a conductivity lower than the conductivity of the load absorber. In some embodiments of any of the methods described herein, 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, an additional mixed mode chromatography, etc.) has a conductivity less than about any of 0 mS / cm, 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4 .0 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm or 7.0 mS / cm. The Conductivity may be between approximately any of 0 mS / cm and 7 mS / cm, 1 mS / cm and 7 mS / cm, 2 mS / cm and 7 mS / cm, mS / cm and 7 mS / cm, or 4 mS / cm. cm and 7 mS / cm, 0 mS / cm and 5.0 mS / cm, mS / cm and 5 mS / cm, 2 mS / cm and 5 mS / cm, 3 mS / cm and 5 mS / cm or mS / cm and 5 mS / cm. In some embodiments, the conductivity 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, 115 additional exclusion, mS / cm, 2.5mS / cm, 5.0 mS / cm, by size, a mixed mode chromatography etc.) is approximately any of 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4 mS / cm, 4.5 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm or 7.0 mS / cm. In some embodiments, the elution buffer has a conductivity greater than the conductivity of the loading buffer. In some embodiments of any of the methods described herein, the elution buffer (such as the elution buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography , such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a conductivity greater than about any of 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 mS / cm, 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, mS / cm, 17.0 mS / cm, 18.0 mS / cm, 19.0 mS / cm, 20.0 mS / cm, 21.0 mS / cm, 22.0 mS / cm, 23.0 mS / cm, 24.0 mS / cm, 25.0 mS / cm, 26.0 mS / cm, 27.0 mS / cm, 28.0 mS / cm, 29.0 mS / cm or 30.0 mS / cm. The conductivity may be between approximately any of 5.5 mS / cm and 30 mS / cm, 116 6.0 mS / cm and 30 mS / cm, 7 mS / cm and 30 mS / cm, 8 mS / cm and 30 mS / cm, 9 mS / cm and 30 mS / cm or 10 mS / cm and 30 mS / cm. In some embodiments, the conductivity of the damper elution is approximately any of 5.5 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm, 11 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 17.0 mS / cm 18.0 mS / cm , 19.0 mS / cm, 20.0 mS / cm, 21.0 22.0 mS / cm, 23.0 mS / cm, 24.0 mS / cm, 25.0 mS / cm, 26.0 27 .0 mS / cm, 28.0 mS / cm, 29.0 mS / cm or 30.0 mS / cm. In mS / cm, aspects of any of the embodiments, the conductivity of the elution buffer (such as the elution buffer for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) the loading and / or washing buffer is changed by step gradient or by linear gradient. In some embodiments, the solution comprising the multispecific antibody is loaded into the first mixed-mode chromatography material in a loading buffer with a conductivity of about <6.5 mS / cm and the polypeptide is eluted from the first chromatography material. 117 mixed mode in an elution buffer with a conductivity of approximately 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 additional embodiments of the above embodiments, the first mixed mode chromatography material is a Capto™Adhere resin. In additional embodiments of the above embodiments, the first mixed mode chromatography material is a resin Capto™ MMC. In some aspects of any of the above embodiments, the conductivity of the elution buffer is changed from the loading and / or washing buffer by step gradient or by linear gradient. In some embodiments, the composition comprising a multispecific antibody is loaded into a first mixed-mode chromatography (e.g., a Capto™Adhere chromatography or a 118 Capto™MMC chromatography) at <6.5 mS / cm and the multispecific antibody is eluted from the first mixed mode chromatography by a step conductivity gradient at approximately 1.5 mS / cm. In some embodiments, the solution comprising the multispecific antibody is loaded into the second mixed-mode chromatography material in a loading buffer with a conductivity of about <6.5 mS / cm and the polypeptide is eluted from the second mixed-mode chromatography material. mixed mode in an elution buffer with a conductivity of approximately 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 additional embodiments of the above embodiments, the second mixed mode chromatography material is a Capto™Adhere resin. In additional embodiments of the previous embodiments, the 119 second mixed mode chromatography material is a Capto™ MMC resin. In some aspects of any of the above embodiments, the conductivity of the elution buffer is changed from the loading and / or washing buffer by step gradient or by linear gradient. In some embodiments, the composition comprising a multispecific antibody is loaded into a second mixed mode chromatography (e.g., a Capto™Adhere chromatography or a Capto™MMC chromatography) at <6.5 mS / cm and the multispecific antibody eluted from the second mixed mode chromatography by a stepwise conductivity gradient at approximately 1.5 mS / cm. In some embodiments, the composition comprising a multispecific antibody is loaded into an anion exchange chromatography (e.g., a QSFF chromatography) at <2.5 mS / cm and the multispecific antibody is eluted from the anion exchange chromatography by a stepwise conductivity gradient at approximately 8.6 mS / cm. In some embodiments, the composition comprising a multispecific antibody is loaded into a cation exchange chromatography (e.g., a POROS chromatography). 50HS) at <5.0 mS / cm and the multispecific antibody was 120 elutes from cation exchange chromatography by a stepwise conductivity gradient in approximately 27.5 mS / cm. In some embodiments of any of the methods described herein, the loading buffer (such as the loading buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography , such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH lower than about any of 10.9, 8, 7, 6 or 5, including any range between these values. In some embodiments of any of the methods described herein, the loading buffer (such as the loading buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography , such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH greater than about any of 4, 5, 6, 7, 8 or 9, even any interval between these 121 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, an additional mixed mode chromatography, etc.) can have a pH of between approximately any of 4 and 9, 4 and 8, 4 and 7, 5 and 9, 5 and 8, 5 and 7, 5 and 6, even any interval between these values. In some embodiments, the pH of the loading buffer (such as the loading buffer used for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography , cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH of approximately any of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, including any range between these values. The pH may be the pH of the loading buffer, the equilibration buffer or the washing buffer (such as a loading buffer, an equilibration buffer and / or a washing buffer used for capture chromatography, first chromatography mixed mode, the second chromatography of 122 mixed mode and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.). In some embodiments, the pH of one or more of the loading buffer, the equilibration buffer, and / or the 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 the wash buffer. In some embodiments, the elution buffer (such as an 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, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH lower than the pH of the loading buffer. In some embodiments of any of the methods described herein, the elution buffer has a pH lower than about any of 8, 7, 6, 5, 4, 3o2, including any range between these values. The pH of the elution buffer can be between approximately any of 4 and 9, 4 and 8, 4 and 7, and 6, 4 and 5, 5 and 9, 5 and 8, 5 and 7, 5 and 6, 6 and 9, 6 and 8, 6 and 123 7, including any interval between these values. In some embodiments, the pH of the elution buffer (such as an 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, an additional mixed mode chromatography, etc.) is approximately any of 4.0, 4.5, 5.0, 5.5, 6.0, 6 .5, 7.0, 7.5, 8.0, 8.5 or 9.0, including any range between these values. In some embodiments, the elution buffer (such as an 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, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH higher than the pH of the loading buffer. In some embodiments of any of the methods described herein, the elution buffer (such as an elution buffer for the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as exchange chromatography anionic, 124 cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) has a pH greater than about any of 5, 6, 7, 8 or 9, including any range between these values. In some embodiments of any of the methods described herein, the elution buffer (such as an elution buffer for capture chromatography) has a pH greater than about any of 2.4o. 4, including any interval between these values. The pH of the elution buffer (such as an 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, mixed mode chromatography additional, etc. ) can be between approximately any of 2 and 9, 3 and 9, 4 and 9, 2 and 8, 3 and 8, 4 and 8, 2 and 7, 3 and 7, 4 and 7, 2 and 6, 3 and 6, and 4 and 6, including any interval between these values. In some embodiments, the pH of the elution buffer is approximately any of 2.0, 2.5, 3.0, 3.5, 4.0, including any range between these values. In some embodiments, the solution comprising a 125 multispecific antibody or antibody arm is loaded onto an affinity chromatography (e.g., protein A chromatography) at approximately pH 7 and the multispecific antibody or antibody arm is eluted from the affinity chromatography by a step gradient in pH of approximately 2.9. In some aspects of any of the embodiments, the pH of the elution buffer (such as an elution buffer for capture chromatography, the first mixed mode chromatography, the second mixed mode chromatography and / or any additional chromatography, such as anion exchange chromatography, cation exchange chromatography, HIC chromatography, size exclusion chromatography, an additional mixed mode chromatography, etc.) the loading and / or washing buffer is changed by step gradient or by linear gradient. In some embodiments of any of the methods described herein, the flow rate is less than about any of 50 CV / h, 40 CV / h or 30 CV / h. The flow rate may be between approximately any of 5 CV / h and 50 CV / h, 10 CV / h and 40 CV / h or 18 CV / h and 36 CV / h. In some embodiments, the flow rate is approximately any of 9 CV / h, 18 CV / h, 25 CV / h, 30 CV / h, 36 CV / h or 40 CV / h. In some modalities of 126 In any of the methods described herein, the flow rate is less than about any of 100 cm / h, 75 cm / h or 50 cm / h. The flow rate may be between approximately any of 25 cm / h and 150 cm / h, 25 cm / h and 100 cm / h, 50 cm / h and 100 cm / h or 65 cm / h and 85 cm / h. Bed height is the height of the chromatography material used. In some embodiments of any of the methods described herein, the bed height is greater than about any of 5 cm, 10 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm or 50 cm. cm. In some embodiments, the bed height is between about 5 cm and 50 cm. In some embodiments, the bed height is determined based on the amount of polypeptide or contaminants in the feed. In some embodiments, the chromatography is in a column or vessel with a volume greater than about 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, 4 0 mL, 50 mL, 75 mL , 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9L, 10L, 25 L, 50L, 100L, 200L, 300L, 400L, 500L, 600L, 700L, 800 L, 900 L or 1000 L. 127 In some embodiments, chromatography fractions are collected. In some embodiments, the fractions collected are greater than about 0.01 CV, 0.02 CV, 0.03 CV, 0.04 CV, 0.05 CV, 0.06 CV, 0.07 CV, 0.08 CV, 0.09 HP, 0.1 HP, 0.2 HP, 0.3 HP, 0.4 HP, 0.5 HP, 0.6 HP, 0.7 HP, 0.8 HP, 0.9 HP, 1.0 HP, 2.0 HP, 3.0 HP, 4.0 HP, 5.0 HP, 6.0 HP, 7.0 HP, 8.0 HP, 9.0 HP or 10.0 HP. In certain embodiments, fractions containing the purified or partially purified product, e.g. e.g. , the multispecific antibody (tai as a bispecific antibody or divalent F(ab')2), antibody arm or Fab, they group together. One skilled in the art can determine how much polypeptide in a fraction can be determined; For example, the amount of polypeptide in a fraction can be determined by UV spectroscopy. In certain embodiments, fractions are collected when the OD280 is greater than about any of 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0. In certain embodiments, fractions are collected when the OD280 is between approximately any of 0.5 and 1.0, 0.6 and 1.0, 0.7 and 1.0, 0.8 and 1.0 or 0.9 and 1.0. In certain embodiments, fractions containing detectable multispecific antibody (e.g., bispecific antibody) or antibody arm are pooled. 128 In certain embodiments of any of the methods described herein, the impurity is a product-specific impurity. Examples of specific product impurities include, but are not limited to, unpaired half-antibody, unpaired antibody light chains, unpaired heavy chains, antibody fragments, homodimers (e.g., paired half dimers of a bispecific antibody comprising the same heavy and light chain), aggregates, high molecular weight species (MHWS) (such as very high molecular weight species (vHMWS)), multispecific antibodies with mismatched disulfides, light chain dimers, heavy chain dimers, species low molecular weight (LMWS) and charge variants (such as acidic variants and basic variants of the antibody). In certain embodiments, the methods provided herein eliminate or reduce the level of unpaired half-antibody from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and unpaired half-antibody. Methods for measuring the presence or level of unpaired half-antibody in a composition are known in the art; for example, by mass spectrometry (such as liquid chromatography-mass spectrometry), CE-SDS, Reversed phase HPLC, HIC HPLC. In certain modalities 129 of any of the methods described herein, the amount of unpaired antibody medium in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any of 5%, 10% , he 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, or 99%, including any range between these values. In certain embodiments, the amount of unpaired half-antibody in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between approximately any one of 10 and 95%; 10% and 99%; 20% and 95%; 20% and 99%; 30% and 95%; 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95%; 60% and 99%; 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of unpaired antibody medium in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of unpaired half-antibody is determined by comparing the amount of unpaired half-antibody in the composition (such as a 130 chromatography fraction) recovered from purification steps with the amount of unpaired antibody medium in the composition before the purification steps purification. In certain embodiments, the methods provided herein eliminate or reduce the homodimer level of a composition comprising a multispecific antibody (e.g., a bispecific antibody) and homodimer. Methods for measuring the presence or level of homodimer in a composition are known in the art; for example, by mass spectrometry (such as liquid chromatography-mass spectrometry, reverse phase HPLC and HIC HPLC. In certain embodiments of any of the methods described herein, the amount of homodimer in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any of 5%, 10%. , he 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 O, r 75 %, 80%, 85%, 90%, 95% or even 99 any interval between these values. In certain embodiments, the amount of homodimer in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between about any of 10 and 95%; 10% and 131 99%; 20% and 95%; 20% and 99%; 30% and 95%; he 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95% r 60% and 99 O. ° r 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of homodimer in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of homodimer is determined by comparing the amount of homodimer in the composition (such as a chromatography fraction) recovered from purification steps with the amount of homodimer in the composition before the steps. purification stages. In certain embodiments, the methods provided herein remove or reduce the level of high molecular weight species (HMWS) protein from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and HMWS protein. The HMWS protein may comprise, e.g. e.g. , aggregated polypeptide (such as aggregated multispecific antibody, aggregated half-antibody, aggregated homodimer, etc.). In certain embodiments, the added polypeptide comprises heavy chain multimers, light chain multimers and / or 132 multimers of the multispecific antibody. The protein of HMWS may comprise 2, 3, 4, 5, 6, 7 or 8 or more monomers of a heavy chain or light chain, or 2, 3, 4, 5, 6, 7 or or more aggregated multispecific antibodies. Methods for measuring aggregated protein (e.g., HMWS protein) are known in the art and are described in, e.g. e.g. WO 2011 / 150110. Such methods include, e.g. e.g., 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 a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any of 5%, the 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 85%, 90%, 95% or 99%, including any range between these values. In certain embodiments, the amount of HMWS protein in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between about any of 10 and 95%; 10% and 99%; 20% and 95%; 20% and 99%; 30% and 95%; 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95%; 60% and 99%; 133 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of HMWS protein in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of HMWS protein is determined by comparing the amount of HMWS protein in the composition (such as a chromatography fraction) recovered from purification steps with the amount of I protein. HMWS in the composition before the stages of i? purification. In certain embodiments, the methods provided herein remove or reduce the level of low molecular weight species (LMWS) protein from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and LMWS protein. The protein of LMWS may comprise fragmented polypeptide. In certain embodiments, the fragmented polypeptide is a multispecific antibody fragment, a fragment of an antibody arm, a heavy chain fragment, or a light chain fragment. Examples of protein LMWS include, among others, a Fab (i.e. fragment antigen binding), Fe 134 (fragment, crystallizable), regions or combinations of both, or any random fragmented part of a multispecific antibody, heavy chain or light chain of interest, or antibodies (containing a light chain / heavy chain pair of antibody) or ¾ antibodies (containing a heterodimer or a homodimer of antibody heavy chains and a single antibody light chain; also indicated in this HHL). Methods for measuring cleaved protein (e.g., LMWS protein) are known in the art and are described in, e.g. e.g., WO 2011 / 150110. Such methods include, e.g. e.g., 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 a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70 or ° 1 75%, 80%, 85%, 90%, 95% or %, including any range between these values. In certain embodiments, the amount of LMWS protein in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced. 135 between approximately any of 10 and 95%; 10% and 99%; 20% and 95%; 20% and 99%; 30% and 95%; 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95%; 60% and 99%; 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of LMWS protein in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of LMWS protein is determined by comparing the amount of LMWS protein in the composition (such as a chromatography fraction) recovered from purification steps with the amount of LMWS protein. in the composition before the purification steps. In certain embodiments, the methods provided herein eliminate or reduce the level of acidic and / or basic variants of a composition comprising a multispecific antibody (e.g., a bispecific antibody) and acidic and / or basic variants. Acidic variants of an antibody (such as a multispecific antibody, e.g., a bispecific antibody) are variants in which the pl of the antibody is lower than the pl of the intact antibody. 136 an antibody (such as natural. The basic variants of a multispecific antibody, e.g., a bispecific antibody) are variants in which the pl of the antibody is higher than the pl of the natural intact antibody. Such charge variants (e.g., acidic and basic variants) can be the result of natural processes, such as oxidation, deamidation, C-terminal processing of lysine residues, N-terminal pyroglutamate formation, and antibody glycation. Methods for measuring load variants are known in the art; for example, imaging capillary isoelectrofocusing (iCIEF). In certain embodiments of any of the methods described herein, the amount of variants loaded into a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any of 5%, %, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 85%, 90%, 95%, or 99%, including any range between these values. In certain embodiments, the amount of variants loaded into a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between about any of 10 and 95%; 10% and 99%; 20% and 95%; 20% and 99%; 30% and 95%; 137 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99% r 60% and 95%; 60% and 99% r 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of variants loaded in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of loaded variants is determined by comparing the amount of loaded variants in the composition (such as a chromatography fraction) recovered from purification steps with the amount of loaded variants in the composition. before the purification steps. In certain embodiments of any of the methods described herein, the impurity is a process-specific impurity. For example, the process-specific impurity may comprise one or more of: leached Ά protein; host cell materials; nucleic acids; other polypeptides; endotoxin; viral contaminants; components of cell culture media, carboxypeptidase B, gentamicin, etc. In certain embodiments, the process-specific impurity may be a host cell protein (HCP) from, e.g. e.g., a 138 prokaryotic cell, a bacterial cell (such as an E. coli cell), an insect cell, a eukaryotic cell, a fungal cell, a yeast cell, an avian cell or a mammalian cell, e.g. e j. , a cell CHO. In certain embodiments, the methods provided herein remove or reduce the level of leached protein A from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and leached protein A. Leached protein A is protein A separated or washed from a solid phase to which it is bound. For example, leached protein A can be leached from protein A chromatography column. The amount of protein A can be measured, for example, by ELISA, as described in WO 2011 / 150110. In certain embodiments, the reduction in the presence or level of leached A protein is determined by comparing the amount of leached A protein in the composition (such as a chromatography fraction) recovered from purification steps with the amount of protein of A leached into the composition before the purification steps. In certain embodiments, the methods provided herein eliminate or reduce the level of proteins of 139 host cells (HCP) of a composition comprising a multispecific antibody (eg, a bispecific antibody) and HCP. HCPs are host cell proteins in which the antibody is produced. multispecific (such as a bispecific antibody). In certain embodiments, the HCP proteins are prokaryotic cell proteins. In certain modalities, the HCPs are E. coli cell proteins (i.e., E. coli proteins or ECPs). Examples of prokaryotic HCPs (such as ECP) ​​include, among others, prokaryotic chaperones, such as FkpA, DsbA and DsbC. In certain embodiments, HCPs are eukaryotic host cell proteins, such as those described elsewhere herein. In certain embodiments, the HCPs are mammalian cell proteins, such as CHO cell proteins (i.e., Chinese hamster ovary proteins or CHOP). In certain embodiments, the amount of HCP (e.g., ECP, FkpA, DsbA, DsbC or e.g. e.g. , CHOP) is measured by enzyme-linked immunosorbent assay (ELISA). For example, antibodies can be generated against ultrapure compositions of FkpA, DsbA or DsbC. In certain embodiments, the amount of FkpA, DsbA and / or DsbC is determined by mass spectrometry. In some embodiments of any of the methods described herein, the amount of HCP (e.g., ECP, FkpA, DsbA, DsbC or, e.g., CHOP). In certain modalities of any 140 of the methods described herein, the amount of HCP (e.g., ECP, FkpA, DsbA, DsbC or, e.g., CHOP) in a composition (such as a chromatography fraction) recovered from one or more Purification steps are reduced to less than about 100 ppm, 75 ppm, 50 ppm, 25 ppm, ppm, 10 ppm, 5 ppm, 2 ppm or 1 ppm, including any range between these values. In some embodiments, the amount of HCP (e.g., ECP, FkpA, DsbA, DsbC or, e.g., CHOP) in a composition (such as a chromatography fraction) is reduced to less than about 100 ppm, 75 ppm, 50 ppm, 25 ppm, 20 ppm, 10 ppm, 5, ppm, 2 ppm or 1 ppm, including any range between these values. In certain embodiments, the reduction in the presence or level of HCP (e.g., ECP, FkpA, DsbA, DsbC or, e.g., CHOP) is determined by comparing the amount of HCP in the composition (such as a chromatography fraction) recovered from purification steps with the amount of HCP in the composition before the purification steps. In certain embodiments, the provided methods remove or reduce the level of nucleic acid (such as host cell DNA and / or RNA) from a composition comprising a multispecific antibody (e.g., a bispecific antibody) and nucleic acid. Methods for measuring nucleic acid (such as DNA and / or RNA from cells 141 hosts) are known in the art and are described in, e.g. e.g., WO 2011 / 150110. Such methods include, e.g. e.g., PCR for host cell DNA or RNA. In certain embodiments of any of the methods described herein, the amount of nucleic acid in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced by more than about any of 5%, %, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 85%, 90%, 95%, or 99%, including any range between these values. In certain embodiments, the amount of nucleic acid in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between about any of 10 and 95%; 10% and 99%; · 20% and 95%; · 20% and · 99%; · 30% and 95%; 30% and 99%; 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95%; 60% and 99%; 70% and 95%; 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of nucleic acid in a composition (such as a chromatography fraction) is reduced by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain modalities, the reduction in 142 of nucleic acid is determined by presence or level by comparing the amount of nucleic acid in the composition (such as a chromatography fraction) recovered from purification steps with the amount of nucleic acid in the composition before the purification steps. In certain embodiments, the methods provided herein remove or reduce the level of a cell culture medium component of a composition comprising a multispecific antibody (e.g., a bispecific antibody) and cell culture medium component. Cell culture medium component refers to a component present in a cell culture medium. In certain embodiments, cell culture medium refers to the cell culture medium at which host cells expressing the multispecific antibody (e.g., bispecific antibody) or arms thereof are harvested. 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 a cell culture medium component in a composition (such as a chromatography fraction) recovered from one or more steps 143 purification is reduced by more than approximately any 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 or 1 60 o ° f 65%, 70%, 75%, 80%, 85%, 90% or 95% or 99%, including any interval between these values. In certain embodiments, the amount of a cell culture medium component in a composition (such as a chromatography fraction) recovered from one or more purification steps is reduced between about any of 10 and 95%; 10% and 99%; 20% and 95%; 20% and 99%; 30% and 95%; 30% and 99%; he 40% and 95%; 40% and 99%; 50% and 95%; 50% and 99%; 60% and 95%; 60% and 99%; 70% and 95%; he 70% and 99%; 80% and 95%; 80% and 99%; 90% and 95%, or 90% and 99%. In some embodiments, the amount of a cell culture medium component in a composition (such as a chromatography fraction) is reduced by approximately any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In certain embodiments, the reduction in the presence or level of a cell culture medium component is determined by the composition of the amount of the cell culture medium component in the composition (such as a chromatography fraction). 144 recovered from purification steps with the amount of the cell culture medium component in the composition before the purification steps. In certain embodiments, a method is provided herein for purifying a bispecific antibody comprising a first arm and a second arm, wherein the first and second arms are produced separately, wherein the method comprises: subjecting the first and second arms arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) performed in binding and elution mode to produce the first and second capture eluate; forming a mixture comprising the first and second capture eluates under conditions sufficient to produce a composition comprising the multispecific antibody, subjecting the composition comprising the multispecific antibody to anion exchange chromatography (e.g., Q Sepharose® chromatography fast flow (QSFF)) in binding and elution mode to produce an anion exchange eluate, wherein the elution is a gradient elution; subjecting the anion exchange eluate to anion exchange mixed mode chromatography (e.g., Capto™Adhere chromatography) in binding and elution mode to produce a first mixed mode eluate, where the elution is an elution of 145 gradient; and subjecting the first mixed mode eluate to cation exchange mixed mode chromatography (e.g., Capto™MMC chromatography) in binding and elution mode to produce a second mixed mode eluate, where the elution is an elution of gradient, and collecting a fraction comprising the bispecific antibody, wherein the method reduces an amount of an impurity in the fraction with respect to the mixture comprising the first and second arms. In certain embodiments, a method is provided herein for purifying a bispecific antibody comprising a first arm and a second arm, wherein the first and second arms are produced separately, wherein the method comprises: subjecting the first and second arms arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) in binding and elution mode to produce the first and second capture eluate; forming a mixture comprising the first and second capture eluates under conditions sufficient to produce a composition comprising the multispecific antibody, subjecting the composition comprising the multispecific antibody to cation exchange mixed-mode chromatography (e.g., Capto chromatography ™MMC) in binding and elution mode to produce a first eluate of 146 mixed mode; wherein the step elution is a pH and salt elution, and subjecting the first mixed mode eluate to anion exchange mixed mode chromatography (e.g. e.g., Capto™Adhere chromatography) in continuous flow mode to produce a second mixed mode eluate, and collect a fraction comprising the bispecific antibody, wherein the method reduces an amount of an impurity in the fraction with respect to the mixture which includes the first and second arms. In certain embodiments, a method is provided herein for purifying a bispecific antibody comprising a first arm and a second arm, wherein the first and second arms are produced separately, wherein the method comprises: subjecting the first and second arms arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) in binding and elution mode to produce the first and second capture eluate; forming a mixture comprising the first and second capture eluates under conditions sufficient to produce a composition comprising the multispecific antibody, subjecting the composition comprising the multispecific antibody to anion exchange mixed-mode chromatography (e.g., Capto chromatography ™Adhere) in binding and elution mode, where elution is a 147 elution in steps, to produce a first mixed mode eluate; and subjecting the first mixed mode eluate to cation exchange mixed mode chromatography (e.g. e.g. , Capto™MMC chromatography) in binding and elution mode to produce a second mixed-mode eluate, where the elution is a step elution, subjecting the second mixed-mode eluate to hydrophobic interaction chromatography (e.g., Hexyl-650C) in continuous flow mode to produce a hydrophobic interaction eluate; and collecting a fraction comprising the bispecific antibody, wherein the method reduces an amount of an impurity in the fraction with respect to the mixture comprising the first and second arms. In certain embodiments, provided herein is a method for purifying a bispecific antibody (such as a bispecific F(ab')2) comprising a first arm and a second arm, wherein the first and second arms are produced separately. , where the method comprises: subjecting the first arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) in binding and elution mode to produce a first capture eluate; Subject the first capture eluate to cation exchange mixed-mode chromatography (e.g., Capto™ MMC chromatography) a mode of 148 binding and elution to produce a first mixed mode eluate; subjecting the second arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) in binding and elution mode to produce a second capture eluate; forming a mixture comprising the first mixed mode eluate and the second capture eluate under conditions sufficient to produce a composition comprising the multispecific antibody, subjecting the composition comprising the multispecific antibody to anion exchange mixed mode chromatography (e.g. ., such as Capto™ chromatography Adhere) to produce a second mixed mode eluate; and subjecting the second mixed mode eluate to cation exchange chromatography (e.g., such as POROS® 50 HS) in binding and elution mode to produce a cation exchange eluate; subjecting the cation exchange eluate to subsequent cation exchange mixed mode chromatography in binding and elution mode to produce a third mixed mode eluate; and collecting a fraction comprising the bispecific antibody, wherein the method reduces an amount of an impurity in the fraction with respect to the mixture comprising the first and second arms. In certain embodiments, herein provided 149 a method for purifying a bispecific antibody (such as a bispecific F(ab')2) comprising a first arm and a second arm, wherein the first and second arms are produced separately, wherein the method comprises: subjecting the first arm to capture chromatography (such as any one or a combination of the capture chromatography steps described elsewhere herein) in binding and elution mode 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; subjecting the second arm to capture chromatography (such as any one or a combination of the steps of I capture chromatography described elsewhere herein) in binding and elution mode to produce a second capture eluate; form a mixture comprising i the first mixed mode eluate and the second capture eluate under conditions sufficient to produce a composition comprising the multispecific antibody, subjecting the composition comprising the antibody ( multispecific to anion exchange mixed-mode chromatography (e.g., such as Capto™ chromatography Adhere) to produce a second mixed mode eluate; and subjecting the second mixed-mode eluate to subsequent cation exchange mixed-mode chromatography (e.g., 150φ Capto™ MMC chromatography) in binding and elution mode to produce a third mixed mode eluate; and collecting a fraction comprising the bispecific antibody, wherein the method reduces an amount of an impurity in the fraction with respect to the mixture comprising the first and second arms. In some embodiments, the multispecific antibody (such as bispecific antibody) is further purified by viral filtration. Viral filtration is the removal of viral contaminants in a polypeptide purification feed stream. Examples of viral leakage include e.g. e.g. , ultrafiltration and microfiltration. In some embodiments, the polypeptide is purified using a parvovirus filter. In some embodiments, the multispecific antibody is concentrated after chromatography (e.g., after the second mixed mode chromatography or after one or more chromatography steps performed after the second mixed mode chromatography). Examples of concentration methods are known in the art and include, but are not limited to, e.g. e.g., ultrafiltration and diafiltration (UFDF). In some embodiments, the multispecific antibody is concentrated by a first ultrafiltration, a diafiltration and a second 151 ultrafiltration. In some embodiments, ultrafiltration and / or diafiltration utilize a filter with a lower cutoff of approximately any of 5 kDal, 10 kDal, 15 kDal, kDal, 25 kDal or 30 kDal. In some embodiments, the retentate from the first ultrafiltration is diafiltered into a pharmaceutical formulation. In some embodiments, the concentration of multispecific antibody after concentration is approximately any of 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL or 300 mg / mL. In some embodiments, the concentration of the multispecific antibody is between approximately any of 10 mg / mL and 20 mg / mL, 20 mg / mL and 30 mg / mL, 30 mg / mL and 40 mg / mL, 40 mg / mL and 50 mg / mL, 50 mg / mL and 60 mg / mL, 60 mg / mL and 7 0 mg / mL, 70 mg / mL and 80 mg / mL, 80 mg / mL and 90 mg / mL, 90 mg / mL and 100 mg / mL, 100 mg / mL and 110 mg / mL, 110 mg / mL and 120 mg / mL, 120 mg / mL and 130 mg / mL, 130 mg / mL and 140 mg / mL, 140 mg / mL and 150 mg / mL, 150 mg / mL and 160 mg / mL, 160 mg / mL and 170 mg / mL, 170 mg / mL and 180 mg / mL, 180 mg / mL and 190 mg / mL, 190 mg / mL and 200 mg / mL, 200 mg / mL or 300 mg / mL. In some embodiments of any of the methods described herein, the methods further comprise 152 combine the purified polypeptide from the purification methods with a pharmaceutically acceptable carrier. In some embodiments, the multispecific antibody is formulated into a pharmaceutical formulation by ultrafiltration / diafiltration. In certain methods, the methods provided herein produce a composition comprising a multispecific antibody that is more than about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% pure. In certain embodiments, the multispecific antibody in the composition is more than about any of 96%, 97%, 98% or 99% pure. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% unpaired antibody arms. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing no more 153 0.3%, approximately any of 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% homodimer. In certain embodiments, the methods provided in the present produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% added protein. In certain methods, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, the 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% of HMWS. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% of LMWS. In certain embodiments, the methods provided herein produce a composition comprising the 154 multispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and 1 0.8%, 0.9%, 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, , 6%, 6.5%, 7 %, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35 %, 40%, 45% or 50% of variants acidic. In certain methods, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% basic variants. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3 .5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm or 10 ppm leached protein A. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3 .5ppm, 4ppm, 4.5 155 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, 10 ppm, 15 ppm, 20 ppm, 2 5 ppm, 3 0 ppm or 35 ppm HCP. In certain embodiments, the methods provided herein produce a composition comprising the multispecific antibody containing less than about any of 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5ppm, 6ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm or ppm nucleic acid. In certain embodiments, the composition comprising the multispecific antibody comprises no more than 0 ppm nucleic acid. In certain embodiments, the nucleic acid in the composition comprising the multispecific antibody is below the level of detection. In certain methods, the methods provided herein produce a composition comprising the multispecific antibody containing no more than about any of 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% cell culture medium component. In certain methods, a composition is provided comprising a multispecific antibody purified according to any of the methods described herein. In certain embodiments, the multispecific antibody 156 of approximately any of the in the composition is more 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% pure. In certain embodiments, the multispecific antibody in the composition is more than about any of 96%, 97%, 98% or 99% pure. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% unpaired antibody arms. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% homodimer. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 157 5% added protein. In certain methods, the composition comprising the multispecific antibody contains no more than about any of 0.1%, the 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% of HMWS. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1%, the 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% , 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% of LMWS. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0. .6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% acidic variants. In certain methods, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% basic variants. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1 ppm, 158 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 1.5 ppm, 2 ppm , 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm or 10 ppm leached protein A. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 1.5 ppm, 2 ppm , 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm or 35 ppm HCP. In certain embodiments, the composition comprising the multispecific antibody contains no more than about any of 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6ppm, 0.7ppm, 0.8ppm, 0.9ppm, 1ppm, 1.5ppm, 2ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm , 9 ppm, 9.5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm or 35 ppm nucleic acid. In certain methods, the composition comprising the multispecific antibody contains no more than about any of 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% cell culture medium component. In some embodiments, a composition is provided that 159 comprises a multispecific antibody, wherein the composition contains: a) at least about 95% 100% multispecific antibody; b) less than approximately 1%-5% unpaired antibody arms; c) less than about 1%-5% homodimers of the antibody; d) not more than approximately % or 2% of HMWS; e) no more than approximately 1% or 2% of LMWS; and / or f) no more than approximately 5% % antibodies. In certain methods, a composition is provided comprising a bispecific antibody purified according to any of the methods described herein. In certain embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g., a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody. In certain embodiments, a composition is provided comprising a bispecific antibody that is more than about any of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95% pure. In certain embodiments, the bispecific antibody in the composition is more than about any of 96%, 97%, 98% or 99% pure. In certain embodiments, the bispecific antibody is a button antibody in 160 a KiH bispecific antibody. Buttonhole (KiH), p. e.g. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody. In certain embodiments, a composition is provided comprising the bispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8 %, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5 %, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10 % of arms of unpaired antibody. In certain embodiments, a composition is provided comprising the bispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, he 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% homodimer. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, he 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% added protein. In certain embodiments, a composition is provided comprising a bispecific antibody that 161 contains no more than about any of 0.1% the 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% HMWS. In certain embodiments, a composition is provided comprising the bispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, he 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% of LMWS. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3 .5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% acidic variants. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1%, 0.5%, 1%, 5%, 10%, 20%. , 25%, 30% or 35% basic variants. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1 ppm, 0.2ppm, 0.3ppm, 0.4ppm, 0.5ppm, 0.6ppm, 0.7ppm, 0.8 162 ppm, 0.9 ppm, 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm , 6.5ppm, 7ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm or 10 ppm leached protein A. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6ppm, 0.7ppm, 0.8ppm, 0.9ppm, 1ppm, 1.5ppm, 2ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm , 9 ppm, 9.5 ppm, 10 ppm, 15 ppm, 2 0 ppm, 2 5 ppm, 30 ppm or 3 5 ppm of HCP. In certain embodiments, a composition is provided comprising a bispecific antibody containing less than about any of 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm or ppm nucleic acid. In certain embodiments, the composition comprising the bispecific antibody comprises no more than 0 ppm nucleic acid. In certain embodiments, the nucleic acid in the composition comprising the bispecific antibody is below the level of detection. In certain embodiments, a composition is provided comprising a bispecific antibody containing no more than about any of 0.1%, 0.5%, 1%, 10%, 15%, 20%. , 25%, 30% or 35% 163 of cell culture medium component. In certain embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g., a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody. In some embodiments, a composition is provided comprising a bispecific antibody, wherein the composition contains: a) at least about 95% 100% bispecific antibody; b) less than approximately 1%-5% unpaired antibody arms; c) less than about 1%-5% homodimers of the antibody; d) not more than approximately % or 2% of HMWS; e) no more than approximately 1% or 2% of LMWS; and / or f) no more than approximately 5% % antibodies. In certain embodiments, the bispecific antibody is a buttonhole (KiH) antibody, e.g. e.g. , a KiH bispecific antibody. In some embodiments, the bispecific antibody is a CrossMab bispecific antibody. One aspect as described herein is a method for purifying an Fe region-containing heterodimeric protein / polypeptide with a multi-step chromatography method, wherein the method comprises an affinity chromatography step followed by two different steps. 164 of multimodal ion exchange chromatography, and thus purify the heterodimeric protein / polypeptide containing Fe region. 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. One aspect as described herein is a method for producing an Fe region-containing heterodimeric protein / polypeptide comprising the following steps i. culturing a cell comprising a nucleic acid encoding the heterodimeric protein / polypeptide containing Fe region, ii. recovering the Fe region-containing heterodimeric protein / polypeptide from the cell or culture medium, iii. purifying the Fe region-containing heterodimeric protein / polypeptide with a method as described herein, and thereby producing the Fe region-containing heterodimeric protein. It has been discovered that the yield of an antibody purification process depends on the sequence of the 165 employees. By choosing a certain sequence / order of chromatography steps, an improved process can be obtained. The methods provided herein are based, at least in part, on the finding that performing a multimodal anion exchange chromatography step (directly) after the affinity chromatography step (initial) and before a chromatography step multimodal cation exchange, an ultrafiltration / diafiltration step can be omitted. This step is necessary if the multimodal cation exchange chromatography step is performed before the multimodal anion exchange chromatography step. In certain embodiments, the multi-stage chromatography method comprises an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by a multimodal cation exchange chromatography step. It has been found that with the method described herein, good purity and yield can be achieved with only three chromatography steps. In certain embodiments, the chromatography method of 166 multi-stage comprises exactly three stages of chromatography. It has been found that the removal of proteins from host cells can be improved if the multimodal anion exchange chromatography method / step is performed in continuous flow mode. In certain embodiments, the multimodal anion exchange chromatography method / step is performed in continuous flow mode. The loading pH of the multimodal anion exchange chromatography step has been found to influence the removal of HCP, byproduct, and DNA. In an all-around preferred embodiment, the multimodal anion exchange chromatography step is performed at a pH of about 7.0. Table 1 SEC HCP Caliper DNA Yield Σ HMW Prepic HHL to os pH Conductivity [%] (%] [ng / mg] [%] [%] [pg / mg] chromatography [mS / cm] 6.5 5.75 85 3 .34 139 8.132 2.428 <0.3 7.0 5.76 69 1.51 55 6.186 1.830 <0.4 7.5 6.73 67 1.23 35 5.671 1.555 22.1 The conductivity of a solution can have a influence 167 in different parameters during a purification process. Here it has been discovered that low conductivity values ​​in the filler (i.e., the solution comprising the heterodimeric polypeptide containing region Fe that is applied to the chromatography material) of the multimodal anion exchange chromatography step leads to improved HCP and DNA removal. Table 2 Charge conductivity [mS / cm] HCP [ng / mg] DNA tpg / mg] 16.92 303 78.6 5.86 35 22.1 3.64 14 0 In certain embodiments, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in a solution with a conductivity value of less than 7 mS / cm. In certain embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with a conductivity value of less than 6 mS / cm. In certain embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a 168 solution with a conductivity value in the range between approximately 6 mS / cm and approximately 2 mS / cm. In certain embodiments, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in a solution with a conductivity value in the range between about 5 mS / cm and about 4 mS / cm. In certain embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in a solution with a conductivity value of about 4.5 mS / cm. In certain embodiments, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in a solution with a conductivity of about 4.5 mS / cm and a pH of about 7. The methods provided herein are based, at least in part, on the finding that the amount of protein loading of the multimodal anion exchange chromatography step also influences the performance of the purification process. If the loading is in a defined range, the overall purification process improves, e.g. e.g., removal of DNA contamination. 169 Table 3: Initial amount of DNA: 80 pg / zog Polypeptide loading per ImpRes Capto adhere material DNA content after chromatography step 220 g / L 180 g / L 0.8 pg / mg 0 pg / mg 150 g / L 120 g / L pg / mg 0 pg / mg In certain embodiments, in the multimodal anion exchange chromatography step, the heterodimeric polypeptide containing Fe region is applied in the range of between about 100 g and about 300 g per liter of chromatography material, i.e. the loading is in the range of approx. 100 g / L and approximately 300 g / L. In certain embodiments, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in the range of between about 120 g and about 240 g per liter of chromatography material. In certain embodiments, in the multimodal anion exchange chromatography step, the Fe region-containing heterodimeric polypeptide is applied in the range of between about 160 g and about 200 g per liter of 170 chromatography material. Certain multimodal resin materials have been found to be especially useful when applied in the method as described herein. 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 flux agarose matrix, a strong multimodal anion exchanger as a ligand, an average particle size of 36-44 pm and an ionic capacity of between 0.08 and 0.11 mmol in Cl- / mL medium. In certain embodiments, the multimodal anion exchange chromatography material is Capto adheres ImpRes. 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 flux agarose matrix, a multimodal weak cation exchanger as a ligand, an average particle size of 36-44 pm and an ionic capacity of between 25 and 39 pmol / mL. 171 In certain embodiments, the multimodal cation exchange chromatography medium is Capto MMC ImpRes. In certain embodiments, the multimodal cation exchange chromatography method / step is performed in binding and elution mode. In certain embodiments, the affinity chromatography step is a protein A chromatography step, a protein G affinity chromatography, a single chain Fv ligand affinity chromatography, a KappaSelect chromatography material chromatography step, a chromatography with chromatography material CaptureSelect or a chromatography step with 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. In certain embodiments, the Fe region-containing heterodimeric protein / polypeptide is an antibody, a bispecific antibody, or Fe fusion proteins. In certain embodiments, the protein / polypeptide 172 heterodimeric containing Fe region is a bispecific antibody. In certain embodiments, the Fe region-containing heterodimeric protein / polypeptide is a CrossMab. In certain embodiments, the Fe region-containing heterodimeric protein / polypeptide is an Fe fusion protein. In certain embodiments, the Fe region-containing heterodimeric protein / polypeptide is a bispecific antibody comprising a) a heavy chain and a light chain of a first full-length antibody that specifically binds to a first antigen; and b) a modified heavy chain and a modified light chain of a full-length antibody that specifically binds to a second antigen, wherein the CL and CH1 constant domains replace each other. In certain embodiments, the bispecific antibody is a bispecific antibody that binds ANG2 and VEGF. In certain embodiments, the Fe region-containing heterodimeric protein / polypeptide is a CrossMab that binds ANG2 and VEGF. In certain embodiments, the bispecific antibody is vanucizumab. In certain embodiments, the bispecific antibody comprises a first antigen binding site that comprises, as a heavy chain variable (VH) domain, the 173 SEQ ID NO: 1 and, as a light chain variable domain (VL), SEQ ID NO: 2; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 3 and, as a light chain variable domain (VL), SEQ ID NO: 4. In certain embodiments, The bispecific antibody comprises a first heavy chain with the amino acid sequence of the SEQ ID NO: 9 and a second heavy chain with the amino acid sequence of SEQ ID NO: 10 and a first light chain with the amino acid sequence of SEQ ID NO: 11 and a second light chain with the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the bispecific antibody comprises a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 5 and, as a light chain variable domain ( VL), SEQ ID NO: 6; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 7 and, as a light chain variable domain (VL), SEQ ID NO: 8. In certain embodiments, The bispecific antibody comprises a first heavy chain with the amino acid sequence of SEQ ID NO: 13 and a second heavy chain with the amino acid sequence of SEQ ID NO: 14 and a first light chain with the amino acid sequence of SEQ ID NO: 15 and a second light chain with the amino acid sequence of SEQ ID NO: 16. The sequences of 174 amino acids of SEQ ID NO: 1-16 are provided in the table 4 below: Table 4 EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA SEQ ID NO: PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY 1 LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQK P SEQ ID NO: 2 GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIK QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA SEQ ID NO: PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY 3 MELSRLRSDD TAVYYCARSP NPYYYDSSGY YYPGAFDIWG QGTMVTVS QPGLTQPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQKPG SEQ ID NO: 4 QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEADYYCQVW DSSSDHYVFG TGTKVTVL EV QLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA SEQ ID NO: PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY 5 LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSS DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP SEQ ID NO: 6 GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP w EDFATYYCQQ YSTVPWTFGQ GTKVEIK 175 QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA SEQ ID NO: PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY 7 MELSRLRSDD TAVYYCARSP NPYYYDSSGY YYPGAFDIWG QGTMVTVSS SYVLTQPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQKPG SEQ ID NO: QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEAD YYCQVW DSSSDHWVFG GGTKLTVLGQ QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARSP NPYYYDSSGY YYPGAFDIWG QGTMVTVSSA SVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST SEQ ID NO: LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGECDKTH 9 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVCTLPPS R DELTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY SEQ ID NO: LQMNSLRA ED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT 10 VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL 176 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK PGK QPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQKPG QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG SEQ ID NO: DEADYYCQVW DSSSDHYVFG TGTKVTVLSS ASTKGPSVFP 11 LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSC DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP SEQ ID NO: EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP 12 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT SEQ ID NO: VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 13 TVSWNSGALT SGVHTFPAVL QSSG LYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMAS RTPEVTCVVV DVSHEDPEVK 177 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLAQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVYTLPPC RDELTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN AYTQKSLSLS PGK QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA PGQG LEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARSP NPYYYDSSGY YYPGAFDIWG QGTMVTVSSA SVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST SEQ ID NO: LTLSKADYEK HKVYACEVTH QGLSSPVTK S FNRGECDKTH 14 TCPPCPAPEA AGGPSVFLFP PKPKDTLMAS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLAQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVCTLPPS RDELTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN AYTQKSLSLS PGK DIQLTQSPSS LSASVGDRVT ITCSASQ DIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP SEQ ID NO: EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP 15 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQ G LSSPVTKSFN RGEC SEQ ID NO: SYVLTQPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQKPG 16 QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG 178 DEADYYCQVW DSSSDHWVFG GGTKLTVLSS ASTKGPSVFP LAPSSKSTSG HTFPAVLQSS NTKVDKKVEP GTAALGCLVK GLYSLSSVVT KSC DYFPEPVTVS VPSSSLGTQT WNSGALTSGV YICNVNHKPS In certain embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about 5% antibodies. In certain embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about ¾% antibodies. In certain embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about 3% ¾ antibodies. In certain embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about 2% ¾ antibodies. In certain embodiments, the purified Fe region-containing heterodimeric polypeptide contains no more than about 1% antibodies. One aspect as described herein is a method for purifying a bispecific antibody that binds ANG2 and VEGF with a multi-step chromatography method, κ wherein the method comprises an affinity chromatography step, followed by a multimodal anion exchange chromatography step, followed by an I 179 multimodal cation exchange chromatography and thus purifying the bispecific antibody that binds to ANG2 and VEGF, wherein the bispecific antibody that binds ANG2 and VEGF comprise a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 1 and, as a light chain variable domain (VL), SEQ ID NO: 2; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 3 and, as a light chain variable domain (VL), SEQ ID NO: 4 or comprising a first antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: 5 and, as a light chain variable domain (VL), SEQ ID NO: 6; and a second antigen binding site comprising, as a heavy chain variable domain (VH), SEQ ID NO: and, as a variable light chain (VL) domain, the SEQ ID NO: 8. In one embodiment, the bispecific antibody that binds ANG2 and VEGF comprise a) the heavy chain and the light chain of a first full-length antibody comprising the first antigen binding site; and b) the modified heavy chain and the modified light chain of a full-length antibody comprising the second antigen binding site, wherein the CL and CHl constant domains replace each other. 180 One aspect as described herein is the use of the method as described herein for the purification of a heterodimeric polypeptide containing Faith. One aspect as described herein is the use of the method as described herein for the reduction of impurities related to the Fe-containing heterodimeric polypeptide. One aspect as described herein is an Fe-containing heterodimeric polypeptide obtained with the method as described herein for the manufacture of a medicament for the treatment of cancer or ocular disease. One aspect as described herein is an Fe-containing heterodimeric polypeptide obtained with the method as described herein for use in the treatment of cancer or ocular disease. Polypép ticLos Monoclonal antibodies 181 φ In some embodiments, the antibodies are monoclonal antibodies. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that arise during the production of the monoclonal antibody, such variants They are generally present in smaller quantities. Therefore, the monoclonal modifier indicates that the nature of the antibody is not a mixture of discrete or polyclonal antibodies. For example, monoclonal antibodies can be made using the hybridoma procedure first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (US Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal is immunized as described herein to obtain lymphocytes that produce or are capable of producing antibodies that specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as 182 polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pages. 59-103 (Academic Press, 1986)). Hybridoma cells prepared in this way are cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of unfused myeloma stem cells. For example, if stem myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of cells deficient in HGPRT. In some embodiments, myeloma cells are those that fuse efficiently, support stable high-level production of an antibody from selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, in some embodiments, the myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors. MOPC-21 and MPC-11 from Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63Ag8-653 cells available in the American Type Culture Collection, 183 Rockville, Maryland, USA. Mouse-human heteromyeloma and human myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987) ) . The culture medium in which the hybridoma cells grow is tested to produce monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980). After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, clones can be subcloned by dilution limiting procedures and cultured by standard methods (Goding, Monoclonal 184 • Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture medium for these purposes includes, for example, D-MEM medium or RPMI-1640. Furthermore, it is possible to culture the hybridoma cells in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, polypeptide A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis , affinity chromatography or ion exchange chromatography. The DNA encoding monoclonal antibodies is easily isolated and sequenced by conventional procedures (for example, with oligonucleotide probes that are capable of specifically binding to genes encoding murine antibody heavy and light chains). In some embodiments, hybridoma cells serve as a source of said DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E cells. coli, simian COS cells, human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce 185 to obtain the immunoglobulin polypeptide, for the purpose of synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Plückthun, Immunol. Rev., 130:151-188 (1992). In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using 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) describes the isolation of human and murine antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain transposition (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies to the construction of very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies. 186 DNA can also be modified, for example, by replacing the coding sequence for the human heavy and light chain constant domains in place of the homologous murine sequences (US Patent No. 4,816,567; Morrison et al., Proc. Nati Academic Sci. USA 81:6851 (1984)) or by covalently attaching to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides are replaced by the constant domains of an antibody, or are replaced by the variable domains of an antigen-combining site of an antibody to create a chimeric bivalent antibody comprising an antigen-combining site having specificity for one antigen and another antigen combining site that has specificity for a different antigen. In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG or IgM. In certain embodiments, the antibody is an IgG monoclonal antibody. Antibody fragments 187 In some embodiments, the antibody is an antibody fragment. Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemistry 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 fragments Fab'-SH can be recovered directly from E. coli and can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled artisan. In other embodiments, the antibody of choice is a single chain Fv (scFv) fragment. See WO 93 / 16185; US Patent No. 5,571,894; and US Patent No. 5,587,458. The antibody fragment may also be a linear antibody, e.g. e.g., as described in US Patent 5,641,870 for example. These linear antibody fragments can be 188 monospecific or bispecific. In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFv, an Fv and a diabody. Polypeptide variants and modifications In certain embodiments, amino acid sequence variants of the proteins herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the protein. Amino acid sequence variants of a protein can be prepared by introducing appropriate modifications in the nucleotide sequence that encodes the protein or by peptide synthesis. Such modifications include, for example, deletions of residues and / or insertions therein and / or substitutions thereof in the amino acid sequences of the protein. Any combination of deletion, insertion, and substitution can be performed to arrive at the final construct, as long as the final construct has the characteristics 189 desired, Polypeptide variant means a polypeptide, e.g., an active polypeptide, as defined herein, that has at least about 80% amino acid sequence identity with a full-length native sequence of the polypeptide, a polypeptide sequence that lacks the signal peptide, an extracellular domain of a polypeptide, with or without the signal peptide. Such polypeptide variants include, for example, polypeptides where one or more amino acid residues are added or deleted at the N or C terminus of the native full-length amino acid sequence. Generally, a polypeptide variant has at least about % amino acid sequence identity, alternatively at least about any of %, 90%, 95%, 96%, 97%, 98% or 99%. of amino acid sequence identity, with a full-length native sequence polypeptide sequence, a polypeptide sequence lacking the signal peptide, an extracellular domain of a polypeptide, with or without the signal peptide. Optionally, the variant polypeptides will have no more than one conservative amino acid substitution compared to the native polypeptide sequence, alternatively no more than about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 190 conservative amino acid substitutions compared to the native polypeptide sequence. The variant polypeptide may be truncated at the N-terminus or C-terminus or may have no internal residues, for example, compared to a full-length natural polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for a desired biological activity. These variant polypeptides with truncations, deletions and insertions can be prepared through any number of conventional techniques. Desired variant polypeptides can be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding a desired variant polypeptide, by polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the nucleic acid fragment are used in the 5' and 3' primers in the PCR. Preferably, the variant polypeptides share at least one biological and / or immunological activity with the natural polypeptide described herein. Amino acid sequence insertions include fusions of amino and / or carboxyl termini varying in length from one residue to polypeptides containing one hundred or more residues, as well as insertions 191 intrasequence of a single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion to the N or C terminus of the antibody to an enzyme or polypeptide that increases the serum half-life of the antibody. For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide are prepared by introducing appropriate nucleotide changes into the nucleic acid of the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues and / or insertions therein and / or substitutions thereof in the amino acid sequences of the polypeptide. Any combination of deletion, insertion, and substitution is performed to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processing of the polypeptide (e.g., antibody), such as by changing the number or position of glycosylation sites. 192 amino acid residue Guidelines for determining what 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 made in the regions of high homology. A useful method for identifying certain residues or regions of the polypeptide (e.g., antibody) that are preferred locations for mutagenesis is called alanine scanning mutagenesis, as described by Cunningham and Wells, Science, 244:1081-1085 (1989). Here a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction. of amino acids with antigen. Those amino acid locations that demonstrate functional sensitivity to substitutions are then refined by introducing more or different variants at or for the substitution sites. Therefore, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation in a 193 Given site, random mutagenesis is carried out on the expressed antibody codon or variants to determine the desired activity. wing or target region and are scanned to Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of greatest interest for substitution mutagenesis include hypervariable regions, but alterations of FR. If such substitutions result in a change in biological activity, then more substantial changes, referred to as exemplary substitutions in Table 5, or as described in more detail below with reference to classes of amino acids, can be introduced and the products can be analyze. Table 5 Residue substitutions Examples of conservative original substitutions Ala (A) Val; Leu; lie Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu 194 Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Leu; Vale; Met; To the; Phe; lie (I) Leu Norleucine Norleucine; lie; Vale; Met; To the; Leu (L) Phe lie Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Trp; Leu; Vale; lie; To the; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; To the; Leu Norleucine Substantial modifications in the biological properties of the polypeptide are achieved by selecting substitutions that differ significantly in their effect of maintaining (a) the backbone structure of the polypeptide in the area of ​​substitution, e.g. 195 as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, Biochemistry, second edition, pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M) (2) non-polar charged: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) acids: Asp (D), Glu (E) (4) basic: Lys (K), Arg (R), His(H) Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acids: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatics: Trp, Tyr, Phe. Non-conservative substitutions will involve the exchange of a member of one of these classes for another 196 class. Any cisterna residues not involved in maintaining the proper conformation of the antibody can also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, one or more cysteine ​​bonds may be added to the polypeptide to improve its stability (particularly in cases where the antibody is an antibody fragment such as an Fv fragment). An example of a substitutive variant involves the substitution of one or more residues of hypervariable regions of an original antibody (e.g., a humanized antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties compared to the original antibody from which they are generated. A convenient way to generate such replacement variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are expressed monovalently from filamentous phage particles as fusions to 197 M13 gene III product packaged within each particle. The phage-displayed variants are then analyzed for their biological activity (e.g., binding affinity), as described herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the target. These contact residues and nearby residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to analysis as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development. Another type of amino acid variant of the polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may comprise residues other than amino acids. For example, the polypeptide may be glycosylated. Said glycosylation can occur naturally during the expression of the polypeptide in the 198 host cell or host organism or may be a deliberate modification arising from human intervention. Toggle means to delete one or more carbohydrate moieties that are found in the polypeptide and / or add one or more glycosylation sites that are not present in the polypeptide. Polypeptide glycosylation is commonly N-linked or 0-linked. N-linked refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-Xserine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. 0-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or 199 more than the tripeptide sequences described above (for glycosylation sites linked to N). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the original antibody sequence (for 0-linked glycosylation sites). The removal of carbohydrate residues present in the polypeptide can be achieved chemically or enzymatically or by mutational substitution of codons that encode amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved through the use of various endo- and exoglycosidases. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of γamino groups of side chains of lysine, arginine and histidine, acetylation of the N-terminal amine and amidation of any of the C-terminal carboxyl group. Chimeric polypeptides 200 The polypeptide described herein may be modified in such a manner to form chimeric molecules comprising the polypeptide fused to another, the heterologous polypeptide, or the amino acid sequence. In some embodiments, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tag polypeptide. Additionally, providing the epitope tag allows the polypeptide to be easily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. Multispecific antibodies In certain embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies with binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for c-met and the other is for 201 any other antigen. In certain embodiments, the bispecific antibodies can bind to two different c-met epitopes. Bispecific antibodies can also be used to localize cytotoxic agents from c-met-expressing cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and buttonhole modification (see, for example, US Patent No. 5,731,168). Multispecific antibodies can also be created by modifying electrostatic targeting effects to create heterodimeric antibody Fe molecules (WO 2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., US Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., <J. Immunol., 148(5):1547-1553 (1992)); using diabody technology to generate antibody fragments 202 bispecific (see, e.g., Hollinger et al., Proc. Nati. Academic Sci. USA, 90:6444-6448 (1993)); and the use of dimers single-chain Fv (sFv) (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of antibodies trispecifics as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). The antibody or fragment herein also includes the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 and WO 2010 / 145793. Also included herein are genetically modified antibodies with three or more functional antigen binding sites, including octopus antibodies, (see, for example, US 2006 / 0025576A1). The antibody or fragment herein also includes a Dual-acting Fab (DAF) comprising an antigen binding site that binds to a first epitope (e.g., on a first antigen) as well as a different epitope (e.g., on the first antigen or on a second different antigen) (see, e.g., US 2008 / 0069820; Bostrom et al. (2009) Science, 5921:1610-1614). 203 Methods for producing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, in which the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Due to the random distribution of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually carried out by affinity chromatography steps, is mostly cumbersome and its products have low yield. Similar procedures are described in WO 93 / 08829 published on May 13, 1993 and in Traunecker et al., EMBOJ., 10: 3655 (1991). According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody and antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably occurs with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3. It is preferred that the first string constant region 204 heavy (CHl) that contains the point necessary for light chain union is present in at least one of the fusions. DNAs encoding immunoglobulin heavy chain and, if desired, immunoglobulin light chain fusions are inserted into separate expression vectors and cotransfected into a suitable host organism. This provides great flexibility to adjust the mutual ratios of the three polypeptide fragments in embodiments in which unequal ratios of the three polypeptide chains in the construct produce optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when expression of at least two polypeptide chains in equal proportions produces high yields or when the proportions are not particularly significant. In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one group and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity). in the other group. This asymmetric structure has been observed to facilitate the separation of the desired bispecific compound from chain combinations. 205 presence of an unwanted immunoglobulin, since the immunoglobulin light chain in only one half of the bispecific molecule provides an easy means of separation. This approach is described in WO 94 / 04690. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Buttonhole button technology According to another approach, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers that are extracted from a recombinant cell culture. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with longer (e.g., tyrosine or tryptophan) side chains (knobs or protrusions). Compensation cavities (eyelets) of identical or similar size to the long side chains are created at the interface of the second antibody molecule by replacing the long amino acid side chains with shorter chains (e.g., alanine or threonine). This provides a mechanism to increase the yield of the heterodimer above that of other end products. 206 unwanted such as homodimers. Buttons and buttonholes are further described herein. The use of buttonholes as a method of producing multispecific antibodies and / or single-arm antibodies and / or immunoadhesins is known in the art. Refer to US Patent No. 5,731,168 issued March 24, 1998 and assigned to Genentech, pub. PCT No. WO2009089004 published on July 16, 2009 and assigned to Amgen and pub. of pat. US No. 20090182127 issued July 16, 2009 and assigned to Novo Nordisk ACE. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9. A brief description is provided herein. A bump refers to at least one chain amino acid lateral that projects from the interface of a first polypeptide and can therefore be placed in a compensation cavity at the adjacent interface (i.e., the interface of a second polypeptide) to stabilize the heteromultimer and thus favor the formation of heteromultimers with respect to the formation of homomultimers, for example. The protrusion may exist at the original interface or may be introduced synthetically (e.g., by altering the nucleic acid that encodes 207 the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is altered to encode the protrusion. To achieve this, the nucleic acid encoding at least one original amino acid residue at the interface of the first polypeptide is replaced with nucleic acid encoding at least one imported amino acid residue that has a larger side chain volume than the original amino acid residue. . It will be noted that there may be more than one corresponding original and imported waste. The upper limit for the number of original residues that are replaced is the total number of residues at the interface of the first polypeptide. The side chain volumes of the various amino acids are shown in the table below. Table 6: Properties of amino acids Amino acid One-letter abbreviation MASSa (daltons) VOLUME13 (Angstrom 3) Accessible surface area0 (Angstrom2) Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225 Asparagine (Asn) N 114.11 117.7 160 Aspartic acid (Asp) D 115.09 111.1 150 208 area of Amino acid One-letter abbreviation VOLUME13 MASSa (daltons) (Angstrom 3) accessible surface0 (Angstrom2) Cysteine ​​(Cys) C 103.14 108.5 135 Glutamine (Gln) Q 128.14 143.9 180 Glutamic acid (Glu) E 129 .12 138.4 190 Glycine (Gly) G 57.06 60.1 75 Histidine (His) H 137.15 153.2 195 Isoleucine (lie) I 113.17 166.7 175 Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18 168.6 200 Methionine (Met) M 131.21 162.9 185 Phenylalanine (Phe) F 147.18 189.9 210 Proline (Pro) P 97.12 122. 7 145 Serine (Ser) S 87.08 89.0 115 Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21 227.8 255 Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0 155 aMolecular weight of the amino acid minus that of water. Values ​​from Handbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical Rubber Publishing Co., 1961. 209 bValues ​​of A.A. Zamyatnin, Prog. Biophys. Mol. Biol. 24:107-123, 1972. cValues ​​of C. Chothia, J. Mol. Biol. 105:1-14, 1975. An accessible surface area is defined as defined in Figures 6-20 of said reference. The preferred import residues for the formation of a protrusion are generally naturally occurring amino acid residues and are preferentially selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). The most preferred are tryptophan and tyrosine. In one embodiment, the original residue for bulge formation has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. Examples of amino acid substitutions in the CH3 domain to form the bulge include, but are not limited to, the T366W substitution. A cavity refers to at least one amino acid side chain that is indented from the interface of a second polypeptide and, therefore, can receive a corresponding protrusion at the adjacent interface of a first polypeptide. The cavity can exist in the original interface or can be introduced synthetically (for example, by altering the nucleic acid that encodes the interface). Normally, the nucleic acid that codes for 210 interface of the second polypeptide is altered to encode the cavity. To achieve this, 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 that has a smaller side chain volume than the original amino acid residue. It will be noted that there may be more than one corresponding original and imported waste. The upper limit for the number of original residues that are replaced is the total number of residues at the interface of the second polypeptide. The side chain volumes of the various amino acids are shown in the table above. The preferred import residues for cavity formation are normally naturally occurring amino acid residues and are preferentially selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine. In one embodiment, the original residue for cavity formation has a considerable side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. Examples of amino acid substitutions in the CH3 domain to generate the cavity include, but are not limited to, the T366S, L368A, and Y407A substitutions. An original amino acid residue is one that is replaced with an import residue that may have a volume 211 side chain smaller or larger than the original residue. The import amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, but is preferably the former. Naturally occurring amino acid residues are the residues encoded by the genetic code and are listed in the table above. Non-natural amino acid residue means a residue not encoded by the genetic code, but which can be covalently linked to one or more adjacent amino acid residues in the polypeptide chain. Examples of amino acid residues of non-natural origin are norleucine, ornithine, norvaline, homoserine and other analogues of amino acid residues such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991), for example. To generate said amino acid residues of non-natural origin, the procedures of Noren et al. can be used. Science 244: 182 (1989) and Ellman et al., supra. In summary, this involves the chemical activation of a suppressor tRNA with a non-naturally occurring amino acid residue, followed by transcription and translation of the RNA in vitro. The methods provided herein involve replacing at least one original amino acid residue, but more than one original residue may be replaced. Typically, no more than the total residues at the interface of the first or second polypeptide will comprise original amino acid residues that are 212 they replace. Typically the original waste preferred for replacement is buried. Buried means that the residue is essentially inaccessible to the solvent. Generally, the import residue is not cysteine ​​to avoid possible oxidation or mispairing of disulfide bonds. The bump is “placeable” in the cavity, meaning that the spatial location of the bump and cavity at the interface of a first polypeptide and a second polypeptide, respectively, and the sizes of the bump and cavity are such that the protrusion can be located in the cavity without significantly disturbing the normal association of the first and second polypeptide at the interface. Since protrusions such as Tyr, Phe, and Trp do not typically extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of a protrusion with a corresponding cavity depends on the realization of a model of the protrusion / protuberance pair. cavity based on a three-dimensional structure such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using various methods accepted in the art. 213 Original or template nucleic acid means the nucleic acid encoding a polypeptide of interest that can be altered (i.e., mutated or genetically modified) to encode a protuberance or cavity. The original or initial nucleic acid may be a naturally occurring nucleic acid or may comprise a nucleic acid subjected to prior modification (eg, a humanized antibody fragment). Altering nucleic acid means that the original nucleic acid mutates by inserting, deleting or replacing at least one codon that encodes an amino acid residue of interest. Typically, a codon encoding an original residue is replaced with a codon encoding an import residue. The techniques to genetically modify DNA in this way are analyzed in Mutagenesis: a Practical Approach, M.J. McPherson, Ed., (IRL Press, Oxford, United Kingdom. (1991), and includes site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis, for example. By mutating an original / template nucleic acid, an original / template polypeptide encoded by the original / template nucleic acid is correspondingly modified. The protrusion or cavity can be introduced at the interface of a first or second polypeptide with means 214 p. e.g., through recombinant techniques, synthetics, in vitro peptide synthesis, the techniques to introduce amino acid residues of non-natural origin previously described, through enzymatic or chemical coupling of peptides or some combination of these techniques. Therefore, the bump or cavity that is introduced is of unnatural or non-natural origin, meaning that it does not exist in nature or in the original polypeptide (e.g., a humanized monoclonal antibody). Generally, the import amino acid residue to form the protrusion has a relatively small number of rotamers (e.g., about 3-6). A rotamer is an energetically favorable conformation of an amino acid side chain. The number of rotamers of the various amino acid residues is analyzed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987). In one embodiment, a first Fe polypeptide and a second Fe polypeptide cross / interact at an interface. In some embodiments where the first and second Fe polypeptides intersect at an interface, the interface of the second Fe polypeptide (sequence) comprises a protrusion (also called a "button") that can be placed in a cavity (also called a "eyelet") at the interface of the first Fe polypeptide (sequence). In a 215 embodiment, the first Fe polypeptide was modified from a template / original polypeptide to encode the cavity or the second Fe polypeptide was modified from a template / original polypeptide to encode the protrusion or both. In one embodiment, the first Fe polypeptide was modified from a template / original polypeptide to encode the cavity and the second Fe polypeptide was modified from a template / original polypeptide to encode the bulge. In one embodiment, the interface of the second polypeptide comprises a protrusion that can be placed in a cavity at the interface of the first Fe polypeptide, wherein the cavity or protrusion or both are introduced into the interface of the first and second Fe polypeptide, respectively. . In some embodiments, wherein the first and second polypeptides Fe intersect at an interface, the interface of the first Fe polypeptide (sequence) comprises a protrusion that can be placed in a cavity at the interface of the second Fe polypeptide (sequence). In one embodiment, the second Fe polypeptide was modified from a template / original polypeptide to encode the cavity or the first Fe polypeptide was modified from a template / original polypeptide to encode the protrusion or both. In one embodiment, the second Fe polypeptide was modified from a template / original polypeptide to encode the cavity and the first Fe polypeptide was modified from a 216 template / original polypeptide to encode the protrusion. In one embodiment, the interface of the first polypeptide comprises a protrusion that can be positioned in a cavity at the interface of the second Fe polypeptide, wherein the protrusion or cavity or both are introduced into the interface of the first and second Fe polypeptide, respectively. . In one embodiment, each of the protuberance and cavity comprises a naturally occurring amino acid residue. In one embodiment, the Fe polypeptide comprising the overhang is generated by replacing an original residue at the interface of a template / original polypeptide with an import residue with a side chain volume greater than that of the original residue. In one embodiment, the Fe polypeptide comprising the protrusion is generated with a method comprising a step in which the polynucleotide encoding an original residue of the interface of said polypeptide is replaced with a polynucleotide encoding an import residue with a volume side chain greater than the original. 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 217 (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, a protrusion is generated by replacing two or more residues in a template / original polypeptide. In one embodiment, the Fe polypeptide comprising a protuberance comprises the replacement of threonine at position 366 with tryptophan, with 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., volume 1 (1991; NIH, Bethesda, MD). In some embodiments, the Fe polypeptide comprising a cavity is generated by replacing an original residue at the interface of a template / original polypeptide with an import residue with a side chain volume less than that of the original residue. For example, the polypeptide Fe comprising the cavity can be generated with a method comprising a step in which the polynucleotide encoding an original residue of the interface of said polypeptide is replaced with a polynucleotide encoding an import residue with a side chain volume less than from the original. 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 218 • 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). A cavity can be generated by replacing one or more original residues of a template / original polypeptide. For example, in one embodiment, the Fe polypeptide comprising a cavity comprises replacing two or more parent amino acids selected from the group consisting of threonine, leucine and tyrosine. In one embodiment, the cavity-comprising Fe polypeptide comprises two or more import residues selected from the group consisting of alanine, serine, threonine and valine. In some embodiments, the Fe polypeptide comprising a cavity comprises the replacement of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine and wherein said original amino acids are replaced with import residues selected from the group consisting of alanine, serine, threonine and valine. In some embodiments, an original amino acid that is replaced is T366, L368 and / or Y407. In one embodiment, the Fe polypeptide comprising a cavity comprises the replacement of threonine at the position 366 with serine, with amino acid numbering according to the EU numbering scheme of Kabat et al. supra. In one embodiment, the Fe polypeptide comprising a cavity 219 φ comprises the replacement of leucine at position 368 with alanine, with amino acid numbering according to the EU numbering scheme of Kabat et al. supra. In one embodiment, the Fe polypeptide comprising a cavity comprises the replacement of tyrosine at position 407 with valine, with amino acid numbering according to the EU numbering scheme of Kabat et al. supra. In one embodiment, the polyFe comprising a cavity comprises two or more amino acid replacements selected from the group consisting of T366S, L368A and Y407V, with amino acid numbering according to the EU numbering scheme of Kabat et al. supra. In some embodiments of these antibody fragments, the Fe polypeptide comprising the protrusion comprises the replacement of threonine at position 366 with tryptophan, with amino acid numbering according to the EU numbering scheme of Kabat et al. supra. In one embodiment, the antibody comprises Fe mutations that constitute "buttons" and "eyelets", as described in WO2005 / 063816. For example, a hole mutation may be one or more of T366A, L368A and / or Y407V in a polypeptide. Fe, and a button mutation can be T366W in an IgGl or IgG4 structure. Those skilled in the art can produce equivalent mutations in other immunoglobulin isotypes. Furthermore, the person skilled in the art will easily understand that it is preferred that the two means 220φ antibodies used for the bispecific are of the same isotype. CrossMab Technology Schaefer et al. (Roche Diagnostics GmbH), describe a method for expressing two light and two heavy chains, which are derived from two existing antibodies, as human bivalent bispecific IgG antibodies without using artificial linkers (PNAS (2011) 108(27): 11187-11192 and US 2009 / 0232811). The method involves exchanging one or more light chain and heavy chain domains within the antigen-binding fragment (Fab) of one half of the bispecific antibody (CrossMab). Correct association of light chains and their cognate heavy chains is achieved by exchanging the heavy chain and light chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This crossover preserves the antigen-binding affinity, but makes the two arms so different that light chain mispairing can no longer occur. See W02009 / 080251, WO2009 / 080252, W02009 / 080253, W02009 / 080254, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 and WO 2010 / 145793, which are incorporated herein by this reference in their entirety. Despite these recent advantages, e.g. e.g. 221 buttons in Due to the development of methodologies such as eyelets (KiH) or CrossMab technology, the expression of multispecies antibodies may still result in the unwanted formation of product-specific impurities specifically associated with their production. These product-specific impurities, for example, may include antibodies (comprising a single heavy chain / light chain pair), % antibodies (comprising a complete antibody lacking a single light chain), or a 5 / 4 antibody byproduct (which comprises an additional heavy or light chain variable domain). BiTE technology Another format, used for bispecific T cell binding component (BiTE) molecules (see, p. e.g. , Wolf et al. (2005) Drug Discovery Today 10:12371244)), is based on single-chain variable fragment (scFv) modules. An scFv consists of the light and heavy chain variable regions of an antibody fused via a flexible linker, which can generally be folded appropriately and so that the regions can be attached to the cognate antigen. A BiTE concatenates two scFvs of different specificities in tandem into a single strand. This configuration rules out the production of molecules with two copies of the same region 222 heavy chain variable. Additionally, the connector configuration is designed to ensure correct pairing of the respective light and heavy chains. Other bispecific antibody formats Strop et al. (Rinat-Pfizer Inc.), describe a method to produce stable bispecific antibodies by expressing and purifying two antibodies of interest separately, and then mixing them under specified redox conditions (J. Mol. Biol. (2012) 420:204 -19). Another heterodimerization domain that has a marked preference for forming heterodimers rather than homodimers can be incorporated into the present multispecific antigen binding proteins. Some illustrative examples include, but are not limited to, e.g. W02007147901 (Kjaargaard et al. - Novo Nordisk: describing ionic interactions); WO 2009089004 (Kannan et al. Amgen: describing electrostatic targeting effects); WO 2010 / 034605 (Christensen et al. Genentech; which describes supercoiled helices). See also, for example, Pack, P. and Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing the leucine zipper or Pack et al., Bio / Technology 11, 1271-1277 (1993) which describes the helix-turn-helix motif. 223 The phrase heteromultimerization domain and heterodimerization domain are used interchangeably herein. In certain embodiments, the multispecific antigen binding protein comprises one or more heterodimerization domains. Zhu et al. (Genentech) genetically engineered mutations at the VL / VH interface of a diabody construct consisting of variable domain antibody fragments completely devoid of constant domains, and generated a heterodimeric diabody (Protein Science (1997) 6:781-788). Similarly, Igawa et al. (Chugai) also genetically engineered mutations at the VL / VH interface of a single-stranded diabody to promote selective expression and inhibit conformational isomerization of the diabody (Protein Engineering, Design & Selection (2010) 23:667-677). US Patent Publication No. 2009 / 0182127 (Novo Nordisk, Inc.) describes the generation of bispecific antibodies by modifying amino acid residues at the Fe interface and at the CH1:CL interface of heavy-light chain pairs which reduce the ability of the light chain of one pair to interact with the heavy chain of the other pair. 224 Antibodies include cross-linked bispecific antibodies or heteroconjugates. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed for targeting unwanted cells of immune system cells (US Patent No. 4,676,980), and for the treatment of HIV infection (WO 91 / 00360, WO 92 / 200373 and EP 03089). Heteroconjugated antibodies can be produced by using any convenient cross-linking method. Suitable cross-linking agents are known in the art and are described in US Patent No. 4,676,980, along with a number of cross-linking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, it is possible to prepare bispecific antibodies through chemical linkages. Brennan et al., Science 229: 81 (1985) describes a procedure in which intact antibodies are cleaved proteolytically to generate F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide. The generated Fab' fragments are then 225 converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for selective immobilization of enzymes. Several techniques have also been described to produce and isolate bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies with leucine zippers have been produced. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then oxidized again to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. Diabody technology, described by Hollinger et al., Proc. Nati. Academic Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (V H) connected to a light chain variable domain (V L) 226 using an eniazer that is too short to allow pairing between the two domains on the same chain. Consequently, the VH and VL domains of one fragment are forced to couple with the complementary VL and VH domains of another fragment, whereby two antigen-binding sites are formed. Another strategy to make bispecific antibody fragments through the use of single-chain Fv (scFv) dimers was also reported. See Gruber et al., J. Immunol. 152:5368 (1994). Reviews of various bispecific and multispecific antibody formats are provided in Klein et al., (2012) mAbs 4:6, 653-663; Spiess et al. (2015) Alternative molecular formats and therapeutic 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. Polynucleotides, vectors, host cells and recombinant methods The multispecific antibodies used in the purification methods described herein can be obtained using methods known in the art, including 227 or recombination methods. The following sections provide guidance on these methods. Polynucleotides "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The polynucleotides encoding the polypeptides can be obtained from any source including, but not limited to, a cDNA library prepared from tissue believed to possess the polypeptide mRNA and express it at a detectable level. Accordingly, polynucleotides encoding the polypeptide can conveniently be obtained from a cDNA library prepared from human tissue. The gene encoding the antibody can also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). For example, the polynucleotide may encode a chain of a complete immunoglobulin molecule, such as a light chain or a heavy chain. a heavy chain 228 or complete includes not only a heavy chain variable (VH) region, but also a heavy chain constant (CH) region, which will typically comprise three constant domains: CH1, CH2 and CH3; and a hinge region. In some situations, the presence of a constant region is desired. Other polypeptides that can be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies (dAb), Fv, scFv, Fab' and F(ab')2, and minibodies. Minibodies are (typically) bivalent antibody fragments from which CH1 and the CKo CL domain have been removed. As minibodies are smaller than conventional antibodies, they should achieve greater tissue penetration in clinical / diagnostic use, but being bivalent they should maintain higher binding affinity than monovalent antibody fragments such as dAbs. Accordingly, unless the context indicates otherwise, the term antibody, as used herein, encompasses not only complete antibody molecules, but also antigen-binding antibody fragments of the type presented above. Preferably, each framework region present in the encoded polypeptide will comprise at least one amino acid substitution with respect to the acceptor framework. 229 © corresponding human. Thus, for example, the framework regions may comprise, in total, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions with respect to the acceptor framework regions. . Suitably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides. The term isolated polynucleotide indicates that the molecule has been removed or separated from its normal or natural environment or has been produced in a way that is not present in its normal or natural environment. In some embodiments, the polynucleotides are purified polynucleotides. The term purified indicates that at least some contaminating molecules or substances have been removed. Suitably, the polynucleotides are substantially purified so that the important polynucleotides constitute the dominant (i.e., most abundant) polynucleotides present in a composition. Polynucleotide expression 230φ The description below relates primarily to the production of polypeptides by culturing cells transformed or transfected with a vector containing polynucleotides encoding polypeptides. Of course, the possibility of employing alternative methods known in the art to prepare polypeptides is contemplated. For example, the appropriate amino acid sequence or portions thereof can be produced by direct synthesis of peptides using solid phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, N.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Automatic synthesis can be achieved, for example, by using a peptide synthesizer Applied Biosystems Peptide Synthesizer (Foster City, Qual.) according to the manufacturer's instructions. It is possible to chemically synthesize various parts of the polypeptide independently and combine them by chemical or enzymatic methods to produce the desired polypeptide. The polynucleotides described herein are inserted into an expression vector for production of the polypeptides. The term "control sequences" refers to DNA sequences necessary to express a sequence. 231 coding binds operatively in a particular host organism. Control sequences include, but are not limited to, promoters (e.g., heterologous or naturally associated promoters), signal sequences, enhancer elements, and transcription termination sequences. A polynucleotide is operably linked when it is in a functional relationship with another polynucleotide sequence. For example, nucleic acids for a presequence or secretory leader are operably linked to the nucleic acids of a polypeptide if they are expressed as a preprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence or a ribosome binding site is operably linked to a coding sequence if it is located so as to facilitate translation. In general, operably linked means that the nucleic acid sequences that are linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, power-ups are not necessarily contiguous. Binding is achieved by binding at convenient restriction sites. In case these sites do not exist, synthetic oligonucleotide linkers or adapters are used according to 232φ conventional practice. For antibodies, the heavy and light chains can be cloned into the same or different expression vectors. Nucleic acid segments encoding immunoglobulin chains are operatively linked to control sequences in the expression vector(s) ensuring expression of immunoglobulin polypeptides. For CrossMabs comprising four different polypeptide chains, four expression cassettes are used. These can be cloned into two to four different expression vectors. Each of the nucleic acid segments encoding the immunoglobulin chains is operatively linked to the control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. If two or more expression cassettes are comprised in the same expression vector, they can be arranged unidirectionally or bidirectionally. Vectors containing the polynucleotide sequences (e.g., variable light and / or variable heavy chain coding sequences and optional expression control sequences) can be transferred into 233 © the host cell by well-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, biolistics, or viral transfection can be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Coid Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For the production of transgenic animals, transgenes can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells and the nuclei of these cells are transferred into enucleated oocytes. Vectors The term vector includes expression vectors and transformation vectors and carrier vectors. The term expression vector means a construct capable of expression in vivo or in vitro. The term transformation vector means a 234 construction capable of being transferred from one entity to another entity; which may be of the same species or may be of a different species. If the construction is capable of being transferred from one species to another; such as from an Escherichia coli plasmid to a bacteria, such as from the genus Bacillus, then the transformation vector is often called a carrier vector. It may even be a construct capable of being transferred from an E plasmid. coli to an Agrobacteríum to a plant. The vectors can be transformed into a suitable host cell as described below to provide expression of a polypeptide. Several vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle or phage. The appropriate nucleic acid sequence can be inserted into the vector by various methods. In general, DNA is inserted into a site or sites of suitable restriction endonuclease using techniques known in the art. The construction of suitable vectors containing one or more of these components employs standard ligation techniques that are known to those skilled in the art. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, 235 ¢) optionally a promoter for the expression of said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes that are known in the art. These expression vectors typically replicate in host organisms as episomes or as an integral part of the host chromosomal DNA. For the production of multispecific antibodies, nucleic acids encoding the multispecific antibody (or an arm of the multispecific antibody, i.e., a heavy chain / light chain pair) are typically isolated and inserted into replicable vectors for cloning, amplification and / or or additional expression. The DNA encoding the antibody is easily isolated and sequenced by conventional procedures (for example, by oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). There are many vectors available. The choice of vector depends in part on the host cell used. It will be noted that constant regions of any isotype can be used for this purpose, including constant regions of IgG, IgM, IgA and IgE and that said constant regions can be obtained from any animal or human species. 236 Host cells The host cell may be a bacteria, yeast or other fungal cell, insect cell, a plant cell or a mammalian cell, for example. A transgenic multicellular host organism that has been genetically engineered to produce a polypeptide can be used. The organism may be, for example, a transgenic mammalian organism (e.g., a transgenic goat or mouse line). Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-positive or Gram-negative organisms, for example, Enterobacteriaceae such as E. coli. Several strains of E. coli are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446 ); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escheríchia, p. e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. e.g. , Salmonella typhimurium, Serratia, p. e.g. , Serratia marcescans and Shigella, as well as Bacílli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P), 237 φ Pseudomonas such as P. aeruginosa and Streptomyces. These examples are illustrative rather than exhaustive. Strain W3110 is a particularly preferred host or primary host because it is a common host strain for fermentations of recombinant polynucleotide products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, the strain W3110 can be modified to effect a genetic mutation in genes encoding endogenous polypeptides in the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the full tonA genotype; E. coli W3110 strain 9E4, which has the complete tonA ptr3 genotype; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan'; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation; and an E. coli strain with a mutant periplasmic protease. Alternatively, in vitro methods for cloning are suitable, eg, PCR or other nucleic acid polymerase reactions. In some embodiments, the prokaryotic host cell (e.g. e.g. , an E. coli host cell) expresses one or more chaperones to facilitate folding and assembly of the antibody. In some embodiments, the chaperone is a £238 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., an E. coli FkpA gene, an E. coli DsbA gene, and / or an E. coli DsbC gene. coli). In these prokaryotic hosts, one can make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). Additionally, any number of various known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system or a phage lambda promoter system. Promoters will typically control expression, optionally with an operator sequence, and will have ribosome binding site sequences and the like, to initiate and complete transcription and translation. Eukaryotic microbes can be used for expression. Eukaryotic microbes, such as filamentous fungi or yeast, are suitable expression or cloning hosts for vectors encoding polypeptides. Saccharomyces cerevisiae is a host microorganism 239 commonly used minor eukaryote. Other hosts include Schizosaccharomyces pombe; Kluyveromyces such as e.g. e.g., K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906), K. thermotolerans and K. marxianus; yarrowia (EP 402.226); Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as e.g. e.g., Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. Niger. Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growing in methanol that is selected from the genus consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. Saccharomyces is a preferred yeast host, with suitable vectors having expression control sequences (e.g. promoters), an origin of replication, termination sequences and the like, if desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Yeast inducible promoters include, among others, promoters for alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. 240 In addition to microorganisms, mammalian tissue cell culture can also be used to express and produce the polypeptides described herein and, in some cases, are preferred (see Winnacker, From Genes to Clones VCH Pubishers, N.Y., N.Y. (1987) For some embodiments, eukaryotic cells may be preferred, as a number of host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art and include CHO cell lines, various lines of Cos cells, HeLa cells, preferably myeloma cell lines, transformed B cells or hybridomas. In some embodiments, the mammalian host cell is a CHO cell. In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines are the monkey kidney line CV1 transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR(CHO or CHO-DP-12 line); cells of Mouse Sertolí; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells 241 (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Generation of zoultie&specific antibodies using prokaryotic host cells Vector construction Polynucleotide sequences encoding polypeptide components of the multispecific antibody to be purified can be obtained according to a method provided herein using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences that encode the polypeptides (such as two or more heavy chains and / or two or more light chains) are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell (such as a host cell). 242j of E. coli). Many vectors that are 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 will depend mainly on the size of the nucleic acids that are inserted into the vector and the particular host cell that is 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 particular host cell in which it is located. Vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the nucleic acid inserted heterologous and a transcription termination sequence. In general, plasmid vectors containing a replicon and control sequences that are derived from a species compatible with the host cell are used in connection with these hosts. The vector commonly contains a replication site as well as marker sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes that encode 243 resistance to ampicillin (Amp) and tetracycline (Tet) and therefore provides a simple means of identifying transformed cells. pBR322, its derivatives and other microbial plasmids or bacteriophages may also contain, or may be modified to contain, promoters that can be used by the microbial organism to express endogenous proteins. Examples of pBR322 derivatives used to express particular antibodies are described in detail in Carter et al., US Patent No. 5,648,237. Furthermore, phage vectors containing a replicon and control sequences compatible with the host microorganism can be used as transformation vectors in connection with these hosts. For example, a bacteriophage such as GEM™-11 can be used in the production of a recombinant vector that can be used to transform susceptible host cells, such as E. coli LE392. An expression vector may comprise two or more promoter-cistron pairs, each encoding polypeptide components. A promoter is a non-translating regulatory sequence located upstream (5') of a cistron that modulates its expression. Prokaryotic promoters are typically divided into two classes, 244 inducible and constitutive. An inducible promoter is a promoter that initiates higher levels of cistron transcription under its control in response to changes in culture conditions, e.g. e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by various possible host cells are known. The selected promoter can be operably linked to a cistron DNA encoding the light or heavy chain by removing the promoter from the parent DNA by restriction enzyme digestion and inserting the isolated promoter sequence into a vector. of native promoter as various heterologous promoters to direct the amplification and / or expression of target genes. In some embodiments, heterologous promoters are used as they generally allow for greater transcription and higher yields of the expressed target gene compared to the natural target polypeptide promoter. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the lactose and lactamase promoter systems, a tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters functional in bacteria (such as other promoters) are also suitable. 245 known bacterial or phage). Their nucleotide sequences are published, allowing experts to operatively join them to cistrons encoding the target light and heavy chain (Siebenlist et al. (1980) Cell 20: 269) using linkers or adapters to provide any point of necessary restriction. The translation initiation region (TIR) ​​is a major determinant of the overall translation level of a protein. The TIR includes the polynucleotide encoding the signal sequence and extends from immediately upstream of the Shine-Dalgarno sequence to approximately twenty nucleotides downstream of the start codon. Generally, the vector comprises a TIR and TIRs and variant TIRs are known in the art, as are methods for generating TIRs. A series of nucleic acid sequence variants can be created with a spectrum of translational resistances, thereby providing a convenient means by which to tune this factor for optimal secretion of a variety of different polypeptides. The use of a reporter gene fused to these variants, such as PhoA, provides a method to quantify the relative translation resistances of different translation initiation regions. Variant or mutant TIRs can be provided as a reference plasmid vector, thereby providing a set of plasmids in which 246 can insert a gene of interest and its expression can be measured, to establish an optimal spectrum of translation resistances for maximum expression of a mature polypeptide. Variant IRRs are described in USP 8,241,901. In one aspect, each cistron in the recombinant vector comprises a secretory signal sequence component that directs translocation of the expressed polypeptides across the membrane. In general, the signal sequence may be a component of the vector or may be a part of the target polypeptide DNA that is inserted into the vector. The signaling sequence that is selected for the purposes of the present invention must be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In prokaryotic host cells that do not recognize and process the natural signal sequences of heterologous polypeptides, the signal sequence is replaced with a prokaryotic signal sequence. Such sequences are known in the art. Likewise, the vector may comprise a signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Lpp or heat stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. 247 vectors In one aspect, one or more polynucleotides (e.g., expression) 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 the light chain and heavy chain of the antibody. In some embodiments, one or more polynucleotides (e.g., expression vectors) collectively encode an antibody of one arm. In one embodiment, a single polynucleotide encodes (a) the light and heavy chain of the one-arm antibody and (b) the Fe polypeptide. In one embodiment, a single polynucleotide encodes the light and heavy chain of the one-arm antibody and a polynucleotide. separate encodes the polypeptide Fe. In one embodiment, the separate polynucleotides encode the light chain component of the one-arm antibody, the heavy chain component of the one-arm antibody, and the Fe polypeptide, respectively. The production of an antibody from an arm is described, for example, in W02005063816. Prokaryotic host cells suitable for expressing antibodies include Archaebacteria and Eubacteria, such as Gram-negative and Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g. E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, 248 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, volume 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC repository no. 27,325) and derivatives thereof, including strain 33D3 with genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kanR (US Patent No. 5,639,635) and strains 63C1 and 64B4. In some embodiments, the E. coli strain is a derivative of W3110 designated 62A7 (AfhuA (AtonA) ptr3, laclq, lacL8, ompTA(nmpc-fepE) AdegP ilvG repaired). Other strains and derivatives thereof are also suitable, such as E. coli 294 (ATCC 31,446), E. coli Β, E. coli λ 1776 (ATCC 31,537) and E. coli RV308(ATCC 31.608). These examples are illustrative rather than exhaustive. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select appropriate bacteria taking into account the ability of the replicon to replicate in the cells of a bacteria. For example, the species E. coli, 249 φ Serratia or Salmonella can be appropriately used as hosts when known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to provide the replicon. Typically, host cells should secrete minimal amounts of proteolytic enzymes and additional protease inhibitors may desirably be incorporated into the cell culture. To improve the production yield and quality of polypeptides in bacterial cultures, bacterial cells can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, the bacterial host cell may comprise additional vectors that express chaperone proteins, such as FkpA and Dsb proteins (DsbB, DsbC, DsbD and / or DsbG), which They can be used for co-transformation of host prokaryotic cells. Chaperone proteins have been shown to facilitate the solubility and proper folding of heterologous proteins produced in bacterial host cells. Multispecific antibody production Host cells are transformed with the above expression vectors and cultured in nutrient media. 250 conventional modified as appropriate to induce promoters, select transformers or amplify genes that encode the desired sequences. Transformation means introducing DNA into the prokaryotic host so that the DNA replicates, either as an extrachromosomal element or via a chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Generally, calcium treatment with calcium chloride is used for bacterial cells that contain substantial cell wall barriers. Another transformation method uses polyethylene glycol / DMSO. Another technique that is used is electroporation. Prokaryotic cells used to produce the purified polypeptides according to the methods provided herein are cultured in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria broth (LB), plus necessary nutritional supplements. In some embodiments, the medium also contains a selection agent, selected based on the construction of the expression vector, to allow the growth of 251 the expression vector of form £ prokaryotic cells with selective. For example, ampicillin is added to the medium to grow cells that express the ampicillin resistance gene. Any necessary supplements may also be included in addition to carbon, nitrogen, and inorganic phosphate sources at appropriate concentrations alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol. Prokaryotic host cells are grown at appropriate temperatures. For growth of E. coli, for example, the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C. °C. The pH of the medium can be any pH ranging from about 5 to about 9, mainly depending on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4 and, more preferably, about 7.0. If an inducible promoter is used in the vector 252 expression, protein expression is induced under conditions suitable for promoter activation. In one aspect, PhoA promoters are used to control transcription of the polypeptides. Therefore, transformed host cells are cultured in phosphate-limited medium for induction. Preferably, the phosphate-limited medium is C.R.A.P medium. (refer, e.g., to Simmons et al., J. Immunol. Methods (2002), 263:133-147) or means described in W02002 / 061090. As is known in the art, a variety of inductors can be used, depending on the vector construction used. In one embodiment, the expressed polypeptides to be purified using methods provided herein are secreted into and extracted from the periplasm of the host cells. Protein extraction typically involves disrupting the microorganism, usually through means such as osmotic shock, ultrasound homogenization, or lysis. Once the cells are disturbed, cell pellets or whole cells can be removed by centrifugation or filtration. The proteins can be further purified, for example, by resin affinity chromatography. Alternatively, proteins can be transported to the culture medium and isolated therein. The cells can be 253 remove from the culture and filter the culture supernatant and concentrate for further purification of the proteins produced. Expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay. In one aspect, the production of antibodies is carried out in large quantities through a fermentation process. There are various large-scale batch fermentation procedures for production of recombinant polypeptides. Large-scale fermentations have a capacity of at least 1000 liters, preferably a capacity of around 1,000 to 100,000 liters. These fermenters use agitator propellants to distribute oxygen and nutrients, especially glucose (the preferred energy / carbon source). Small scale fermentation broadly refers to fermentation in a fermenter with a volumetric capacity of no more than about 100 liters and which can range from about 1 liter to about 100 liters. In a fermentation process, the induction of protein expression typically begins once the cells are 254 grown under suitable conditions to a desired density, e.g. e.g. , an OD550 of around 180-220, when the cells are in the early static phase. As is known in the art and described above, various inductors can be used, depending on the vector construction used. Cells can be cultured for shorter periods before induction. Cells are typically induced for about 12-50 hours, although longer or shorter induction times can be used. To improve the production yield and quality of polypeptides, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors that express chaperone proteins, such as FkpA, DsbA and / or DsbC for cotransformation of host prokaryotic cells. Chaperone proteins have been shown to facilitate the solubility and proper folding of heterologous proteins produced in bacterial host cells. In some embodiments, FkpA, DsbA and / or DsbC are expressed in the bacterial host cell. To minimize proteolysis of heterologous proteins 255 expressed (especially those that are proteolytically sensitive), certain host strains deficient in proteolytic enzymes can be used. For example, host cell strains can be modified to make one or more genetic mutations in genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations of these. Some strains of E. coli with protease deficiency can be purchased and are described, for example, in Joly et al., (1998), supra; Georgiou et al., US patent #5,264,365; Georgiou et al., US Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). In one embodiment, E. coli strains deficient in proteolytic enzymes and transformed with plasmids expressing one or more chaperone proteins are used as host cells in the expression system. Generation of multispecific antibodies using eukaryotic host cells Signal sequence component A vector for use in a eukaryotic host cell 256 it may optionally contain a signal sequence or other polypeptide with a specific cleavage site at the N terminus of the mature protein or polypeptide of interest. Preferably, the selected heterologous signal sequence is one recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signaling sequences are available, as well as viral secretion leaders, for example, the herpes simplex signal gD. The DNA for said precursor region is linked in frame to the DNA encoding the desired heteromultimeric protein (e.g., antibodies). Replication origin In general, an origin of replication component is not necessary for mammalian expression vectors. For example, the SV40 origin can usually be used, but only because it contains the early promoter. Selection gene co-opponent The expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode 257 proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies where relevant, or (c) supply critical nutrients not available from complex media. An example of a targeting scheme uses a drug to stop the growth of a host cell. These cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such dominant ...

Claims

1. A method for purifying a multispecific antibody from a composition comprising the multispecific antibody and an impurity, wherein the multispecific antibody comprises multiple arms, each arm comprising a VH / VL unit, wherein each arm of the multispecific antibody is produced separately, characterized in that the method comprises the sequential steps of a) subjecting each arm of the multispecific antibody to capture chromatography to produce capture eluates for each arm of the multispecific antibody, b) forming a mixture comprising 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 generate a first mixed-mode eluate; d) subjecting the first mixed-mode eluate to a second mixed-mode chromatography to generate a second mixed-mode eluate; e) collecting a fraction comprising the multispecific antibody, wherein the method reduces the amount of a specific impurity from the product of the composition, wherein the specific impurity is one or more unpaired antibody arms and antibody homodimers;wherein a) the first mixed-mode chromatography is a cation-exchange mixed-mode chromatography and the second mixed-mode chromatography is an anion-exchange mixed-mode chromatography; or wherein b) the first mixed-mode chromatography is an anion-exchange mixed-mode chromatography and the second mixed-mode chromatography is a cation-exchange mixed-mode chromatography; wherein the anion-exchange mixed-mode chromatography comprises a quaternary amine and a hydrophobic moiety and the cation-exchange mixed-mode chromatography comprises an N-benzyl-N-methylethanolamine, wherein the multispecific antibody is a bispecific antibody; wherein the bispecific antibody is a button-in-button (KiH) bispecific antibody. Five claims follow.