METHODS FOR ANALYZING INCORRECT CHAIN ​​MATCHING IN MULTISPECIFIC BINDING PROTEINS

Abstract

This document provides methods for monitoring the production of a multispecific binding protein and one or more incorrect species by a cell line, as well as related production and screening methods. In some embodiments, the methods comprise the detection of a quantity (e.g., a relative quantity) of a multispecific binding protein and one or more incorrect species in a cell culture medium using ultra-performance size-exclusion liquid chromatography-mass spectrometry (SE-UPLC-MS). In some embodiments, the multispecific binding protein is a multispecific antibody, an antibody fragment, or an Fc fusion protein.

Description

METHODS FOR ANALYZING INCORRECT CHAIN ​​MATCHING IN MULTI-SPECIFIC BINDING PROTEINS Cross-reference to related applications This application claims priority over U.S. Provisional Application No. 62 / 926,313, filed on October 25, 2019, and EP Application No. EP20315271.5, filed on May 28, 2020, the disclosures of which are incorporated herein by reference in their entirety. Presentation of the sequence list in an ASCII text file The contents of the following ASCII text file presentation are incorporated herein by reference in their entirety: a computer-readable form (CRF) of the sequence list (filename: 183952032840SEQLIST.TXT, date of registration: May 29, 2020, size: 2 KB). Field of invention The disclosure refers to methods for monitoring the production of a multispecific binding protein and one or more mismatched species by a host cell, as well as related production and screening methods. The disclosure also refers to methods for monitoring the production of an antibody or antibody derivative and one or more weight variant species by a host cell, as well as related production and screening methods. Background of the invention Multispecific antibodies that bind two or more different epitopes on the same or different antigens have become attractive therapeutic options in immuno-oncology in recent years (Baeuerle, PA & Reinhardt, C., Cancer phies 2009, 69 (12), 4941-4). Multitargeting has been used for various purposes, such as achieving enhanced drug specificity or mimicking natural ligands in signaling pathways, e.g., in hemophilia treatments through simultaneous binding to pairs of receptors on the surface of the same cell (Kitazawa, T. et al. Nat Med 2012, 18 (10), 1570-4). A prominent application is the T cell docking (TCE) concept where one arm of the molecule activates T cells via binding to the CD3 / CD28 receptor and the other arm targets a tumor antigen to kill tumors (Krah, S. et al. N Biotechnol 2017, 39 (Pt B), 167-173; Correnti, CE et al. Leukemia 2018, 32(5), 1239-1243). Trispecific IgG-like antibodies (tsAbs) are composed of two different heavy chains and two different light chains, commonly expressed in a single host cell followed by an intracellular chain assembly. While this production method eliminates the need for two separate cell lines and purification processes, it can generate unwanted, incorrectly paired species in addition to the desired tsAb. In the absence of a rational design and with random subunit association, the theoretical yield of correctly paired species is only 12.5% ​​(Figure 1B). Forced heterodimerization of the heavy chains has been LPOfrnn / zznz / E / YiAi has been successfully achieved through protein engineering approaches, such as knob-in-hole design (Ridgway, JB et al. Protein Eng 1996, 9 (7), 617-21). However, cognate pairing of the light chains with the correct heavy chains remains a major challenge in tsAb production. As such, methods for monitoring or analyzing the production of a multispecific binding protein and one or more mismatched species remain necessary. Furthermore, methods for monitoring or analyzing the production of antibodies or antibody derivatives comprising multiple species that vary in molecular weight (e.g., with different chemical modifications, such as chemically modified cysteine ​​or other residues, or with different glycoforms) remain necessary. All references cited herein, including patent applications, patent publications, and UnIProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference. Brief description of the invention To meet these and other needs, this document provides methods for monitoring the production of a multispecific binding protein and one or more mismatched species (e.g., by a cell line). These methods provide, among other things, a high-throughput analytical platform based on the denaturation of intact SEC-LC-MS for the identification and relative quantification of mismatched chain and other IgG-related species. Advantageously, these methods allow intact MS analysis of mAb-related species (e.g., multispecific binding proteins, antibodies, Fe fusion proteins, antibody fragments, etc.) directly from a clarified cell harvest, avoiding costly and time-consuming purification (e.g., protein A affinity chromatography) and buffer exchange steps.As such, these methods can be used to rapidly screen a large number of clones for potential production cell lines. In some embodiments, methods are provided herein for monitoring the production of a multispecific binding protein and one or more mismatched species, comprising the methods: detecting, by mass spectrometry and ultra-performance size exclusion liquid chromatography (SE-UPLC-MS), an amount of a multispecific binding protein and one or more mismatched species in a cell culture medium comprising the multispecific binding protein and one or more mismatched species.In some embodiments, the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, and one or more mismatched species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the binding protein. Multispecies LQOfrnn / zznz / E / YiAi. In some embodiments, the multispecific binding protein is a multispecific antibody comprising a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain. In some embodiments, the multispecific binding protein is a bispecific antibody, an antibody fragment, or an Fe fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody, an antibody fragment, or an Fe fusion protein.In some embodiments, the one or more mismatched species comprises one or more of: an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody light chains; and an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains forming the three antigen-binding sites, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula: Vl2-Li-Vli-L2-Cl [I] and a second polypeptide chain of the binding protein comprises a structure represented by the formula: VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [II] and a third polypeptide chain of the binding protein comprises a structure represented by the formula: Vn3-Cm-b¡sagra-Cn2-CH3 [III] and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula: VL3-Cl [IV] where: V li is a first immunoglobulin light chain variable domain; V l2 is a second immunoglobulin light chain variable domain; V l3 is a third immunoglobulin light chain variable domain; Vm is a first immunoglobulin heavy chain variable domain; V n2 is a second immunoglobulin heavy chain variable domain; V n3 is a third immunoglobulin heavy chain variable domain; Cl is an immunoglobulin light chain constant domain; Chi is a Chi immunoglobulin heavy chain constant domain; LPOfrnn / zznz / E / YiAi Ch2 is a constant domain of immunoglobulin heavy chain Chz; Chs is a constant domain of immunoglobulin Chs heavy chain; hinge is an immunoglobulin hinge region that connects the Chi and Ch2 domains; and Li, L2, L3 and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light-heavy chain pair, wherein Vw and Vli form a first antigen-binding site, wherein Vh2 and Vi_2 form a second antigen-binding site, and wherein Vhs and Vl3 form a third antigen-binding site. In some embodiments, the one or more mismatched species comprise one or more of: an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula IV. In some embodiments, the detection of the quantity of the multispecific binding protein and of one or more mismatched species comprises the deconvolution of one or more MS spectra obtained by MS. In some embodiments, a relative quantity of the multispecific binding protein is detected compared to the quantity of one or more mismatched species. In some embodiments, a relative quantity of the multispecific binding protein is detected compared to the quantity of one or more mismatched individual species. In some embodiments, a relative quantity of the multispecific binding protein is detected compared to the total quantity of mismatched species. In some embodiments, the methods further comprise, prior to detection, providing or obtaining (e.g.The methods involve separating the cell culture medium from a cell line that produces the multispecific binding protein and one or more mismatched species. In some embodiments, the methods further comprise, prior to detection, separating the cell culture medium from a cell line that produces the multispecific binding protein and one or more mismatched species. In some embodiments, prior to detection, the cell culture medium is separated from the cell line by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography. In some embodiments, the MS is intact. In some embodiments, the MS is quadrupole time-of-flight (QToF) MS.In some methods, SE-UPLC is used to denature SE-UPLC. In some methods, SE-UPLC is directly coupled to MS. In some methods, the further components include, prior to SE-UPLC-MS, contacting the multispecific binding protein and one or more mismatched species with a protease. In some methods, the protease is IdeS or IdeZ. In some methods, SE-UPLC is performed with an initial flow rate of less than approximately 0.4 mL / min. In some methods, SE-UPLC is performed with a flow rate of [missing information]. LPOfrnn / zznz / E / YiAi initial flow rate of approximately 0.1 mL / min. In some modes, SE-UPLC is performed at a flow rate of approximately 0.1 mL / min for the first 25 minutes, followed by a flow rate of approximately 0.4 mL / min (e.g., for minutes 25–33). In some modes, SE-UPLC is performed by isocratic elution with a mobile phase. In some modes, the mobile phase comprises a solution of acetonitrile:water 30:70. In some modes, the mobile phase comprises formic acid and trifluoroacetic acid (TFA). In some modes, the mobile phase comprises approximately 0.05% formic acid and approximately 0.05% trifluoroacetic acid (TFA). In some modes, detection of the amount of multispecific binding protein and one or more mispaired species is achieved in approximately 33 minutes or less. In some forms, the procedure takes approximately 33 minutes or less.In some modalities, MS is able to resolve a mass difference of approximately 300 Da between the multispecific binding protein and one or more mismatched species, or between two mismatched species. In some modalities, MS is able to resolve a mass difference of approximately 162 Da between the multispecific binding protein or mismatched species and one or more glycoforms. In some modality, the cell line is a mammalian cell line. In some modality, the cell line is a Chinese hamster ovary (CHO) cell line. In some modality, prior to screening, the cell line is cultured with the cell culture medium in continuous cell culture, e.g., in a stirred-tank bioreactor. In some modality, prior to screening, the cell line is cultured with the cell culture medium in batch cell culture. In some embodiments, methods for producing a multispecific binding protein are provided herein, comprising the methods: (a) growing a cell line comprising one or more polynucleotides encoding the multispecific binding protein in a cell culture medium under conditions suitable for the production of the multispecific binding protein and one or more mismatched species by the cell line; (b) separating, from the cell line, the cell culture medium comprising the multispecific binding protein and one or more mismatched species; (c) detecting an amount of the multispecific binding protein and one or more mismatched species in the cell culture medium by size-exclusion ultra-performance liquid chromatography-mass spectrometry (SE-UPLC-MS);and (d) removing at least a portion of one or more of the mismatched species from the multispecific binding protein produced by the cell line, or determining one or both of the quality and purity of the multispecific binding protein produced by the cell line. In some embodiments, the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain. In some embodiments, the one or more mismatched species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association different from that of the multispecific binding protein. In some forms, the multispecific binding protein is a multispecific antibody comprising a first antibody heavy chain, a first antibody light chain, a LPOfrnn / zznz / E / YiAi second antibody heavy chain different from the first antibody heavy chain and a second antibody light chain different from the first antibody light chain. In some modalities, the multispecific binding protein is a bispecific antibody or Fe fusion protein. In some modalities, the multispecific binding protein is a trispecific antibody or Fe fusion protein.In some embodiments, the one or more mismatched species comprises one or more of: an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody light chains; and an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains forming the three antigen-binding sites, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula: Vl2-Li-Vli-L2-Cl[I] and a second polypeptide chain of the binding protein comprises a structure represented by the formula: Vhi -L3-V H2-L4-CH1 -hinge-CH2-CH3[II] and a third polypeptide chain of the binding protein comprises a structure represented by the formula: VH3-Cm-hinge-CH2-CH3[III] and a fourth polypeptide chain of the linker protein comprises a structure represented by the formula: Vls-Cl[IV] where: V li is a first immunoglobulin light chain variable domain; V i_2 is a second immunoglobulin light chain variable domain; Vls is a third variable domain of immunoglobulin light chain; Vm is a first immunoglobulin heavy chain variable domain; V n2 is a second immunoglobulin heavy chain variable domain; Vhs is a third variable domain of immunoglobulin heavy chain; Cl is an immunoglobulin light chain constant domain; Chi is a Chi immunoglobulin heavy chain constant domain; Ch2 is a Ch2 immunoglobulin heavy chain constant domain; Chs is a constant domain of immunoglobulin Chs heavy chain; hinge is an immunoglobulin hinge region that connects the Chi and Ch2 domains; and L1, L2, L3 and L4 are amino acid linkers; LPOfrnn / zznz / E / YiAi wherein the polypeptide of formula I and the polypeptide of formula II form a crossed light-heavy chain pair, wherein Vhi and Vli form a first antigen-binding site, wherein Vh2 and Vl2 form a second antigen-binding site, and wherein Vh3 and Vl3 form a third antigen-binding site. In some embodiments, the one or more mismatched species comprise one or more of: an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula IV. In some methods, the detection of the amount of multispecific binding protein and one or more mismatched species involves the deconvolution of one or more MS spectra obtained by MS. In some methods, the detection of multispecific binding protein and one or more mismatched species involves the evaluation of the overall titer of multispecific binding protein produced by the cell line. In some methods, the detection of multispecific binding protein and one or more mismatched species involves the evaluation of the overall titer of the mismatched species produced by the cell line. In some methods, a relative amount of multispecific binding protein is detected compared to the one or more mismatched species.In some modalities, a relative amount of the multispecific binding protein is detected compared to an amount of one or more mismatched individual species. In some modalities, a relative amount of the multispecific binding protein is detected compared to a total amount of mismatched species. In some modalities, the cell culture medium is separated from the cell line by centrifugation. In some modalities, the cell culture medium is subjected to SE-UPLC in (c) without prior chromatographic separation. In some modalities, the cell culture medium is subjected to SE-UPLC in (c) without prior protein A affinity chromatography. In some modalities, the MS is intact. In some modalities, the MS is quadrupole time-of-flight (QToF) MS. In some modalities, the SE-UPLC is denaturing SE-UPLC. In some modalities, the SE-UPLC is directly coupled to the MS.In some embodiments, the methods further comprise, after culturing the cell line and before detection, contacting the multispecific binding protein and one or more mismatched species with a protease. In some embodiments, the protease is IdeS or IdeZ. In some embodiments, SE-UPLC is performed with an initial flow rate of less than approximately 0.4 mL / min. In some embodiments, SE-UPLC is performed with an initial flow rate of approximately 0.1 mL / min. In some embodiments, SE-UPLC is performed with a flow rate of approximately 0.1 mL / min for the first 25 minutes, followed by a flow rate of approximately 0.4 mL / min. In some embodiments, SE-UPLC is performed using isocratic elution with a mobile phase. In some embodiments, the mobile phase comprises a solution comprising acetonitrile:water 30:70. In some embodiments, the mobile phase comprises formic acid and trifluoroacetic acid (TFA). In LPOfrnn / zznz / E / YiAi modalities, the mobile phase comprises approximately 0.05% formic acid and approximately 0.05% trifluoroacetic acid (TFA). In some modalities, detection is performed in approximately 33 minutes or less. In some modalities, the method is performed in approximately 33 minutes or less. In some modalities, MS is able to resolve a mass difference of approximately 300 Da between the multispecific binding protein and one or more mismatched species, or between two mismatched species. In some modalities, MS is able to resolve a mass difference of approximately 162 Da between the multispecific binding protein or mismatched species and one of several glycoforms. In some embodiments, the cell line is a mammalian cell line. In some embodiments, the cell line is a Chinese hamster ovary (CHO) cell line. In some embodiments, the cell line is grown in continuous cell culture (e.g., in a stirred-tank bioreactor). In some embodiments, the cell line is grown in batch cell culture. In some embodiments, methods are provided herein for screening a plurality of cell lines for the production of a multispecific binding protein, comprising the methods: detecting an amount of a multispecific binding protein produced by a first cell line of the plurality according to the method of any one of the above embodiments; and detecting an amount of the multispecific binding protein produced by a second cell line of the plurality according to the method of any one of the above embodiments. In some embodiments, the methods further comprise: comparing the amount of multispecific binding protein produced by the first cell line with the amount of multispecific binding protein produced by the second cell line; and, based on the comparison, selecting the cell line that produced the greatest amount of multispecific binding protein. In some embodiments, the methods further comprise: detecting an amount of one or more incorrectly paired species produced by the first cell line according to the method of any one of the above embodiments; and detecting an amount of one or more incorrectly paired species produced by the second cell line according to the method of any one of the above embodiments.In some embodiments, the methods further comprise: after detecting the quantity of one or more incorrectly paired species produced by the first and second cell lines; comparing the quantity of one or more incorrectly paired species produced by the first cell line with the quantity of one or more incorrectly paired species produced by the second cell line; and, based on the comparison, selecting the cell line that produced the highest proportion of multispecific binding protein to one or more incorrectly paired species. In some embodiments, the cell line is selected based on a higher proportion of multispecific binding protein to the quantity of one or more individual incorrectly paired species. In some embodiments, the cell line is selected based on a higher proportion of multispecific binding protein to the total quantity of incorrectly paired species. In some embodiments, methods are provided herein for monitoring the production of an antibody or antibody derivative and one or more weight variant species, LPOfrnn / zznz / E / YiAi comprising methods: detecting, by ultra-performance size-exclusion liquid chromatography and mass spectrometry (SE-UPLC-MS), a quantity of an antibody or antibody derivative and one or more weight variant species in a cell culture medium comprising the antibody or antibody derivative and one or more weight variant species, wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight. In some embodiments, methods for producing an antibody or antibody derivative are provided herein, comprising methods: (a) growing a cell line comprising one or more polynucleotides encoding an antibody or antibody derivative in a cell culture medium under conditions suitable for the production of the antibody or antibody derivative and one or more weight variant species by the cell line;(b) separating, from the cell line, the cell culture medium comprising the antibody or antibody derivative and one or more weight variant species; (c) detecting a quantity of the antibody or antibody derivative and one or more weight variant species in the cell culture medium by size exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS); and (d) removing at least a portion of one or more of the weight variant species from the antibody or antibody derivative produced by the cell line, or determining one or both of the quality and purity of the antibody or antibody derivative produced by the cell line. In some embodiments, this document provides methods for screening a plurality of cell lines for the production of an antibody or antibody derivative, comprising the methods of: detecting an amount of an antibody or antibody derivative produced by a first cell line of the plurality according to the method of any one of the above embodiments; and detecting an amount of the antibody or antibody derivative produced by a second cell line of the plurality according to the method of any one of the above embodiments. In some embodiments, the methods further comprise: comparing the amount of antibody or antibody derivative produced by the first cell line with the amount of antibody or antibody derivative produced by the second cell line; and, based on the comparison, selecting the cell line that produced the greatest amount of the antibody or antibody derivative.In some modalities, the methods further comprise: detecting a quantity of one or more species of weight variants produced by the first cell line according to the method of any one of the above modalities; and detecting a quantity of one or more species of weight variants produced by the second cell line according to the method of any one of the above modalities.In some embodiments, the methods further comprise: after detecting the quantity of one or more weight variant species produced by the first and second cell lines; comparing the quantity of one or more weight variant species produced by the first cell line with the quantity of one or more weight variant species produced by the second cell line; and based on the comparison, selecting the cell line that produced the highest proportion of antibodies or antibody derivatives against one or more weight variant species, or selecting the cell line that produced the highest relative proportion of a single weight variant species in relation to the total quantity of antibodies or antibody derivatives and one or more weight variant species produced. In some embodiments, the quantity of an antibody or antibody derivative and / or one or more... LPOfrnn / zznz / E / YiAi weight variant species according to any of the modalities contained herein refers to a relative quantity (e.g., compared to one or more different species, or compared to a total quantity of antibodies / species produced). In some embodiments, the antibody or antibody derivative comprises a cysteine ​​residue at position 293 (Cys293). In some embodiments, the antibody or antibody derivative and one or more weight variant species comprise species with a free cysteine ​​(e.g., not disulfide bonded to another cysteine ​​of the antibody or antibody derivative) that has been cysteinylated, N-acetylcysteinylated, or glutathionylated. In some embodiments, the antibody or antibody derivative is not N-glycosylated (e.g., in the Fe region of the antibody). In some embodiments, the antibody or antibody derivative comprises a mutation in the Fe region that reduces or eliminates N-glycosylation. In some embodiments, the antibody or antibody derivative comprises an N300A mutation (EU index). In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g., in the Fe region of the antibody), and the method further comprises (e.g.)(prior to SE-UPLC-MS), the removal of the antibody N-glycosylation. In some embodiments, the removal of the antibody N-glycosylation comprises treating the antibody with a peptide:N-glucosidase enzyme (e.g., PNGase F). In some embodiments, the detection of the quantity of the antibody or antibody derivative and one or more weight-variant species comprises the deconvolution of one or more MS spectra obtained by MS. In some embodiments, the methods further comprise, prior to detection, providing or obtaining (e.g., from a cell line that produces the antibody or antibody derivative and one or more weight-variant species) a cell culture medium comprising the antibody or antibody derivative and one or more weight-variant species.In some embodiments, the methods further comprise, prior to detection, separating the cell culture medium from a cell line that produces the antibody or antibody derivative and one or more weight variant species. In some embodiments, prior to detection, the cell culture medium is separated from the cell line by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography. In some embodiments, the MS is intact MS. In some embodiments, the MS is quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is denatured SE-UPLC. In some embodiments, the SE-UPLC is directly coupled to the MS.In some embodiments, one or more weight variant species represent antibody or antibody derivative species comprising a chemically modified cysteine ​​residue. In some embodiments, the antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 119 Da. In some embodiments, MS is able to resolve a mass difference between cysteinylated (mass shift of 119 Da), N-acetylcysteinylated (mass shift of 161 Da), and glutathionylated (mass shift of 305 Da) species. The antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 162 Da. In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g., in the Fe region of the antibody), and one or more weight variant species represent glycoforms of the antibody. LPOfrnn / zznz / E / YiAi antibody or antibody derivative. In some modalities, MS is able to resolve a mass difference of approximately 162 Da between the antibody or antibody derivative and one or more of the weight variant species representing glycoforms of the antibody or antibody derivative. In some modalities, the antibody or antibody derivative is a multispecific antibody. It should be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to a person skilled in the art. These and other embodiments of the invention are described in more detail in the detailed description that follows. Brief description of the figures Figure 1A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites for three target proteins: CD28, CD3, and HER2. A first pair of polypeptides possesses dual variable domains with a cross orientation (VH1-VH2 and VL2-VL1), forming two antigen-binding sites that recognize CD3 and CD28 (CODV). A second pair of polypeptides possesses a single variable domain (VH3 and VL3), forming a single antigen-binding site that recognizes HER2 (Fab). The trispecific binding protein shown in Figure 1A utilizes a constant region with a knob-into-hole mutation, where the knob is located in the second pair of polypeptides with a single variable domain. Figure 1B illustrates the species resulting from the mismatch of the heavy chain (left) or light chain (right). On the left are the species resulting from the mismatch of two Fab heavy chains (top) or two CODV heavy chains (bottom). On the right are the species resulting from the mismatch of two Fab light chains (bottom left), two CODV light chains (bottom right), or the mismatch of Fab and CODV light chains to the wrong heavy chains (top right), as well as the correctly matched trispecific binding protein (top left). Figures 2A and 2B show deconvoluted mass spectrometry (MS) spectra for intact (Figure 2A) and IdeS-digested trispecific binding proteins with F(ab')2 fragments (Figure 2B), with different labeled species. The species represented include correctly paired trispecific binding proteins (H1L1 / H2L2), incorrectly paired species with two Fab light chains (H1L1 / H2L1), and incorrectly paired species with two CODV light chains (H1L2 / H2L2). The annotations show experimental mass versus theoretical mass (in parentheses). Figures 3A and 3B show the production of trispecific binding protein from a series of clones. Figure 3A shows a ranking of anti-CD38 trispecific binding protein-producing clones by mass percentage correct of tsAb (purity). Figure 3B shows the productivity of each clone (normalized titer) in the same order as shown in Figure 3A (i.e., ranked by purity). LPOfrnn / zznz / E / YiAi Figure 3C shows pie charts representing the contributions of mismatched impurities and antibody media, as well as correctly matched species, in a randomly selected subset of batch-culture-generated anti-CD38 trispecific binding protein-producing clones. Figures 4A–4C show intact SEC-MS analysis of trispecific binding proteins. Figure 4A shows a representative base peak chromatogram for denaturing intact SEC-MS analysis of a clarified tsAb collected fluid. Figure 4B shows pooled spectra under each chromatographic peak. Figure 4C shows an enhanced deconvoluted mass spectrum for a mixture of anti-CD38 and anti-HER2 trispecific binding proteins that resolves mismatched light chain species from two constructs with Amase = 302 Da. Figure 5A shows the relative quantification of correctly matched and incorrectly matched species obtained for different column loadings (protein pg) of 4.4–21.8 pg for anti-HER2 trispecific binding protein and 11.0–55.0 pg for anti-CD38 trispecific binding protein, demonstrating the robustness and reliability of this method for analyzing low- and high-titer collection samples. For anti-HER2 trispecific binding protein, the column loadings are shown as 4.4 pg, 8.7 pg, 13.1 pg, 17.4 pg, and 21.8 pg (from left to right). For anti-CD38 trispecific binding protein, the column loadings are shown as 11.0 pg, 22.0 pg, 33.0 pg, 44.0 pg, and 55.0 pg (from left to right). Figures 5B and 5C show raw and deconvoluted mass spectra obtained by SEC-LC-MS analysis of clarified collection (Figure 5B) and ProA-purified trispecific anti-CD38 binding protein (Figure 5C), demonstrating the comparability of the two methods. Annotations on the deconvoluted spectra show the experimental masses and percentage intensities for each species. Figures 5D and 5E show the classification of anti-HER2 trispecific binding protein producing clones based on the percentage of correct tsAb mass (purity) (Figure 5D) and the productivity measured by titer for the same clones (Figure 5E). Figures 6A and 6B show the correct mass yield of tsAb in different clones of anti-CD38 trispecific binding protein (Figure 6A) and anti-HER2 trispecific binding protein (Figure 6B) grown under different cell culture conditions (amber or batch, as indicated). Figures 6C-6E show the effects of different growth conditions on chain mismatch and antibody medium levels in anti-CD38 TCE clones grown under two different ambr conditions (Figures 6C and 6D) and batch culture conditions (Figure 6E). Figures 6F and 6G show the effects of different growth conditions on chain mismatch levels in anti-HER2 TCE clones grown under amber (Figure 6F) or discontinuous (Figure 6G) culture conditions. Figure 7 illustrates an illustrative workflow for high-performance intact MS, according to some LPOfrnn / zznz / E / YiAi modalities. In addition to high-throughput capability, additional advantages include: (1) no need for protein A purification, saving time and costs; (2) more direct information on cell line performance by providing information on potential wasted biomass in free chains, antibody media, and other subspecies that may not pass protein A purification; and (3) applicability to Fe-free modalities such as Fabs, scFvs, and other antibody fragments. Figures 8A and 8B show the intact deconvoluted mass spectra obtained for anti-HIV\CD28\CD3 trispecific antibody-producing cell clones with the highest and lowest levels of chain mismatch, respectively. Figures 9A and 9B show the productivity (assessed by titer, pg / mL; Figure 9A) and correctly matched product production (% correct mass; Figure 9B) of 50 trispecific anti-HIV\CD28\CD3 producing cell clones grown under batch culture conditions. Figures 10A and 10B show the detailed glycan profiles obtained for the reference mass (H1L1 / H2L2) in a selected clone that produces trispecific antiHIV\CD28\CD3 antibodies. The clone was grown under two different conditions: in a spin tube (batch condition; Figure 10A) and in an ambr15 bioreactor (batch-fed condition; Figure 10B). Figures 11A and 11B show the MS analysis of antibody crops containing a modified cysteine ​​(Cys293) for terminally protected cysteine ​​status. Figure 11A shows the identification of the terminally protected status of Cys293 in species by direct analysis of intact MS. This antibody contained an N300A mutation that ablated the N-glycosylation of the Fe region. Figure 11B shows the identification of the terminally protected status of Cys293 using direct analysis of intact MS of an antibody treated with PNGase F to remove N-glycans prior to analysis. Detailed description of the invention The disclosure provides, among other things, methods for monitoring the production of a multispecific binding protein and one or more mismatched species, e.g., a multispecific antibody, antibody fragment, Fe fusion protein, or other multispecific binding protein comprising an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first, and one or more mismatched species comprising two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.Similar methods can be applied to the production of a multispecific binding protein, screening of cell lines for the production of a multispecific binding protein, or in the production of antibodies or Fe fusion proteins, as well as to the production of an antibody or antibody derivative (e.g., produced with one or more weight vanant species), or to the screening of cell lines for the production of an antibody or antibody derivative (e.g., produced with one or more weight vanant species). Advantageously, these methods allow intact MS analysis of mAb-related species directly in the LPOfrnn / zznz / E / YiAi collected and clarified, avoiding costly and time-consuming purification (e.g., protein A affinity chromatography) and buffer exchange steps. As such, these methods can be used to rapidly screen large numbers of clones for potential production cell lines. The following description sets out illustrative methods, parameters, and the like. However, it should be recognized that such a description is not intended to limit the scope of this disclosure, but is provided as a description of illustrative modalities. Definitions As used in this disclosure, the following terms, unless otherwise stated, shall be understood to have the following meanings. Unless the context requires otherwise, singular terms shall include plurals and plural terms shall include singular terms. It is understood that the aspects and modalities of disclosure described in this document include comprising, consisting of, and essentially consisting of aspects and modalities. The term polynucleotide, as used herein, refers to single- or double-stranded nucleic acid polymers at least 10 nucleotides in length. In some embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides, or a modified form of any nucleotide type. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-deoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenote, phosphorodiselelenote, phosphoranilothioate, phosphoranthionylate, and phosphoramidate. The term polynucleotide specifically includes both single-stranded and double-stranded DNA forms. An isolated polynucleotide is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide occurs in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. An isolated polypeptide is one that: (1) is free from at least some other polypeptides with which it would normally be found, (2) is essentially free from other polypeptides of the same source, e.g., of the same species, (3) is expressed by a cell of a different species, (4) has been separated from at least approximately 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a polypeptide with which the isolated polypeptide is associated in nature, (6) is operatively associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by DNA, cDNA, mRNA, or other genomic RNA, be of synthetic origin, or any combination thereof.Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise). Natural antibodies generally comprise a tetramer. Each of these tetramers LPOfrnn / zznz / E / YiAi is typically composed of two identical pairs of polypeptide chains, each pair having a full-length light chain (generally with a molecular weight of approximately 25 kDa) and a full-length heavy chain (generally with a molecular weight of approximately 50–70 kDa). The terms heavy chain and light chain, as used herein, refer to any immunoglobulin polypeptide that has sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of approximately 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.Therefore, in a natural antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (Vh) and three constant domains (Chi, Ch2, and Chs), wherein the Vh domain is at the amino end of the polypeptide and the Ch3 domain is at the carboxyl end, and a full-length light chain immunoglobulin polypeptide includes a variable domain (Vl) and a constant domain (Cl), wherein the Vl domain is at the amino end of the polypeptide and the Cl domain is at the carboxyl end. Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses that include, but are not limited to, IgG1 and IgG2. IgA is similarly subdivided into subclasses that include, but are not limited to, IgG1 and IgG2. Within full-length light and heavy chains, the variable and constant domains are generally linked by a J region of approximately 12 or more amino acids, and the heavy chain also includes a D region of approximately 10 more amino acids. See, p. e.g., Fundamental Immunology (Paul, W., ed., Raven Press, 2nd Sed., 1989), which is incorporated by reference in its entirety for all purposes.The variable regions of each light / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies generally exhibit the same general structure of relatively conserved frame regions (FRs) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs of the two chains in each pair are usually aligned by the frame regions, which can enable binding to a specific epitope. From the amino terminus to the carboxy terminus, the light and heavy chain variable domains typically comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The term CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently by different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs can be called Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; LPOfrnn / zznz / E / YiAi Chothia et al. (1989, Nature 342: 877–83) found that some subportions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having high diversity at the amino acid sequence level. These subportions were designated L1, L2, and L3 or H1, H2, and H3, where L and H designate the light chain and heavy chain regions, respectively. These regions can be called Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (1995, FASEB J. 9: 133–39), MacCallum (1996, J. Mol. Biol. 262(5): 732–45), and Lefranc (2003, Dev. Comp. Immunol.). 27: 55-77.However, other definitions of CDR boundaries may not strictly adhere to one of the systems presented here, but will overlap with Kabat's CDRs, although they may be shortened or lengthened based on predictions or experimental findings that certain residues, groups of residues, or even entire CDRs do not significantly affect antigen binding. The methods used herein may employ CDRs defined according to any of these systems, although some methods utilize CDRs defined by Kabat or Chothia. The identification of predicted CDRs using amino acid sequence is well-established in the field, as in Martin, AC Protein sequence and structure analysis of antibody variable domains, in Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. SpringerVerlag, Berlin, pp. 33–51 (2010).The amino acid sequence of the heavy and / or light chain variable domain can also be inspected to identify CDR sequences using other conventional methods, e.g., by comparing them with known amino acid sequences from other heavy and light chain variable regions to determine sequence hypervariability regions. The numbered sequences can be aligned by visual inspection or using an alignment program such as one of the programs in the CLUSTAL suite, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to accurately delineate the framework and CDR regions and thus correct sequence-based assignments. The term Fe, as used herein, refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from antibody digestion or produced by other means, either in monomeric or multimeric form, and may contain the hinge region. The original immunoglobulin source of native Fe is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Fe molecules are composed of monomeric polypeptides that may be joined in dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fe molecules varies from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgGA1, and IgGA2). An example of an Fe is a disulfide-bound dimer resulting from papain digestion of an IgG.The term native Fe as used herein is generic for monomeric, dimeric, and multimeric forms. An F(ab) fragment typically includes a light chain and the Vh and Cm domains of a heavy chain, wherein the Vh-Chi heavy chain portion of the F(ab) fragment cannot form a bond LPOfrnn / zznz / E / YiAi disulfide with another heavy-chain polypeptide. As used herein, an F(ab) fragment may also include a light chain containing two variable domains separated by an amino acid linker and a heavy chain containing two variable domains separated by an amino acid linker and a Cm domain. An F(ab') fragment typically includes a light chain and a portion of a heavy chain containing more than the constant region (between the Chi and Chz domains), so that an interchain disulfide bond can form between two heavy chains to form an F(abj2) molecule.The term "binding protein" as used herein refers to a non-natural (or recombinant or modified) molecule that binds specifically to at least one target antigen. In some embodiments, the binding protein comprises two or more antigen-binding domains. In some embodiments, the binding protein is a multispecific antibody, antibody fragment, or Fe fusion protein. In some embodiments, the binding protein is a bispecific antibody or antibody fragment. In some embodiments, the binding protein is a trispecific antibody or antibody fragment. In some embodiments, the binding protein is a trispecific binding protein, e.g., as described below. In some embodiments, the binding protein comprises one or two Fe regions condensed to one, two, three, or more antigen-binding domains or other polypeptides (e.g., an Fe fusion protein).In some embodiments, the binding protein is a dual variable domain (DVD) immunoglobulin, e.g., as described in WO2012061558. In some embodiments, the binding protein comprises dual variable domains having a cross orientation, e.g., as described in WO2012135345. In some embodiments, the binding protein comprises four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula: Vli-Li-Vl2-L2-Cl [I] and two polypeptide chains have a structure represented by the formula: Vh2-L3-Vhi-L4-Chi-Fc [II] where: V li is a first immunoglobulin light chain variable domain; V i_2 is a second immunoglobulin light chain variable domain; Vm is a first immunoglobulin heavy chain variable domain; V r2 is a second immunoglobulin heavy chain variable domain; Cl is an immunoglobulin light chain constant domain; Chi is the Chi immunoglobulin heavy chain constant domain; Fe is the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains Ch2, Chs; and Li, L2, L3 and L4 are amino acid linkers; and wherein the polypeptides of formula I and the polypeptides of formula II form a crossed light chain-heavy chain pair. A trispecific binding protein of the present disclosure, unless otherwise specified LPOfrnn / zznz / E / YiAi On the contrary, it generally comprises four polypeptide chains forming at least three antigen-binding sites, wherein a first polypeptide chain has a structure represented by the formula: VL2-Li-Vli-L2-Cl [I] and a second polypeptide chain has a structure represented by the formula: VHi-L3-VH2-L4-CHi-hinge-CH2-CH3 [II] and a third polypeptide chain has a structure represented by the formula: VH3-Chi [III] and a fourth polypeptide chain has a structure represented by the formula: Vls-Cl [IV] where: V li is a first immunoglobulin light chain variable domain; V l2 is a second immunoglobulin light chain variable domain; V i_3 is a third immunoglobulin light chain variable domain; Vm is a first immunoglobulin heavy chain variable domain; Vn2 is a second immunoglobulin heavy chain variable domain; Vhs is a third variable domain of immunoglobulin heavy chain; Cl is an immunoglobulin light chain constant domain; Chi is the constant domain of the Chi immunoglobulin heavy chain; and hinge is an immunoglobulin hinge region that connects the Chi and Ons domains; Li, L2, L3 and L4 are amino acid linkers; and where the polypeptide of formula I and the polypeptide of formula II form a crossed light chain heavy chain pair. A recombinant molecule is one that has been prepared, expressed, created, or isolated by recombinant means. One disclosure modality provides binding proteins that have biological and immunological specificity to between one and three target antigens. Another disclosure modality provides nucleic acid molecules comprising nucleotide sequences that encode polypeptide chains forming such binding proteins. Another disclosure modality provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences that encode polypeptide chains forming such binding proteins. Yet another disclosure modality provides host cells that express such binding proteins (i.e., that comprise nucleic acid molecules or vectors encoding polypeptide chains forming such binding proteins). The term interchangeability, as used herein, refers to the interchangeability of variable domains within the binding protein format with retention of final folding and binding affinity. Complete interchangeability refers to the ability to exchange the order of both Vhi and Vh2 domains, and therefore the order of Vli and Vl2 domains, in either the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., reverse the order) i QQbnn / zznz / B / YiAi while maintaining the full functionality of the binding protein, as evidenced by the retention of binding affinity. It should also be noted that the designations Vh and Vl refer only to the domain's location within a particular protein chain in the final format. For example, Vhi and Vh2 could be derived from the Vli and Vi_2 domains in progenitor antibodies and placed in the Vhi and Vh2 positions in the binding protein.Similarly, Vli and Vl2 could be derived from the Vhi and Vh2 domains in progenitor antibodies and placed in the Vw and Vh2 positions on the binding protein. Therefore, the designations Vh and Vl refer to the current location and not the original location on a progenitor antibody. Thus, the Vh and Vl domains are interchangeable. The term antigen, target antigen, or antigen target as used herein refers to a molecule or part of a molecule that is capable of being bound by a binding protein and is also capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen. The term monospecific binding protein refers to a binding protein that binds specifically to one antigen target. The term monovalent binding protein refers to a binding protein that has one antigen-binding site. The term bispecific binding protein refers to a binding protein that binds specifically to two different antigen targets. The term bivalent binding protein refers to a binding protein that has two binding sites. The term trispecific binding protein refers to a binding protein that binds specifically to three different antigen targets. The term trivalent binding protein refers to a binding protein that has three binding sites. In some configurations, the trivalent binding protein can bind to one antigen target. In others, the trivalent binding protein can bind to two antigen targets. In still others, the trivalent binding protein can bind to three antigen targets. An isolated binding protein is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with the diagnostic or therapeutic uses of the binding protein and may include enzymes, hormones, and other protein or non-protein solutes. In some modalities, the binding protein will be purified: (1) to more than 95% by weight of antibodies as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminus or internal amino acid sequence residues by using a rotating cup sequestrant, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining.Isolated binding proteins include the in situ binding protein within recombinant cells, since at least one component of the natural environment of the binding protein will not be present. The terms substantially pure or substantially purified, as used herein, refer to a compound or species that is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition in which the species comprises at least approximately 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species. The term epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In some modalities, epitope determinants include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some modalities, they may have specific three-dimensional structural features and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In some modalities, a binding protein is said to bind specifically to an antigen when it preferentially recognizes its target antigen within a complex mixture of proteins and / or macromolecules.In some modalities, a binding protein is said to bind specifically to an antigen when the equilibrium dissociation constant is < 108M, more preferably when the equilibrium dissociation constant is < 109M, and most preferably when the dissociation constant is < 10'10M. The dissociation constant (Kd) of a binding protein can be determined, for example, by surface plasmon resonance. In general, surface plasmon resonance assays measure real-time binding interactions between the ligand (a target antigen in a biosensor array) and the analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon assays can also be performed by immobilizing the analyte (binding protein in a biosensor array) and presenting the ligand (target antigen). The term Kd, as used herein, refers to the dissociation constant of the interaction between a particular binding protein and a target antigen. The term "binds specifically" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with a Kd of at least approximately 1x10'6M, 1x10'7M, 1x10'8M, 1 x 10'9M, 1 x 10'10M, 1 x 10'11M, 1 x 10'12M, or more, and / or to bind to an epitope with an affinity that is at least twice as high as its affinity for a nonspecific antigen. The term linker as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility LPOfrnn / zznz / E / YiAi so that the light and heavy chain domains fold into double-variable region cross-linked immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of immunoglobulin domains is well understood. The precise location of a domain transition can be determined by locating stretches of peptides that do not form secondary structural elements such as beta sheets or alpha helices, as demonstrated by experimental data or as can be predicted using secondary structure modeling or prediction techniques.The linkers described herein are known as Li, which is located in the light chain between the C-terminus of Vi_2 and the N-terminus of the Vli domain; and L2, which is located in the light chain between the C-terminus of Vli and the N-terminus of the Cl domain. The heavy chain linkers are known as L3, which is located between the C-terminus of Vhi and the N-terminus of the Vh2 domain; and U, which is located between the C-terminus of Vh2 and the N-terminus of the Chi domain. The term vector, as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term vector includes a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. One type of vector is a plasmid, which refers to a circular, double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, into which additional DNA segments can be inserted into the viral genome. Some vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors, which have a bacterial origin of replication, and mammalian episomal vectors). Other vectors (e.g.,Non-episomal mammalian vectors can integrate into the genome of a host cell after their introduction and thus replicate along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operationally linked. Such vectors are referred to herein as recombinant expression vectors (or simply, expression vectors). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms plasmid and vector may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, this disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. The term recombinant host cell (or host cell) as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell but also to the progeny of that cell. As some modifications may occur in successive generations due to mutation or environmental influences, such progeny may, in fact, not be identical to the progenitor cell, but such cells are still included within the scope of the term host cell as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including systems LPOfrnn / zznz / E / YiAi bacterial, yeast, baculoviral, and mammalian expression vectors (as well as phage visualization expression systems) are available. An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein, so that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells grow, from which the binding protein can be recovered. The term transformation, as used herein, refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it is genetically altered from its native state. After transformation, the transforming DNA may recombine with the cell's DNA by physically integrating into a chromosome of the cell, or it may be transiently maintained as an episomal element without being replicated, or it may replicate independently as a plasmid. A cell is considered stably transformed when the DNA replicates with cell division. The term transfection, as used herein, refers to the uptake of foreign or exogenous DNA by a cell, and a cell is transfected when the exogenous DNA has been introduced into the cell membrane.A number of transfection techniques are well known in the field. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells. The term "natural," as used herein and applied to an object, refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by humans is natural. Similarly, "non-natural," as used herein, refers to an object that is not found in nature or that has been structurally modified or synthesized by humans. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; non-natural amino acids and analogues such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids may also be suitable building blocks for the polypeptide chains of binding proteins. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylysine, ophosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, according to standard usage and convention. Natural residues can be divided into classes based on common side chain properties: (1) hydrophobic: Met, Ala, Val, Leu, lie, Phe, Trp, Tyr, Pro; LPOfrnn / zznz / E / YiAi (2) polar hydrophiles: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) aliphatic: Ala, Gly, lie, Leu, Val, Pro; (4) hydrophobic aliphatics: Ala, lie, Leu, Val, Pro; (5) neutral hydrophiles: Cys, Ser, Thr, Asn, Gln; (6) acids: Asp, Glu; (7) basics: His, Lys, Arg; (8) residues that influence chain orientation: Gly, Pro; (9) aromatics: His, Trp, Tyr, Phe; and (10) hydrophobic aromatics: Phe, Trp, Tyr. Conservative amino acid substitutions can involve exchanging one member of one class for another member of the same class. Non-conservative substitutions can involve exchanging one member of one class for a member of another class. A subject matter expert can determine suitable variants of the polypeptide chains of binding proteins using well-established techniques. For example, an expert can identify suitable regions of a polypeptide chain that can be altered without disrupting activity by targeting regions not believed to be important for activity. Alternatively, an expert can identify residues and portions of molecules that are conserved among similar polypeptides. Furthermore, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without disrupting biological activity or negatively impacting the polypeptide structure. Methods Some aspects of this disclosure relate to methods for monitoring the production of a multispecific binding protein and one or more mismatched species. In some embodiments, the methods involve detecting an amount of the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) by size-exclusion ultra-performance liquid chromatography-mass spectrometry (SE-UPLC-MS). In some embodiments, the product and one or more mismatched species are detected in a cell culture medium. Illustrative, non-limiting descriptions of multispecific binding proteins are provided herein. Some aspects of this disclosure relate to methods for monitoring the production of an antibody or antibody derivative and one or more weight variant species. In some embodiments, the methods comprise the detection of a quantity of the product (e.g., one or more weight variant species of an antibody or antibody derivative) by size-exclusion ultra-performance liquid chromatography-mass spectrometry (SE-UPLC-MS). In some embodiments, the antibody or antibody derivative and one or more weight variant species are detected in a cell culture medium. The antibody or antibody derivative and one or more weight variant species may represent, for example, multiple species that vary in molecular weight (e.g., with different chemical modifications, such as chemically modified cysteine ​​or other residues, or with different glycoforms).As demonstrated in the Examples below, the methods described herein with reference to the analysis of an antibody, antibody fragment, Fe fusion protein or multispecific binding protein and one or more incorrectly matched species can be applied to the analysis of an antibody or antibody derivative and one or more weight variant species. In some methods, the cell culture medium is a clarified cell culture collection. For example, the cell culture medium may be one that has been collected from a cell culture and clarified by tangential flow filtration (TFF), depth filtration, and / or centrifugation. In some methods, the further methods include separating the cell culture medium or collecting a cell line that produces the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species). In some methods, the cell culture medium is clarified by centrifugation. In some methods, the cell culture medium is subjected to SE-UPLC without prior chromatographic separation. That is, in some methods, the cell culture medium is one that has not been subjected to prior chromatographic separation.In some methods, the cell culture medium is subjected to SEUPLC without prior protein A affinity chromatography. That is, in some methods, the cell culture medium is one that has not been put in contact with protein A. In some modalities, the detection of the quantity of the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) involves deconvolving one or more MS spectra obtained by MS. Methods for deconvolving MS spectra are known in the art. A non-limiting review of useful software for deconvolution is illustrated below. In some modalities, a relative amount of a multispecies binding protein is detected, e.g., in comparison with the amount of one or more mismatched species. For example, the amount of a multispecies binding protein produced can be compared with the amount of one or more mismatched individual species, and / or with the total amount of mismatched species (e.g., the total amount of all mismatched species, or a certain subset thereof). In some modes, MS is intact MS. In some modes, MS is quadrupole time-of-flight (QToF) MS. A variety of QToF MS methods are known in the art. Illustrative, non-limiting parameters for QToF MS are described in the Examples below. In some modes, MS is able to resolve a mass difference of approximately 1000 Da or less, approximately 900 Da or less, approximately 800 Da or less, approximately 700 Da or less, approximately 600 Da or less, approximately 500 Da or less, approximately 400 Da or less, approximately 300 Da or less, or approximately 200 Da or less. In some modes, MS is able to resolve a mass difference of approximately 300 Da. For example, in some modes, MS is able to resolve a mass difference of approximately 300 Da between a multispecific binding protein and one or more species. LPOfrnn / zznz / E / YiAi incorrectly paired, or between two incorrectly paired species. In some modes, MS is able to resolve a mass difference of approximately 162 Da. For example, in some modes, MS is able to resolve a mass difference of approximately 162 Da between glycoforms, e.g., between a multispecific binding protein or an incorrectly paired species and one of more glycoforms. In some forms, SE-UPLC is denaturing SE-UPLC. In some forms, SE-UPLC is directly coupled to MS. A variety of SE-UPLC methods are known in the art. Illustrative, non-limiting conditions for SE-UPLC are described in the Examples below. In some methods, SE-UPLC is performed with a slower initial flow rate, followed by a faster one. While not adhering to a strict theory, it is believed that a slower initial flow rate allows for more efficient separation based on the size of the cell culture components, while increasing the flow rate after this initial period improves the overall speed of the method. In some methods, SE-UPLC is performed with an initial flow rate of less than approximately 0.4 mL / min, followed by a flow rate greater than or equal to approximately 0.4 mL / min. In some methods, the initial flow rate is less than approximately 0.4 mL / min, less than approximately 0.3 mL / min, less than approximately 0.2 mL / min, or approximately 0.1 mL / min. In some methods, the initial flow rate refers to the first 15, 20, or 25 minutes of SE-UPLC. In some methods, SE-UPLC is performed with a flow rate of less than approximately 0.4 mL / min for the first 25 minutes, followed by a flow rate greater than or equal to approximately 0.4 mL / min (e.g., for 25–33 minutes). In some modalities, SE-UPLC is performed with a flow rate of approximately 0.1 mL / min for the first 25 minutes, followed by a flow rate of approximately 0.4 mL / min (e.g., for 25–33 minutes). In some modalities, SE-UPLC is performed by diverting part(s) of the liquid chromatography (LC) flow without protein elution (e.g., 0-10 minutes and / or 24-33 minutes) to the waste, providing faster analysis for detection and avoiding contamination of the MS source. In some embodiments, SE-UPLC is performed by isocratic elution with a mobile phase. For example, in some embodiments, the mobile phase comprises a solution of acetonitrile:water 30:70. In some embodiments, the mobile phase comprises formic acid (FA) and trifluoroacetic acid (TFA). Without wishing to be bound by theory, it is thought that while TFA is useful for liquid chromatography (LC) separation, it can suppress the MS signal and, therefore, a mobile phase comprising a mixture of FA and TFA can allow effective liquid chromatography (LC) separation while maintaining a signal-to-noise ratio suitable for MS analysis. In some embodiments, the mobile phase comprises approximately 0.05% formic acid and approximately 0.05% trifluoroacetic acid (TFA). Advantageously, the methods provided herein allow for rapid product analysis, enabling high-throughput screening (e.g., of a multitude of cell lines for potential products). For example, in some modalities, detection is achieved in approximately 30 minutes or less, approximately 33 minutes or less, approximately 35 minutes or less, approximately 40 minutes or less, approximately 45 minutes or less, or LPOfrnn / zznz / E / YiAi approximately 60 minutes or less. In some embodiments, the methods also include, prior to SE-UPLC-MS, contacting the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) with a protease. In some embodiments, the protease is IdeS or IdeZ, e.g., for the analysis of antibody fragments. In some embodiments, the antibody or antibody derivative comprises a cysteine ​​residue at position 293 (Cys293). In some embodiments, the antibody or antibody derivative and one or more weight vane species comprise species with a free cysteine ​​(e.g., not disulfide bonded to another cysteine ​​of the antibody or antibody derivative) that has been cysteinylated, N-acetylcysteinylated, or glutathionylated. In some embodiments, the antibody or antibody derivative is not N-glycosylated (e.g., in the Fe region of the antibody). In some embodiments, the antibody or antibody derivative comprises a mutation in the Fe region that reduces or eliminates N-glycosylation. In some embodiments, the antibody or antibody derivative comprises an N300A mutation (EU index). In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g., in the Fe region of the antibody), and the method further comprises (e.g.)(before SE-UPLC-MS), antibody N-glycosylation removal. In some modalities, antibody N-glycosylation removal comprises treatment of the antibody with a peptide:N-glucosidase enzyme (e.g., PNGase F). In some embodiments, one or more weight variant species represent species of the antibody or antibody derivative comprising a chemically modified cysteine ​​residue. In some embodiments, MS is able to resolve a mass difference between cysteinylated (mass shift of 119 Da), N-acetylcysteinylated (mass shift of 161 Da), and glutathionylated (mass shift of 305 Da) species. For example, antibodies and antibody derivatives comprising a free cysteine ​​residue (e.g., a non-disulfide cysteine ​​linked to another cysteine ​​residue of the antibody / antibody derivative) and / or antibodies and antibody derivatives comprising an additional cysteine ​​residue modified in its Fe region may be useful, e.g., for the conjugation of a compound (e.g., a cytotoxic drug to form an antibody-drug conjugate).This extra cysteine ​​is unpaired (free), meaning it does not participate in disulfide bonds with other cysteines in the mAb molecule. Previous work had shown that this extra cysteine ​​(Cys293) can form unwanted disulfide bonds with nearby cysteine ​​residues, also known as disulfide bond fighting, which could lead to antibody structural instability. Uncontrolled sulfhydryl chemistry can also pose a significant manufacturing risk during processing to form the ADC, leading to an undesirable drug conjugation profile. To avoid these risks, it is imperative to have the free cysteine ​​terminally protected with disulfide-linked modifications, such as cysteinylation and glutathionylation, by adjusting cell culture conditions. These modifications can be selectively reduced to generate free cysteine ​​before the drug conjugation step.The methods described herein can be used, e.g., to analyze the production of an antibody or antibody derivative comprising different weight variant species, such as species comprising a chemically modified cysteine ​​such as a cysteine ​​coated with a disulfide-linked modification, such as cysteinylation and glutathionylation. In some models, the antibody or antibody derivative is N-glycosylated (e.g., in the Fe region of the antibody), and one or more weight variant species represent glycoforms of the antibody or antibody derivative. In some models, MS is able to resolve a mass difference of approximately 162 Da between the antibody or antibody derivative and one or more weight variant species representing glycoforms of the antibody or antibody derivative. In some models, the antibody or antibody derivative is a multispecific antibody. In some models, the antibody or antibody derivative is a monoclonal antibody. Other aspects of this disclosure relate to methods for producing an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein. In some embodiments, the methods comprise culturing a cell line comprising one or more polynucleotides encoding the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) under conditions suitable for the production of the product by a cell line; isolating, from the cell line, a cell culture medium comprising the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species); and detecting a quantity of the product (e.g.,(d) separating an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) into the cell culture medium by ultra-performance size exclusion liquid chromatography and mass spectrometry (SE-UPLC-MS); and (d) removing at least a portion of one or more of the mismatched species from the multispecific binding protein produced by the cell line. In some embodiments, the methods comprise culturing a cell line comprising one or more polynucleotides encoding the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) under conditions suitable for the production of the product by a cell line; separating, from the cell line, a cell culture medium comprising the product (e.g.(e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species); detect an amount of the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) in the cell culture medium by ultra-performance size exclusion liquid chromatography and mass spectrometry (SE-UPLC-MS); and (d) determine one or both of the quality and purity of the multispecific binding protein produced by the cell line. In some embodiments, the methods comprise culturing a cell line comprising one or more polynucleotides encoding the product (e.g., an antibody, an antibody fragment, an Fe fusion protein or a multispecific binding protein and one or more mismatched species) under conditions suitable for the production of the product by a cell line; separating, from the cell line, a cell culture medium comprising the product (e.g., an antibody, an antibody fragment,. LPOfrnn / zznz / E / YiAi an Fe fusion protein or a multispecific binding protein and one or more mismatched species); detect a quantity of the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) in the cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS); and (d) determine one or both of the quality and purity of the multispecific binding protein produced by the cell line and remove at least a portion of one or more of the mismatched species from the multispecific binding protein produced by the cell line. Any of the illustrative products described herein may find use in these and other methods. Other aspects of this disclosure relate to methods for screening a plurality of cell lines for the production of a product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species). In some embodiments, the methods comprise detecting an amount of a product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) produced by a first cell line (e.g., using any of the methods described herein) and detecting an amount of the product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) produced by a second cell line (e.g., using any of the methods described herein).(using any of the methods described herein), e.g., other than the first cell line. Any of the illustrative products described herein may find use in these and other methods. In some embodiments, the methods further involve comparing the amount of product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) produced by the first cell line with the amount of product (e.g., an antibody, an antibody fragment, an Fe fusion protein, or a multispecific binding protein and one or more mismatched species) produced by the second cell line; and, based on this comparison, selecting the cell line that produced the greatest amount of product. In some embodiments, two or more, three or more, four or more, five or more, ten or more, twenty or more, thirty or more, forty or more, or fifty or more different cell lines are examined. Advantageously, the methods described herein are rapid and capable of high-throughput screening. In some embodiments, the methods further comprise detecting the quantity of one or more incorrectly paired species produced by the first cell line and detecting the quantity of one or more incorrectly paired species produced by the second cell line. In some embodiments, the methods further comprise, for example, after detecting the quantity of one or more incorrectly paired species produced by the first and second cell lines, comparing the quantity of one or more incorrectly paired species produced by the first cell line with the quantity of one or more incorrectly paired species produced by the second cell line. In some embodiments, based on this comparison, the cell line is selected. LPOfrnn / zznz / E / YiAi that produced the highest proportion of multispecific binding protein to one or more mismatched species. In some modalities, the cell line is selected based on a higher proportion of multispecific binding protein to the amount of one or more mismatched individual species, and / or based on a higher proportion of multispecific binding protein to the total amount of mismatched species. In some modalities, the selected cell line is chosen for use as a production cell line. Multispecific binding proteins Some aspects of this disclosure relate to multispecific binding proteins. In some embodiments, the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain. For example, in some embodiments, the multispecific binding protein is a bispecific antibody or a bispecific Fe fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody or a trispecific Fe fusion protein. In some embodiments, the one or more mismatched species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association different from that of the multispecific binding protein. In some embodiments, the multispecific binding protein is a multispecific antibody comprising a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain.In some embodiments, the one or more incorrectly matched species comprise one or more of: an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody heavy chains; an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody light chains; and an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody light chains. In some forms, multispecific binding proteins comprise four polypeptide chains forming three antigen-binding sites that bind specifically to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains that have a cross orientation and wherein a second pair of polypeptides forming the binding protein possess a single variable domain. In some embodiments, the multispecific binding proteins comprise one or two Fe regions condensed to one, two, three, or more antigen-binding domains or other polypeptides (e.g., an Fe fusion protein). In some embodiments, the multispecific binding protein is a dual variable domain (DVD) immunoglobulin, e.g., as described in WO2012061558. In some embodiments, the multispecific binding protein comprises domains LPOfrnn / zznz / E / YiAi dual variables having a cross orientation, e.g., as described in WO2012135345. In some embodiments, the binding protein comprises four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula: Vli-Li-Vl2-L2-Cl[I] and two polypeptide chains have a structure represented by the formula: Vh2-L3-Vhi-L4-Chi-Fc [II] where: V li is a first immunoglobulin light chain variable domain; V l2 is a second immunoglobulin light chain variable domain; Vhi is a first immunoglobulin heavy chain variable domain; V f¡2 is a second immunoglobulin heavy chain variable domain; Cl is an immunoglobulin light chain constant domain; Chi is the Chi immunoglobulin heavy chain constant domain; Fe is the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains Ch2, Chs; and Li, l_2, l_3 and L4 are amino acid linkers; where the polypeptides of formula I and the polypeptides of formula II form a crossed light chain-heavy chain pair. In some embodiments, each of the three antigen-binding sites binds to a different target (e.g., polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains that form the three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: Vl2-Li-Vli-L2-Cl[I] and a second polypeptide chain comprises a structure represented by the formula: Vhi-L3-\ / H2-L4-CHi-hinge-CH2-CH3[II] and a third polypeptide chain comprises a structure represented by the formula: VH3-CHi-hinge-CH2-CH3[III] and a fourth polypeptide chain comprises a structure represented by the formula: Vls-Cl[IV] where: V li is a first immunoglobulin light chain variable domain; V l2 is a second immunoglobulin light chain variable domain; V l3 is a third immunoglobulin light chain variable domain; Vhi is a first immunoglobulin heavy chain variable domain; V h2 is a second immunoglobulin heavy chain variable domain; Vhs is a third variable domain of immunoglobulin heavy chain; Cl is an immunoglobulin light chain constant domain; LPOfrnn / zznz / E / YiAi Chi is a Chi immunoglobulin heavy chain constant domain; Ch2 is a constant domain of immunoglobulin heavy chain Oη2; Ch3 is a Chs immunoglobulin heavy chain constant domain; hinge is an immunoglobulin hinge region that connects the Chi and Ch2 domains; and Li, L2, La and L4 are amino acid linkers; where the polypeptide of formula I and the polypeptide of formula II form a crossed light chain heavy chain pair. In some embodiments, the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-heavy chain pair. In some embodiments, Vhi and Vli form a first antigen-binding site, where Vh2 and Vl2 form a second antigen-binding site, and where Vh3 and Vls form a third antigen-binding site. In some embodiments, one or more incorrectly paired species comprise one or more of: an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula IV. In some forms, multispecific binding proteins are trispecific and / or trivalent. In some forms, multispecific binding proteins are bispecific and / or bivalent. It is envisaged that any of the antigen-binding sites described herein may be used in a trispecific binding protein of the disclosure hereof, e.g., comprising four polypeptide chains having the structures described above. For example, in some embodiments, a trispecific binding protein of the disclosure hereof comprises a pair of VH1 and VL1 domains forming a first antigen-binding site, a pair of VH2 and VL2 domains forming a second antigen-binding site, and a pair of VH3 and VL3 domains forming a third antigen-binding site. In some embodiments, a trispecific binding protein of the disclosure hereof comprises a pair of VH1 and VL1 domains forming a first antigen-binding site, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site.In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds to a CD28 polypeptide, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site. In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds to a tumor target protein. In some embodiments, QQbnn / zznz / B / YiAi A trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds to a CD28 polypeptide, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds to an HIV target protein. In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds to an HIV target protein, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to an HIV target protein, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds to an HIV target protein.In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds a CD28 polypeptide, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds a tumor target protein. In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds a CD28 polypeptide, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds a CD38 polypeptide.In some embodiments, a trispecific binding protein of the present disclosure comprises a pair of VH1 and VL1 domains forming a first antigen-binding site that binds to a CD28 polypeptide, a pair of VH2 and VL2 domains forming a second antigen-binding site that binds to a CD3 polypeptide, and a pair of VH3 and VL3 domains forming a third antigen-binding site that binds to a HER2 polypeptide. In some embodiments, a binding protein described herein binds to one or more tumor target proteins and one or more T cell target proteins. In some embodiments, the binding protein is capable of specifically binding to one tumor target protein and two different epitopes on a single T cell target protein. In some embodiments, the binding protein is capable of specifically binding to one tumor target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form one antigen-binding site that specifically binds to one tumor target protein. In some embodiments, the target protein is either CD38 or HER2.Additional tumor target proteins are provided below. In some modalities, one or more of the T cell target proteins are one or more of CD3 and CD28. The illustrative multispecific binding proteins that may be used in the methods of this disclosure also include, without limitation, those described in International Publications Nos. WO2017074878, WO2017180913, WO2018151841 and WO2019074973. In some embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds a CD38 polypeptide, an antigen-binding site that binds a CD28 polypeptide, and an antigen-binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the formula I polypeptide and the formula II polypeptide form a cross-linked light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen-binding sites described above represent Vhs and Vl3 and form a third antigen-binding site that binds a CD38 polypeptide.In some modalities, Vm and Vli form a first antigen-binding site that binds to a CD28 polypeptide, Ά and Vl2 form a second antigen-binding site that binds to a CD3 polypeptide, and Vh3 and Vls form a third antigen-binding site that binds to a CD38 polypeptide. In some embodiments, a binding protein comprising an antigen-binding site that binds to a HER2 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, a binding protein comprising an antigen-binding site that binds to a HER2 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites as described above, wherein the pair of Vhs and Vl3 domains forming a third antigen-binding site that binds to a HER2 polypeptide In some embodiments, a binding protein of these disclosures comprises an antigen-binding site that binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some modalities, a tumor target protein in this disclosure includes, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR, BOMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC),CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF),CX3CL1 (also known as, LPOfrnn / zznz / E / YiAi as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, G¡24, GITR, GITRL, GMCSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILIO, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as IL25 receptor), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TNFRSF7, Tp55, TREMI, TSLP (also known as IL7Ra co-receptor), TSLPR, TWEAK, VEGF, VISTA, Vstm3,WUCAM and XCR1 (also known as GPR5 / CCXCR1). In some modalities, one or more of the above antigen targets are human antigen targets. In some embodiments, a binding protein of these disclosures comprises one antigen-binding site that binds to an HIV target protein. In some embodiments, a binding protein of these disclosures comprises three antigen-binding sites that bind to an HIV target protein. In some embodiments, the binding proteins specifically bind to one or more HIV target proteins (e.g., as described below) and one or more target proteins on a T cell, including the T cell receptor complex. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28. In some embodiments, the HIV target protein is glycoprotein 120, glycoprotein 41, or glycoprotein 160. In some embodiments, a binding protein binds to one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160.Illustrative HIV target proteins include, without limitation, the MPER of HIV-1 gp41 protein, a CD4-binding site of HIV-1 gp120 protein, a glycan in the V3 loop of HIV-1 gp120 protein, or a trimer tip of HIV-1 gp120 or gp160 protein. For example, in some embodiments, a binding protein in this disclosure comprises an antigen-binding site that binds to a CD4-binding site of HIV-1 gp120 protein. Illustrative antigen-binding sites that bind to HIV target proteins contemplated for use herein include, without limitation, those described in International Publication No. WO2017 / 074878, such as those of the antibodies CD4BS a, CD4BS b, MPER, MPER100W, V1 / V2 a, V1 / V2 b, and V3. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide and one of which binds to a CD3 polypeptide. In some embodiments, a binding protein comprising a binding site to LPOfrnn / zznz / E / YiAi antigen-binding CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a CD38 polypeptide. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a HER2 polypeptide.In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor target protein. In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds to a tumor target protein (including, without limitation, CD38 or HER2), an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-heavy chain pair (e.g., as described herein).In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above represent Vhi and Vli and form a first antigen-binding site that binds to a CD28 polypeptide. In some embodiments, Vm and Vli form a first antigen-binding site that binds to a CD28 polypeptide, Vh2 and Vl2 form a second antigen-binding site that binds to a CD3 polypeptide, and Vh3 and Vls form a third antigen-binding site that binds to a tumor target protein (including, without limitation, CD38 or HER2). In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to a CD28 polypeptide and one of which binds to a CD3 polypeptide.In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a CD38 polypeptide. In some embodiments, a binding protein comprising an antigen-binding site that binds to a... LPOfrnn / zznz / E / YiAi CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a HER2 polypeptide. In some embodiments, a binding protein comprising an antigen-binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor target protein. In some embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen-binding site that binds to a tumor target protein (including, without limitation, CD38 or HER2), an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-heavy chain pair (e.g., as described herein). In some embodiments, Vh2 and Vl2 form a second antigen-binding site that binds to a CD3 polypeptide.In some modalities, Vm and Vli form a first antigen-binding site that binds to a CD28 polypeptide, Vh2 and Vl2 form a second antigen-binding site that binds to a CD3 polypeptide, and Vhs and Vls form a third antigen-binding site that binds to a tumor target protein (including, without limitation, CD38 or HER2). Connectors In some forms, the L1, L2, L3, and L4 linkers vary from zero amino acids (length = 0) to approximately 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids. The linkers may also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. L1, L2, L3, and L4 in a binding protein may all have the same amino acid sequence or they may all have different amino acid sequences. Examples of suitable linkers include, for instance, GGGGSGGGGS (SEQ ID NO:1), GGGGSGGGGSGGGGS (SEQ ID NO: 2), S, RT, TKGPS (SEQ ID NO:3), GQPKAAP (SEQ ID NO: 4), GGSGSSGSGG (SEQ ID NO: 5), and DKTHT (SEQ ID NO:6), as well as those disclosed in International Publications Nos. WO2017 / 074878 and WO2017 / 180913. The examples listed above are not intended to limit the scope of disclosure in any way, and linkers comprising randomly selected amino acids from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in binding proteins. The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element required. For example, glycine, serine, and alanine are best for linkers that require maximum flexibility. Combinations of glycine, proline, threonine, and serine are useful for more rigid and extended linkers. Any amino acid residue can be considered a linker in combination with... LPOfrnn / zznz / E / YiAi other amino acid residues to build larger peptide linkers as needed depending on the desired properties. In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, L1 is 3 to 12 amino acid residues long, L2 is 3 to 14 amino acid residues long, L3 is 1 to 8 amino acid residues long, and L4 is 1 to 3 amino acid residues long. In some embodiments, L1 is 5 to 10 amino acid residues long, L2 is 5 to 8 amino acid residues long, L3 is 1 to 5 amino acid residues long, and L4 is 1 to 2 amino acid residues long. In some forms, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length. In some embodiments, L1, L2, L3, and L4 are independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGGS (SEQ ID NO: 1), GGGGSGGGGSGGGGGS (SEQ ID NO: 2), S, RT, TKGPS (SEQ ID NO: 3), GQPKAAP (SEQ ID NO: 4), and GGSGSSGSGSGG (SEQ ID NO: 5). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGGS (SEQ ID NO: 1), GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 2), S, RT, TKGPS (SEQ ID NO: 3), GQPKAAP (SEQ ID NO: 4), and GGSGSSGSGG (SEQ ID NO: 5). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 1), GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 2), S, RT, TKGPS (SEQ ID NO: 3), GQPKAAP (SEQ ID NO: 4), and GGSGSSGSGG (SEQ ID NO: 5). 4), L2 comprises the sequence TKGPS (SEQ ID NO: 3), L3 comprises the sequence S and L4 comprises the sequence RT. In some forms, at least one of L1, L2, L3, or L4 comprises the DKTHT sequence (SEQ ID NO: 6). In some forms, L1, L2, L3, and L4 comprise the DKTHT sequence (SEQ ID NO: 6). Fe regions and constant domainsIn some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising a Cm-linked Fe region, the Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Ch3. In some embodiments, a binding protein of the present disclosure comprises a third polypeptide chain further comprising a Chi-linked Fe region, the Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Chs.In some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising a Chi-linked Fe region, the Fe region comprising an immunoglobulin hinge region and Ch2 and Chs immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a Chi-linked Fe region, the Fe region comprising an immunoglobulin hinge region and Ch2 and Chs immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fe region. In some modals, the Fe region is a human Fe region, e.g., a human Fe region lgG1, lgG2, lgG3, or lgG4. In some modals, the Fe region includes a Chi, Ch2, Ch3, and optionally Ch4 antibody hinge domain. In some modals, the Fe region is a human Fe region lgG1. In some modals, the Fe region is a human Fe region lgG4. In some modals, the Fe region includes one or more of the mutations described below. In some embodiments, the Fe region is an Fe region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the light chain constant region is a constant region of one of the light chain polypeptides (e.g., polypeptide 2 or 3).e.g., polypeptide 1 or 4) of a binding protein shown in Table 4. In some embodiments, a binding protein of the present disclosure includes one or two Fe variants. The term Fe variant, as used herein, refers to a molecule or sequence that is modified from native Fe but still comprises a binding site for the rescue receptor, FcRn (neonatal Fe receptor). Illustrative Fe variants and their interaction with the rescue receptor are known in the art. Therefore, the term Fe variant may comprise a molecule or sequence that is humanized from non-human native Fe. Furthermore, native Fe comprises regions that may be deleted because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention.Therefore, the term Fe variant comprises a molecule or sequence that lacks one or more native Fe sites or residues, or in which one or more Fe sites or residues have been modified, affecting or involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminus heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) complement interaction, (6) binding to an Fe receptor other than a rescue receptor, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, a binding protein of the present disclosure (e.g., a trispecific binding protein) comprises a button mutation in the second polypeptide chain and a buttonhole mutation in the third polypeptide chain. In some embodiments, a binding protein of this disclosure comprises a "button" mutation in the third polypeptide chain and a "knot" mutation in the second polypeptide chain. In some embodiments, the button mutation comprises substitution(s) at positions corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the button mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W.In some embodiments, the buttonhole mutation comprises the substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366 and / or 368 of human lgG1 or lgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally. LPOfrnn / zznz / E / YiAi Y349C, T366S, and / or L368A. In some forms, the buttonhole mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some forms, the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human lgG1 or lgG4, according to the EU index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C;and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and / or 368 and of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and / or L368A. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366 and / or 368 and of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and, optionally, Y349C, T366S and / or L368A;and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises the amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C.; In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises the amino acid substitution at the position corresponding to position 366 of human lgG1 or lgG4, according to the EU index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitution(s) at positions corresponding to positions 366, 368 and / or 407 and of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A and / or Y407V. In some forms, the second polypeptide chain also comprises a first region LPOfrnn / zznz / E / YiAi Fe linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitution(s) at positions corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A and / or Y407V; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises the amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V.In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human lgG1 or lgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W.In some modalities, the first and / or second Fe region are human lgG1 Fe regions. In some modalities, the first and / or second Fe region are human lgG4 Fe regions. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, S354C, T366W and R409K; and wherein the third chain The polypeptide LPOfrnn / zznz / E / YiAi further comprises a second Fe region linked to CH1, wherein the second Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human Fe lgG4 region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368,407 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, wherein the second Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, S354C, T366W and R409K. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human lgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, S354C and T366W; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, wherein the second Fe region is a human Fe lgG4 region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366,368 and 407 of human lgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human lgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, wherein the second Fe region is a human Fe lgG4 region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3,where the second Fe region comprises amino acid substitutions at positions, LPOfrnn / zznz / E / YiAi corresponding to positions 234, 235, 354 and 366 of human lgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W. In some embodiments, a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g., Fe receptor-mediated antibody-dependent cellular phagocytosis (ADOR), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC).In some embodiments, the second polypeptide chain further comprises a first Fe region linked to Chi, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Chs; wherein the third polypeptide chain further comprises a second Fe region linked to Chi, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Ch3; ​​wherein the first and second Fe regions are human IgG1 Fe regions; and wherein the first and second Fe regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A.In some forms, the Fe regions of the second and third polypeptide chains are human Fe lgG1 regions, and wherein the Fe regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human lgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A.In some embodiments, the second polypeptide chain further comprises a first Fe region linked to Chi, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Chs; wherein the third polypeptide chain further comprises a second Fe region linked to Chi, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Chs; wherein the first and second Fe regions are human lgG1 Fe regions; and wherein the first and second Fe regions each comprise amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human lgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A.In some embodiments, the Fe regions of the second and third polypeptide chains are human lgG1 Fe regions, and the Fe regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human lgG1 according to the EU index, where the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fe regions of the second and third polypeptide chains are human lgG4 Fe regions, and the Fe regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human lgG4 according to the EU index, where the amino acid substitutions are F234A and L235A.In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fe region linked to Chi, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Chs, and a third polypeptide chain further comprising a second Fe region linked to Chi, the second Fe region comprising an immunoglobulin hinge region and constant domains of. LPOfrnn / zznz / E / YiAi immunoglobulin heavy chain Chí and Ch3; ​​and wherein the first and second Fe regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human lgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1, wherein the second Fe region is a human Fe lgG4 region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368,407 and 409 of human lgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fe region linked to CH1, wherein the first Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fe region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K; and wherein the third polypeptide chain further comprises a second Fe region linked to CH1,wherein the second Fe region is a human lgG4 Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fe region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K. In some embodiments, the Fe region is a human IgG4 Fe region comprising one or more mutations that reduce or eliminate the Fcyl and / or Fcyll binding. In some embodiments, the Fe region is a human IgG4 Fe region comprising one or more mutations that reduce or eliminate the Fcyl and / or Fcyll binding but do not affect the FcRn binding. In some embodiments, the Fe region is a human IgG4 Fe region comprising amino acid substitutions at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P and / or R409K. In some embodiments, the Fe region is a human IgG4 Fe region comprising amino acid substitutions at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In some forms, the amino acid substitutions are F234A and / or L235A.In some forms, the Fe region is a human Fe lgG4 region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235 and / or 409 of. LPOfrnn / zznz / E / YiAi human lgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A and / or R409K. In some embodiments, the Fe region is a human lgG4 Fe region comprising amino acid substitutions at positions corresponding to positions 233-236 of human lgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fe region is a human lgG4 Fe region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236 and / or 409 of human lgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion in 236; and / or R409K. In some forms, the Fe region comprises one or more mutations that reduce or eliminate Fe receptor binding and / or the effector function of the Fe region (e.g., Fe receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)). In some embodiments, the Fe region is a human Fe lgG1 region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fe region is a human Fe lgG1 region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human lgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S. In some embodiments, a binding protein of the present disclosure comprises one or more mutations to enhance the stability, e.g., of the hinge region and / or the dimer interface of lgG4 (See, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K.In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fe region linked to Ow, the first Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch1 and Ch3, and a third polypeptide chain further comprising a second Fe region linked to Chi, the second Fe region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains Ch2 and Ch3; ​​wherein the first and second Fe regions are human lgG4 Fe regions; and wherein the first and second Fe regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human lgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K.In some embodiments, a binding protein of this disclosure comprises button-and-loop mutations and one or more stability-enhancing mutations. In some embodiments, the first and / or second Fe region are human Fe lgG4 regions. In some forms, the Fe region is a human lgG1 Fe region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235 and / or 329 of lgG1 LPOfrnn / zznz / E / YiAi human according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A and / or P329A. In some embodiments, the Fe region is a human lgG1 Fe region comprising amino acid substitutions at positions corresponding to positions 298, 299 and / or 300 of human lgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A and / or Y300S. Nucleic acids and vectors Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides that form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Coid Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications, as is commonly achieved in the art, or as described herein. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, administration, and patient treatment. In some modalities, the isolated nucleic acid is operatively linked to a heterologous promoter for the direct transcription of the nucleic acid sequence encoding the binding protein. A promoter can refer to nucleic acid control sequences that direct the transcription of a nucleic acid. A first nucleic acid sequence is operatively linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operatively linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence.Examples of promoters may include, but are not limited to, promoters derived from viral genomes (such as polyomavirus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus, simian virus 40 (SV40), and the like), heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, heat shock promoters, and the like), the CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), the phosphoglycerate kinase (PGK) promoter, a tetracycline inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, and the tre system, the main lambda phage operator and promoter regions, the 3-phosphoglycerate kinase promoter, the yeast acid phosphatase promoters, and the yeast alpha mating factor promoter.The polynucleotides encoding the binding proteins of this disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or any other suitable promoter readily recognized by a person skilled in the art. LPOfrnn / zznz / E / YiAi In some modalities, the isolated nucleic acid is incorporated into a vector. In some modalities, the vector is an expression vector. Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed. The term regulatory sequence includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, alpha-fetoprotein, insulin, and the like) and enhancer sequences from a eukaryotic virus (such as the SV40 enhancer on the late side of the origin of replication (bp 100–270), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer on the late side of the origin of replication, adenovirus enhancers, and the like).Examples of suitable vectors include plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.). Expression vectors can be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral DNA and plasmid vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest. Host cells Other aspects of this disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. For example, a host cell or cell line may be used to produce a product of this disclosure. In some modalities, an isolated host cell from the present disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. califi cell). In some embodiments, the host cell is a yeast cell (e.g., an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g., 293 or 293 subcloned cells for growth in suspension culture), Exp1293™ cells, and Chinese hamster ovary cells. (CHO), baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, cells FS4, a human hepatoma cell line (e.g., Hep G2) and myeloma cells (e.g., NS0 and Sp2 / 0 cells). LPOfrnn / zznz / E / YiAi In some embodiments, a host cell or cell line of this disclosure is grown in continuous cell culture, e.g., in a stirred-tank bioreactor. In some embodiments, a host cell or cell line of this disclosure is grown in batch cell culture. Suitable batch cell culture equipment, techniques, and stirred tanks are known in the art; illustrative, non-limiting descriptions are provided in the Examples below. Suitable illustrative conditions and techniques for producing any of the products described herein using a host cell or cell line described herein (e.g., above) are known in the art. Other aspects of this disclosure relate to a method for producing any of the binding proteins described herein. In some embodiments, the method includes: a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acids, vectors, and / or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions to express a protein are well known to those skilled in the art. Methods for isolating proteins from cultured host cells are well known to those skilled in the art, including, for example, by affinity chromatography (e.g.,, two-stage affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography). Examples The following examples illustrate specific methods of disclosure and various uses thereof. They are provided for explanatory purposes only and should not be interpreted as limiting the scope of the invention in any way. Example 1: Identification and clonal variation of mismatched strands for asymmetric trispecific binding proteins Multispecific IgG antibodies with asymmetric constructs have become widely used formats for therapeutic applications in recent years. The correct subunit assembly in this class of therapeutic agents is a critical quality attribute (CQA) with a direct impact on biological activity. Therefore, early development efforts (e.g., clone selection) should be guided by information on potential chain mismatches to enable timely decision-making and risk mitigation. Mismatched antibodies may have suboptimal levels of expected biological activity and are considered product-related impurities. Removing such mAb-related impurities requires additional purification steps after the protein A (ProA) column, increasing both the cost and timeline of the process. Therefore, any potential chain mismatches must be identified and monitored during early development, starting with cell line development. This requires a high-throughput analytical platform capable of screening a large number of samples for levels of LPOfrnn / zznz / E / YiAi incorrect pairing with a fast response time. Intact protein mass spectrometry has been used for the identification and relative quantification of mismatched species in bispecific IgG. Several different intact protein MS approaches based on reversed-phase separation or size-exclusion liquid chromatography have been reported for denaturation and native MS analysis (Wang, C. et al. MAbs 2018, 10(8), 1226-1235; Schaefer, W. et al. MAbs 2016, 8 (1), 49-55; Schachner, L. et al. Anal Chem 2016, 88 (24), 12122-12127; Yin, Y. et al. MAbs 2016, 8 (8), 1467-1476). These approaches typically require antibody purification via protein A affinity prior to LC-MS analysis. The following examples describe a high-throughput analytical platform consisting of denaturing size-exclusion liquid chromatography (SEC) coupled to QToF MS for intact MS analysis of mAb species, such as the trispecific binding protein shown in Figure 1A. This intact mass method can be performed directly on clarified collected fluid without prior purification or sample preparation. This method can be performed directly on collected cell culture fluid prior to protein A purification to provide firsthand, unbiased information on the types and distribution of cell-generated mAb-related species, taking into account clone performance and characteristics, and growth conditions.This analytical platform has enabled the screening of a large number of CHO (Chinese hamster ovary) cell clones expressing different tsAb constructs for mismatched pairing and antibody media levels. The results of this intact mass analysis method, particularly at early development, facilitate the selection of CHO clones that produce tsAbs of suitable product quality. Materials and methods Antibody expression and purification Trispecific antibodies (tsAbs) were generated by co-expression of the four subunits in a single CHO host cell system using either batch culture in spin tubes or the fed-batch process in the ambr® bioreactor system. The titer was determined in the collected fluid using Octet with ProA biosensors. Samples collected from clarified cell culture were stored frozen at -80 °C prior to mass spectrometry analysis. Purification of the tsAbs from the clarified cell culture fluids was performed using protein A spin columns in a 96-well plate format with a Tecan liquid handling system, followed by concentration measurement using Octet. IdeS digestion of antibodies To a 10 pL solution of tsAb at a concentration of ~2 mg / mL, 20 pL of 25 mM Tris buffer with pH 7.2 were added. The sample was mixed with a 1.5 pL (66.6 units / pL) IdeS enzyme solution from Genovis Inc. (Cambridge, MA, USA) followed by incubation at 37 °C for two hours. LPOfrnn / zznz / E / YiAi Size exclusion chromatography (SEC) for mass spectrometry (MS) Size exclusion liquid chromatography (LC) separation was performed using a BEH SEC UPLC column, 1.7 pm, 200 Å, 4.6 x 300 mm, controlled by an Acquity UPLC H-Class (Waters Corp., Milford, MA, USA). For purified ProA antibody samples, 0.1% trifluoroacetic acid (TFA) in 30:70 acetonitrile:water was used as the mobile phase at a flow rate of 0.2 mL / min for 15 minutes. For clarified collected samples, 0.05% formic acid (FA) and 0.05% TFA in 30:70 acetonitrile:water were used at flow rates of 0.1 mL / min (from 0 to 25 minutes) and 0.4 mL / min (from 25 to 33 minutes) in isocratic mode. Both FA and TFA were LC-MS grade from Sigma (St. Louis, MO, USA). The LC-MS water and acetonitrile were from Thermo Fisher Scientific (Waltham, MA, USA). Mass spectrometry Liquid chromatography (LC) was coupled to a Xevo G2 QToF mass spectrometer (Waters Corp., Milford, MA, USA) for online intact MS data acquisition in sensitivity mode and in the m / z range of 500-4000 using the following source parameters: capillary voltage of 3 kV, sampling cone voltage of 40 V, extraction cone voltage of 2.5 V, source temperature of 150 °C, desolvation temperature of 500 °C, cone gas flow of 50 L / h, desolvation gas flow of 800 L / h, collision energy of 6 V. Data analysis The acquired raw LC-MS files were discontinuously processed using Byos® (Protein Metrics Inc.) for deconvolution and relative quantification. Results Mismatching of heavy and light chains in trispecific antibodies results in mass shifts from the expected mass of the correctly matched species, which are easily detectable by mass spectrometry of intact proteins. The only exception is potential scrambled light chain molecules, which have the same mass as the correctly matched structure and are therefore indistinguishable by mass spectrometry (Figure 1B). Intact MS analysis of the collected cell culture fluid is hampered by the complex matrix and requires effective chromatographic separation in conjunction with MS detection. Specifically, tsAb species suffer from ion suppression due to the overwhelming amount of free CODV (~36 kDa) and Fab (~23 kDa) light chains in the collectant and their inherently low ionization efficiency due to their large size (up to 210 kDa for some mismatched species). Therefore, optimization of chromatography parameters, including flow rate, mobile phase modifiers and their organic content, SEC column pore size and protein loading, as well as MS source parameters, was carried out during the development of the SE-UPLC-MS Intacta method to obtain high-quality MS data from direct injection of the collection fluid without sample preparation. An initial workflow included automated proA purification of tsAb from the clarified collection, followed by SEC-LC denaturation along with QToF MS for intact MS data acquisition. SEC denaturation allows for rapid (15 minutes) and efficient desalination, as well as the LPOfrnn / zznz / E / YiAi separation of tsAbs from overexpressed light chains that are often co-purified with mAbs. Figure 2A shows the deconvolved mass spectrum of a CD38 TCE tsAb, with the mass at 174188 Da corresponding to the desired set, H1L1 / H2L2. Two other observed masses at 160683 Da and 187692 Da match the theoretical masses of the mispaired light chain species H1L1 / H2L1 and H1L2 / H2L2, respectively. To further verify the structural assignment of species, the tsAb sample was digested with IdeS, a cysteine ​​protease that digests antibodies at a specific site below the hinge, generating a cluster of F(ab')2 and Fc / 2 fragments. As seen in Figure 2B, three different Fab fragments with the same mass shift pattern observed in the intact molecules appear after IdeS digestion, further confirming the mispaired light chain assignment. The development of cell lines for protein-based therapeutic agents, including antibodies, is fundamental to early-stage development. A key objective is to generate a stable monoclonal cell line (commonly CHO cells) that can consistently express the target recombinant protein in high quantities and with the desired product quality attributes, such as glycosylation, charge heterogeneity, etc. (Chusainow, J. et al. Biotechnol Bioeng 2009, 102 (4), 118296). In the context of multispecific antibodies, the potential for mispairing of subunits is a critical quality attribute (CQA) that must be monitored during the selection of final production groups and clones. Intact MS analysis of ProA-purified materials from 38 CHO cell clones expressing anti-CD38 TCE using the denaturing intact SEC-MS method was performed to determine the types and relative levels of impurities associated with the product, including mismatched strands and antibodies co-purified with the desired tsAb form. The relative levels of different species were calculated based on the mass intensities after deconvolution of the combined MS spectra acquired through the tsAb chromatographic peak.Although different IgG-related species can vary widely in mass, especially half-antibodies compared to full-length tsAb structures, and therefore in their ionization efficiencies, MS-based relative species quantification is an effective tool for comparing and ranking clones based on their mismatched and half-antibody levels. Figure 3A shows the purity percentage (i.e., the percentage of correct mass) in different clones of an anti-CD38 TCE construct grown using batch CHO cell culture. A high degree of variability in purity, ranging from 90% to 10%, was observed among the clones. Furthermore, the relative levels of mismatched species and antibody media varied significantly from clone to clone, as shown in Figure 3C. Moreover, alignment of the purity results with clone productivity values ​​(Figure 3B), measured by titer, demonstrates the absence of correlation between the two, meaning that highly productive clones may produce a high degree of mismatched, erroneously matched, and incompletely assembled mAb species. These results indicate the importance of screening tsAb clones to detect levels of mismatched pairing and using that information in conjunction with productivity and other cultivation parameters. LPOfrnn / zznz / E / YiAi cell for the development of cell lines and the selection of the best production clones. Example 2: Direct analysis of intact MS from the clarified harvest In order to decrease the cost and delivery time of clone screening and streamline the workflow, the parameters of the denaturing SEC-MS method were optimized to allow intact MS measurement of mAb species directly in the clarified cell culture fluid, generated during cell line development, avoiding any prior purification or sample handling. Intact MS analysis of cell culture fluid is hampered by the complex matrix, particularly the presence of non-ionic surfactants such as Pluronic F-68, commonly added to the medium to protect cells from hydrodynamic damage. This can lead to co-elution and ion suppression during MS analysis. Furthermore, tsAb species suffer from ion suppression due to the overwhelming amount of free CODV (~36 kDa) and Fab (~23 kDa) light chains overexpressed in the collected sample, as well as the inherently low ionization efficiency of tsAbs due to their large size (up to 210 kDa for some mismatched species). Chromatographic separation of proteins from surfactants in reversed-phase stationary (RP) systems commonly used for intact protein MS can be quite challenging, if not impossible, due to the hydrophobicity of both species.Furthermore, a given RP-LC method exhibits different selectivity behaviors for different tsAbs depending on the amino acid sequences and cannot be used as a platform method for multiple trispecific antibody constructs. In this example, SEC-LC under denaturing conditions was successfully employed for the size-based separation of small-molecule antibody and surfactant species in clarified cell culture fluids. Liquid chromatography parameters, including flow rate, mobile phase modifiers, and organic content, as well as MS source parameters, were optimized for the acquisition of high-quality MS data on tsAb species in the collected sample, comparable to the purified ProA material. The optimized denaturing SEC method efficiently separated mAb-related species, including the overexpressed CODV light chain and tsAb and Fab light chain, from each other and from surfactant species, as seen in the base-peak chromatogram in Figures 4A and 4B. Surfactants are eluted last in the chromatogram, and the flow is diverted to the residues to avoid contamination of the MS source (Figure 4A). This SEC-MS method is a product-nonspecific platform and enables rapid, high-throughput screening of a large number of clones in different tsAb constructs. Furthermore, the developed method can be applied to other mAb-related modalities, including Fe-free antibody formats such as the scFv2 and Fab fusion proteins (Brinkmann, U. and Kontermann, RE MAbs 2017, 9 (2), 182-212), which cannot be purified using automated ProA purification systems. The smallest mass difference between two incorrectly paired species in the anti-CD38 TCE construct is 810 Da, and in the anti-HER2 TCE construct it is 1095 Da, well below the mass resolution power of the method. To demonstrate the method's ability to resolve incorrectly paired species that are closer in mass, the collected samples The clarified LPOfrnn / zznz / E / YiAi samples from both constructs were mixed in a 1:1 ratio to generate a more complex mixture of mismatched species. Both samples contained H1L1 / H2L1 species with a difference in theoretical mass values ​​of 302 Da for CD38 TCE and anti-HER2 TCE. As shown in Figure 4C, online SEC-MS analysis of the collected sample fully resolved these two species and their glycoforms, marked by mass changes of 162 Da. Next, the repeatability, reliability, and robustness of the MS signal were evaluated using clarified collected fluids from two different tsAb constructs: TCE anti-HER2 and TCE anti-CD38, with titers of 435 and 1100 pg / mL, respectively. Figure 5A shows that the relative levels of three different tsAb assemblies are consistent across different column loadings achieved by increasing the injection volume from 10 to 50 pL. These results demonstrate the robustness and reliability of MS quantification in the column loading range of 4–55 pg. Therefore, online SEC-MS with a fixed injection volume of clarified collected fluid can be used for clones with both low and high titers, typically in the 100–1100 pg / mL range. The repeatability of the method evaluated by triplicate LC-MS analysis at each protein loading level through two tsAb constructs in Figures 4A-4C returned %RSD values ​​below 10% (Table A). Table A. Relative levels of incorrectly matched and reference masses and accuracy results for triplicate analysis of collected TCE anti-CD38 and TCE anti-HER2 samples at different column loadings. LPOfrnn / zznz / E / YiAi Ref. mass H1L1 / H2L1 H1L2 / H2L2 tsAb Construction Collected Injection Volume (PL) Column Loading (pg of protein) Median % RSD Median % RSD Median na % RSD TCE antiHER2 10 4.4 57.9 0.5 26.0 0.7 16.2 1.1 20 8.7 55.7 0.4 27.0 1.8 17.3 2.8 30 13.1 55.9 0.8 26.9 1.7 17.2 2.0 40 17.4 55.8 1.0 27.0 1.0 17.1 2.6 50 21.8 57.5 3.5 25.8 6.3 16.7 2.3 TCE antiCD38 10 11.0 83.3 0.7 6.3 2.6 10.4 4.3 20 22.0 83.0 1.2 6.6 10.4 10.2 3.1 30 33.0 81.9 0.7 6.8 9.3 10.0 6.4 40 44.0 82.7 0.7 7.0 6.0 10.0 6.2 50 55.0 82.8 0.9 7.2 8.8 10.0 6.5 Furthermore, the comparison of the results of the quantification of incorrect pairing obtained by the SEC-LC-MS Intacta analysis of the clarified harvested material and the purified ProA materials showed a close agreement between the two methods, further confirming the suitability of direct analysis of intact MS from the harvest for the determination of incorrect pairing (Figures 5B and 5C). The SEC-MS method was used for high-throughput screening of 50 anti-HER2 TCE clones. In this study, the correctly assembled molecule (175,576 Da) was found to be the predominant form, while the mismatched light chain species H1L1 / H2L1 (162,187 Da) and H1L2 / H2L2 (188,965 Da) were detected at varying levels in all samples tested. No or very low levels of half-antibody were detected in the tested clones. Similar to anti-CD38 TCE, no correlation was observed between mismatch levels and clone productivity. (Figures 5D & 5E). Example 3: Effect of cell culture growth conditions on incorrect strand pairing Two different sets of studies were conducted to investigate the effect of cell growth conditions on chain mismatch and antibody half-formation in tsAbs. In the first study, anti-CD38 TCE clones were cultured under a) batch culture and harvested on day 10 and b) and c) two different growth conditions in the ambr® microbioreactor. Intacta SEC-MS analysis (ProA purified materials) showed variations in the distribution of mismatched antibodies and media in each clone cultured under three different conditions (Figures 6C-6E). However, clone classification based on total impurity levels generally remained unchanged regardless of the growth condition, as shown in Figure 6C. Similar findings were obtained in the anti-HER2 TCE study, where clarified collected fluid from selected clones grown under batch culture conditions (day 10 collection) and in the ambr® microbioreactor was analyzed by Intacta SEC-LC-MS for their relative levels of mismatch. While the distribution of mismatched species in each given clone varied between batch culture and ambr conditions (Figures 6F and 6G), clone classification based on total mismatch was generally similar between the two growth conditions (Figure 6B). These results underscore the importance and validity of initial clone selection based on mismatch data despite inevitable changes in process conditions in the later stages of drug development. It is worth noting that, in both studies, there was greater variation in the performance of correctly matched tsAbs among clones cultured in ambr® compared to batch culture; marked by purity ranges of 10–90% vs. 47–80% for CD38 TCE and 46–70% vs. 45–58% for anti-HER2 TCE (Figures 6A and 6B). Interestingly, in both cases, the superior clones appeared to perform better, suggested by a 10–20% higher purity, in the ambr bioreactor than in batch culture. Conclusions Misassembled species are product-related impurities that commonly occur during the manufacture of multispecific antibodies, leading to decreased production yields and requiring costly purification steps, as well as robust analytical methods for early-stage monitoring. In the previous Examples, a high-throughput analytical platform based on denaturing Intacta SEC-LC-MS was described for the identification and relative quantification of chain mispairing and other IgG-related species. The method developed by LPOfrnn / zznz / E / YiAi allows for intact MS analysis of mAb-related species directly in the clarified collected sample, avoiding the slow and costly ProA purification and buffer exchange steps (Figure 7). Analysis of intact MS of two different tsAb constructs—anti-CD38 TCE and anti-HER2 TCE—from different CHO cell clones showed variations in the yield level of correctly matched tsAb from clone to clone, with no apparent correlation between clone productivity (titer) and mismatched matching. Furthermore, different growth conditions can affect the types and distribution of impurities, but not the quality classification of the clones, confirming the validity of the initial clone selection based on mismatched matching data, despite the inevitable changes in process conditions in the later stages of drug development. Example 4: Intact mass analysis of the collected sample for trispecific binding proteins Intact protein mass spectrometry has been used for the identification and relative quantification of biotherapeutics, their variants, and product-related impurities by others in the past. Several different intact protein MS approaches based on inverted phase separation or size-exclusion LC have been reported for denaturing and native MS analysis (Xu, L. et al. (2017) Science 358:85-90; Ridgway, JB et al. (1996) Protein Eng. 9:617-621; Wang, C. et al. (2018) MAbs 10:1226-1235; Schaefer, W. et al. (2016) MAbs 8:49-55). These approaches typically require antibody purification by protein A affinity prior to LC-MS analysis. The preceding examples describe a high-throughput analytical platform consisting of denaturing size-exclusion liquid chromatography (SEC) coupled with QToF mass spectrometry for intact mass spectrometry analysis of chain mismatch in trispecific antibodies (tsAbs). See also Tousi, F. et al. (2020) Anal. Chem. 92:2764-2769. This intact mass spectrometry method can be performed directly on the clarified collected fluid without prior purification or sample preparation. This analytical platform has enabled the screening of a large number of CHO (Chinese hamster ovary) cell clones expressing different tsAb constructs, based on their levels of mismatched species and antibody media. The results of this intact mass spectrometry method, particularly in the early stages of development, facilitate the selection of CHO clones that produce tsAbs of acceptable product quality. Examples 4 and 5 describe applications of the same analytical method to clone selection for different mAb-related constructs: a trispecific anti-HIV\CD28\CD3 antibody (Example 4) and a cysteine-modified monoclonal antibody (mAb) (Example 5). Materials and methods Size exclusion chromatography (SEC) Size exclusion LC separation was performed using a BEH SEC UPLC column, 1.7 pm, 200 Å, 4.6 x 300 mm, controlled by an Acquity UPLC H-Class (Waters Corp., Milford, MA, USA). A 0.05% formic acid (FA) and 0.05% triglyceride (TFA) solution in 30:70 acetonitrile:water was used as the mobile phase at flow rates of 0.1 mL / min (0 to 25 minutes) and 0.4 mL / min (25 to 33 minutes) in isocratic mode. A 50 pL volume of clarified sample was injected directly into the LC-MS system using an Acquity autosampler set to 4 °C. Both FA and TFA were LC-MS grade. LQOfrnn / zznz / E / YiAi from Sigma (St. Louis, MO, USA). The LC-MS water and acetonitrile were from Thermo Fisher Scientific (Waltham, MA, USA). Mass spectrometry (MS) The LC was coupled to a Xevo G2 (or G2XS) QToF mass spectrometer (Waters Corp., Milford, MA, USA) for online intact MS data acquisition in sensitivity mode and in the m / z range of 500-4000. The electrospray ionization (ESI) parameters for the Xevo G2 instrument included: capillary voltage of 3 kV, sampling cone voltage of 40 V, extraction cone voltage of 2.5 V, source temperature of 150 °C, desolvation temperature of 500 °C, cone gas flow rate of 50 L / h, desolvation gas flow rate of 800 L / h, and collision energy of 6 V. The ESI parameters for the Xevo G2XS were as follows: capillary voltage of 3 kV, sampling cone voltage of 200 V, source temperature of 1500 °C, source compensation of 150, desolvation temperature of 250 °C, cone gas flow rate of 50 L / h, desolvation gas flow rate of 800 L / h, and collision energy of 6 V. Data analysis The acquired LC-MS raw files were discontinuously processed using Byos® (Protein Metrics Inc.) for deconvolution, annotation, and relative quantification. Results Samples collected from clarified cell cultures of 50 CHO cell clones expressing anti-HIV\CD28\CD3 tsAb were analyzed using the previously described denaturing Intacta SEC-MS method. Analysis of the acquired raw MS files revealed a high degree of variability in mismatch rates (%) among the tested clones, ranging from as little as 1% to 100%. H1L1 / H2L1 (165–167 kDa) was the dominant mismatched species. A small number of clones contained the mismatched H1L2 / H2L2 form. The H2L2 / H2L2 homodimer was detected in some clones. Half antibodies (H1L1 and H2L2) were present in some clones, but at low levels. Unlike previously tested trispecific antibodies, no significant amount of H1L2 / H2L2 mismatch type was detected in HIV TCE clones.Figures 8A and 8B show the intact deconvolved mass spectra for anti-HIV\CD28\CD3 clones with the highest and lowest levels of strand mismatch, respectively. Figures 9A and 9B show clone productivity, measured by protein A titer and relative levels of correct mass, for 50 cell clones producing trispecific antiHIV\CD28\CD3 antibodies grown under batch culture conditions during the clone selection stage as part of cell line development. The mismatch analysis results, along with the productivity data, allowed for the selection of clones with the highest titers and the lowest levels of chain mismatch. Interestingly, several clones with relatively high productivities showed 0% correctly matched tsAb, as seen at the far right of the column plots in Figure 9B. Excellent chromatographic separation combined with the use of a highly sensitive mass spectrometer allowed for the unraveling of the extremely heterogeneous glycan profile at the intact level, due to the presence of sialized N-glycans in the Fab LC, in addition to the usual heavy-chain Fe N-glycans. This enables rapid monitoring of N-glycosylation profiles in clones without the need for more elaborate glycan analyses using methods that are generally labor-intensive and time-consuming.As an example, Figures 10A and 10B show the detailed glycan profile for the reference mass (H1L1 / H2L2) in a selected clone producing anti-HIV\CD28\CD3 grown under two different conditions: 1) in a spin tube (batch condition) and 2) in an ambr15 bioreactor (fed batch condition), respectively. These figures show the shift toward more galactosylated and sialylated N-glycans, evidenced by the shift toward higher mass values. Due to the extreme glycan heterogeneity added by light-chain glycosylation, the molecular weight of the reference form (H1L1 / H2L2) and the mismatched form H1L1 / H2L1 spans the range of 1000 Da and 2000 Da, respectively. The assignment of glycoforms to the intact anti-HIV\CD28\CD3 reference form is detailed in Table 1. Table 1. Observed glycoform assignment for trispecific antibody. Observed mass (Da) Theoretical mass (Da) △mass (Da) ID 178214 178210 4 H1L1 / H2L2 (G0F / G0F / G0) 178375 178372 3 +Gal 178532 178534 -2 +Gal 178668 178663 5 178372 Da + NeuAc 178827 178825 2 +Gal 178985 178987 -2 +Gal 179121 179116 5 178827Da+NeuAc 179279 179278 1 +Gal 179440 179440 0 +Gal 179592 579586 6 +Fuc Example 5: Intact mass analysis of collected sample for cysteine-modified antibodies This product is a thiomAb, meaning an antibody comprising an additional modified cysteine ​​residue in its Fe region, useful, e.g., for compound conjugation (e.g., a cytotoxic drug to form an antibody-drug conjugate). This additional cysteine ​​is unpaired (free), meaning it does not participate in disulfide bonds with other cysteines in the mAb molecule. Previous work had shown that this additional cysteine ​​(Cys293) can form unwanted disulfide bonds with nearby cysteine ​​residues, also known as disulfide bond revolution, which could lead to structural instability of the antibody. Uncontrolled sulfhydryl chemistry can also pose a significant manufacturing risk during processing to form the ADC, leading to an undesirable drug conjugation profile.To avoid these risks, it is imperative to have the free cysteine ​​terminally protected with disulfide-linked modifications, such as cysteinylation and glutathionylation, by adjusting the cell culture conditions. These modifications can be selectively reduced to generate free cysteine ​​before the drug conjugation step. In this example, the denaturing intact SEC-MS developed from the cell culture collection was used for rapid screening of clones to determine their Cys293 terminal protection status during cell line development. The first tioAb candidate studied had an N300A mutation in the Fe region that ablated Fe N-glycosylation. The absence of glycans allowed for the identification of Cys293 terminal protection by direct analysis of intact MS from the collected sample without any manipulation (Figure 11A). However, the second candidate, thiomAb, with a typical Fe N-glycosylation profile, showed a highly complex pattern in intact MS data due to the presence of multiple terminally protected species, including N-acetyl cysteinylation (+161 Da), which interfered with mass displacement due to the addition of hexose monosaccharide residues (+162 Da) to the N-glycans. To circumvent this problem and facilitate data interpretation, the clarified collectant of the second candidate was treated with PNGase F to remove the N-glycans under native conditions. The deglycosylated collectant was subsequently subjected to Intacta SEC-MS as before.Figure 11B shows the intact deconvoluted mass spectrum obtained for the second candidate after deglycosylation, with three types of terminal protection modifications identified: Cysteinylation (Cys) with a mass shift of +119 Da, N-acetyl cysteinylation (N-acetyl Cys) with a mass shift of +161 Da, and Glutathionylation (Glu) with a mass shift of +305 Da. Screening of 22 CHO cell clones expressing thiomAb with this method showed 100% terminal protection of the modified cysteine ​​in all tested clones.

Claims

1. A method for monitoring the production of a multispecific binding protein and one or more mismatched species, the method comprising: detecting, by size exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of a multispecific binding protein and one or more mismatched species in a cell culture medium comprising the multispecific binding protein and one or more mismatched species, thereby monitoring the production of the multispecific binding protein and one or more mismatched species;wherein the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, and wherein one or more mismatched species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

2. The method according to claim 1, wherein the multispecific binding protein is a multispecific antibody comprising a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain.

3. The method according to claim 2, wherein one or more mismatched species comprises one or more of: (i) an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody heavy chains; (ii) an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody heavy chains; (iii) an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody light chains; and (iv) an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody light chains.

4. The method according to claim 1, wherein the multispecific binding protein comprises four polypeptide chains forming the three antigen-binding sites, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula: VL2-L1-VL1-L2-CL [I] and a second polypeptide chain of the binding protein comprises a structure represented by the formula: LPOfrnn / zznz / E / YiAi VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II] and a third polypeptide chain of the binding protein comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III] and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula: VL3-CL [IV] wherein: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain;VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain CH1; CH2 is an immunoglobulin heavy chain constant domain CH2; CH3 is an immunoglobulin heavy chain constant domain CH3; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3, and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-heavy chain pair, wherein VH1 and VL1 form a first antigen-binding site, wherein VH2 and VL2 form a second antigen-binding site, and wherein VH3 and VL3 form a third antigen-binding site.

5. The method according to claim 4, wherein one or more mismatched species comprise one or more of: (i) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula I; (ii) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula II; (iii) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula III; and (iv) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula IV.

6. The method according to any of claims 1-5, wherein detecting the amount of multispecific binding protein and one or more incorrectly paired species comprises deconvolving one or more MS spectra obtained by MS.

7. The method according to any of claims 1-6, wherein a relative amount of the multispecific binding protein is detected compared to one or more incorrectly paired LPOfrnn / zznz / E / YiAi species.

8. The method according to claim 7, wherein a relative amount of multispecific binding protein is detected compared to an amount of one or more mismatched individual species.

9. The method according to claim 7, wherein a relative amount of multispecific binding protein is detected in comparison to a total amount of incorrectly paired species.

10. The method according to any of claims 1-9, wherein the cell culture medium is a cell culture collectible.

11. The method according to claim 10, further comprising, prior to detection, separating the cell culture medium from a cell line that produces the multispecific binding protein and one or more incorrectly paired species.

12. The method according to claim 11, wherein, prior to (a), the cell culture medium is separated from the cell line by centrifugation.

13. The method according to any of claims 1-12, wherein the cell culture medium is subjected to SE-UPLC without prior chromatographic separation.

14. The method according to any of claims 1-12, wherein the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography.

15. The method according to any of claims 1-14, wherein MS is intact MS.

16. The method according to any of claims 1-15, wherein MS is quadrupole time-of-flight (QToF) MS.

17. The method according to any of claims 1-16, wherein SE-UPLC is SE-UPLC denaturing.

18. The method according to any of claims 1-17, wherein the SE-UPLC is directly coupled to the MS.

19. The method according to any of claims 1-18, wherein SE-UPLC is performed with an initial flow rate of less than approximately 0.4 mL / min.

20. The method according to claim 19, wherein SE-UPLC is performed with an initial flow rate of approximately 0.1 mL / min.

21. The method according to claim 20, wherein SE-UPLC is performed with a flow rate of approximately 0.1 mL / min for the first 25 minutes, followed by a flow rate of approximately 0.4 mL / min.

22. The method according to any of claims 1-21, wherein SE-UPLC is performed by isocratic elution with a mobile phase.

23. The method according to claim 22, wherein the mobile phase comprises a solution comprising acetonitrile:water 30:

70.

24. The method according to claim 22 or claim 23, wherein the mobile phase LPOfrnn / zznz / E / YiAi comprises formic acid and trifluoroacetic acid (TFA).

25. The method according to claim 24, wherein the mobile phase comprises approximately 0.05% formic acid and approximately 0.05% trifluoroacetic acid (TFA).

26. The method according to any of claims 1-25, wherein the detection is carried out in approximately 33 minutes or less.

27. The method according to any of claims 1-26, wherein the MS is capable of resolving a mass difference of approximately 300 Da between the multispecific binding protein and one or more mismatched species, or between two mismatched species.

28. The method according to any of claims 1-27, wherein the MS is capable of resolving a mass difference of approximately 162 Da between the multispecific or mismatched species-binding protein and one of more glycoforms.

29. The method according to any of claims 11-28, wherein the cell line is a mammalian cell line.

30. The method according to claim 29, wherein the mammalian cell line is a Chinese hamster ovary (CHO) cell line.

31. The method according to any of claims 1-30, wherein, prior to detection, the cell line is cultured with the cell culture medium in a continuous stirred-tank bioreactor culture.

32. The method according to any of claims 1-30, wherein, prior to detection, the cell line is cultured with the cell culture medium in a batch cell culture.

33. A method for producing a multispecific binding protein, the method comprising: (a) growing a cell line comprising one or more polynucleotides encoding the multispecific binding protein in a cell culture medium under conditions suitable for the production of the multispecific binding protein and one or more mismatched species by the cell line; (b) separating, from the cell line, the cell culture medium comprising the multispecific binding protein and one or more mismatched species; (c) detecting an amount of the multispecific binding protein and one or more mismatched species in the cell culture medium by ultra-performance size exclusion liquid chromatography and mass spectrometry (SE-UPLC-MS);and (d) removing at least a portion of one or more of the mismatched species from the multispecific binding protein produced by the cell line, or determining one or both of the quality and purity of the multispecific binding protein produced by the cell line; wherein the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, and wherein one or more mismatched species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association distinct from that of the multispecific binding protein.

34. The method according to claim 33, wherein the multispecific binding protein is a multispecific antibody comprising a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain.

35. The method according to claim 34, wherein one or more mismatched species comprises one or more of: (i) an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody heavy chains; (ii) an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody heavy chains; (iii) an association of four polypeptide chains of the multispecific antibody comprising two of the first antibody light chains; and (ii) an association of four polypeptide chains of the multispecific antibody comprising two of the second antibody light chains.

36. The method according to claim 33, wherein the multispecific binding protein comprises four polypeptide chains forming the three antigen-binding sites, wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula: VL2-L1-VL1-L2-CL [I] and a second polypeptide chain of the binding protein comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II] and a third polypeptide chain of the binding protein comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III] and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula: VL3-CL [IV] wherein: VL1 is a first immunoglobulin light chain variable domain; VL2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain;VH1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain CH1; CH2 is an immunoglobulin heavy chain constant domain CH2; CH3 is an immunoglobulin heavy chain constant domain CH3; hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3, and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light-heavy chain pair, wherein VH1 and VL1 form a first antigen-binding site, wherein VH2 and VL2 form a second antigen-binding site, and wherein VH3 and VL3 form a third antigen-binding site.

37. The method according to claim 36, wherein one or more mismatched species comprise one or more of: (i) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula I; (II) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula II; (iii) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula III; and (iv) an association of four polypeptide chains of the binding protein comprising two polypeptide chains according to formula IV.

38. The method according to any of claims 33-37, wherein the detection of the amount of multispecific binding protein and one or more incorrectly paired species comprises the deconvolution of one or more MS spectra obtained by MS.

39. The method according to any of claims 33-38, wherein the detection of the multispecific binding protein and one or more incorrectly matched species comprises the evaluation of the overall titer of the multispecific binding protein produced by the cell line.

40. The method according to any of claims 33-39, wherein the detection of the multispecific binding protein and one or more incorrectly paired species comprises the evaluation of the overall titer of the incorrectly paired species produced by the cell line.

41. The method according to any of claims 33-40, wherein a relative amount of the multispecific binding protein is detected compared to one or more incorrectly paired species.

42. The method according to claim 41, wherein a relative amount of the multispecific binding protein is detected compared to an amount of one or more mismatched individual species.

43. The method according to claim 41, wherein a relative amount of the multispecific binding protein is detected in comparison to a total amount of incorrectly paired species.

44. The method according to any of claims 33-43, wherein the cell culture medium LPOfrnn / zznz / E / YiAi is separated from the cell line by centrifugation.

45. The method according to any of claims 33-44, wherein the cell culture medium is subjected to SE-UPLC in (c) without prior chromatographic separation.

46. ​​The method according to any of claims 33-44, wherein the cell culture medium is subjected to SE-UPLC in (c) without prior protein A affinity chromatography.

47. The method according to any of claims 33-46, wherein MS is intact MS.

48. The method according to any of claims 33-47, wherein MS is quadrupole time-of-flight (QToF) MS.

49. The method according to any of claims 33-48, wherein SE-UPLC is SE-UPLC denaturing.

50. The method according to any of claims 33-49, wherein the SE-UPLC is directly coupled to the MS.

51. The method according to any of claims 33-50, wherein SE-UPLC is performed with an initial flow rate of less than approximately 0.4 mL / min.

52. The method according to claim 51, wherein SE-UPLC is performed with an initial flow rate of approximately 0.1 mL / min.

53. The method according to claim 52, wherein SE-UPLC is performed with a flow rate of approximately 0.1 mL / min for the first 25 minutes, followed by a flow rate of approximately 0.4 mL / min.

54. The method according to any of claims 33-53, wherein SE-UPLC is performed by isocratic elution with a mobile phase.

55. The method according to claim 54, wherein the mobile phase comprises a solution comprising acetonitrile:water 30:

70.

56. The method according to claim 54 or claim 55, wherein the mobile phase comprises formic acid and trifluoroacetic acid (TFA).

57. The method according to claim 56, wherein the mobile phase comprises approximately 0.05% formic acid and approximately 0.05% trifluoroacetic acid (TFA).

58. The method according to any of claims 33-57, wherein (c) is achieved in approximately 33 minutes or less.

59. The method according to any of claims 33-58, wherein the MS is capable of resolving a mass difference of approximately 300 Da between the multispecific binding protein and one or more mismatched species, or between two mismatched species.

60. The method according to any of claims 33-59, wherein the MS is capable of resolving a mass difference of approximately 162 Da between the multispecific or mismatched species-binding protein and one of more glycoforms.

61. The method according to any of claims 33-60, wherein the LPOfrnn / zznz / E / YiAi cell line is a mammalian cell line.

62. The method according to claim 61, wherein the mammalian cell line is a Chinese hamster ovary (CHO) cell line.

63. The method according to any of claims 33-62, wherein the cell line is grown in (a) a continuous culture of stirred tank bioreactors.

64. The method according to any of claims 33-62, wherein the cell line is grown in (a) a batch cell culture.

65. A method for screening a plurality of cell lines for the production of a multispecific binding protein, the method comprising: (a) detecting an amount of a multispecific binding protein produced by a first cell line of the plurality according to the method of any one of claims 33-64, and (b) detecting an amount of the multispecific binding protein produced by a second cell line of the plurality according to the method of any one of claims 33-64.

66. The method according to claim 65, further comprising, after (a) and (b): (c) comparing the amount of multispecific binding protein produced by the first cell line with the amount of multispecific binding protein produced by the second cell line, and (d) on the basis of the comparison, selecting the cell line that produced the greatest amount of the multispecific binding protein.

67. The method according to claim 65 or claim 66, further comprising: the detection of a quantity of one or more incorrectly paired species produced by the first cell line according to the method according to any of claims 33-64; and the detection of a quantity of one or more incorrectly paired species produced by the second cell line according to the method according to any of claims 33-64.

68. The method according to claim 67, further comprising, after detecting the quantity of one or more incorrectly paired species produced by the first and second cell lines: comparing the quantity of one or more incorrectly paired species produced by the first cell line with the quantity of one or more incorrectly paired species produced by the second cell line; and based on the comparison, selecting the cell line that produced the highest proportion of multispecific binding protein to one or more incorrectly paired species.

69. The method according to claim 68, wherein the cell line is selected based on a higher proportion of multispecific binding protein relative to the amount of one or more incorrectly matched individual species.

70. The method according to claim 68, wherein the cell line is selected based on a higher proportion of multispecific binding protein with respect to the total amount of incorrectly paired species.

71. A method for monitoring the production of an antibody or antibody derivative and one or more weight variant species, the method comprising: detecting, by size exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), a quantity of an antibody or antibody derivative and one or more weight variant species in a cell culture medium comprising the antibody or antibody derivative and one or more weight variant species, thereby monitoring the production of the antibody or antibody derivative and one or more weight variant species; wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight.

72. A method for producing a multi-specific binding protein, the method comprising: (a) growing a cell line comprising one or more polynucleotides encoding the antibody or antibody derivative in a cell culture medium under conditions suitable for the production of the antibody or antibody derivative and one or more weight variant species by the cell line; (b) separating, from the cell line, the cell culture medium comprising the antibody or antibody derivative and one or more weight variant species; (c) detecting an amount of the antibody or antibody derivative and one or more weight variant species in the cell culture medium by size exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS);and (d) eliminate at least a portion of one or more of the weight variant species of the antibody or antibody derivative produced by the cell line, or determine one or both of the quality and purity of the antibody or antibody derivative produced by the cell line; wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight.

73. The method according to claim 71 or claim 72, wherein the antibody or antibody derivative and one or more weight variant species comprise species having a free cysteine ​​that has been cysteinylated, N-acetylcysteinylated, or glutathionylated.

74. The method according to claim 71 or claim 72, wherein the antibody or antibody derivative and one or more weight variant species comprise a chemically modified cysteine ​​residue.

75. The method according to any of claims 71-74, wherein the antibody or antibody derivative comprises a cysteine ​​residue in position 293, numbered according to the EU index.

76. The method according to any of claims 71-75, wherein the antibody or antibody derivative is not N-glycosylated in the Fe region.

77. The method according to any of claims 71-76, wherein the antibody or antibody derivative comprises a mutation in the Fe region that eliminates N-glycosylation in the Fe region.

78. The method according to claim 77, wherein the antibody or antibody derivative comprises an N300A mutation, numbered according to the EU index. LPOfrnn / zznz / E / YiAi 79. The method according to any of claims 71-76, further comprising, prior to detection by SE-UPLC-MS, the removal of N-glycosylation in the Fe region.

80. The method according to claim 79, wherein the elimination of N-glycosylation comprises treating the antibody or antibody derivative with a peptide: N-glycosidase enzyme.

81. The method according to any of claims 71-80, wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 119 Da.

82. The method according to any of claims 71-81, wherein the method is capable of resolving a mass difference between the cysteinylated, N-acetylcysteinylated, and glutathionylated species.

83. The method according to claim 71 or claim 72, wherein the antibody or antibody derivative and one or more weight variant species comprise glycoforms of the antibody or antibody derivative.

84. The method of any of claims 71-83, wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 162 Da.

85. The method according to any of claims 71-84, wherein the method is capable of resolving a mass difference between the antibody or antibody derivative and one of more weight variant species representing glycoforms of the antibody or antibody derivative.

86. The method according to any of claims 71-85, wherein the antibody or antibody derivative is a monoclonal antibody.

87. The method according to any of claims 71-85, wherein the antibody or antibody derivative is a multispecific antibody or a binding protein.

88. The method according to any of claims 71-87, wherein the detection of the amount of antibody or antibody derivative and one or more weight variant species comprises the deconvolution of one or more MS spectra obtained by MS.

89. The method according to any of claims 71-88, wherein a relative amount of antibody or antibody derivative is detected in comparison with one or more weight vanant species.

90. The method according to claim 89, wherein a relative amount of antibody or antibody derivative is detected in comparison to an amount of one or more individual weight variant species.

91. The method according to claim 89, wherein a relative amount of antibody or antibody derivative is detected in comparison to a total amount of weight variant species.

92. The method according to claim 71, wherein the cell culture medium is a cell culture collection.

93. The method according to claim 92, further comprising, prior to detection, the separation of the cell culture medium from a cell line producing the antibody or antibody derivative and one or more weight variant species.

94. The method according to claim 93, wherein, prior to (a), the cell culture medium is separated from the cell line by centrifugation.

95. The method according to claim 72, wherein the cell culture medium is separated from the cell line by centrifugation.

96. The method according to any of claims 71-95, wherein the cell culture medium is subjected to SE-UPLC without prior chromatographic separation.

97. The method according to any of claims 71-95, wherein the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography.

98. The method according to any of claims 71-97, wherein MS is intact MS.

99. The method according to any of claims 71-98, wherein MS is quadrupole time-of-flight (QToF) MS.

100. The method according to any of claims 71-99, wherein SE-UPLC is SE-UPLC denaturing.

101. The method according to any of claims 71-100, wherein the SE-UPLC is directly coupled to the MS.

102. The method according to any of claims 71-101, wherein the cell line is a mammalian cell line.

103. The method according to claim 102, wherein the mammalian cell line is a Chinese hamster ovum (CHO) cell line.

104. The method according to any of claims 72-103, wherein the cell line is grown in a continuous culture of stirred tank bioreactors.

105. The method according to any of claims 72-103, wherein the cell line is grown in a batch cell culture.

106. A method for screening a plurality of cell lines for the production of an antibody or antibody derivative, the method comprising: (a) detecting an amount of antibody or antibody derivative produced by a first cell line of the plurality according to the method of any one of claims 72-105, and (b) detecting an amount of antibody or antibody derivative produced by a second cell line of the plurality according to the method of any one of claims 72-105.

107. The method according to claim 106, further comprising, after (a) and (b): (c) comparing the amount of antibody or antibody derivative produced by the first cell line with the amount of antibody or antibody derivative produced by the second cell line, and (d) on the basis of the comparison, selecting the cell line that produced the greatest amount of antibody or antibody derivative. LPOfrnn / zznz / E / YiAi 108. The method according to claim 106 or claim 107, further comprising: detecting a quantity of one or more species of weight variants produced by the first cell line according to the method according to any of claims 72-105; and detecting a quantity of one or more species of weight variants produced by the second cell line according to the method according to any of claims 72-105.

109. The method according to claim 108, further comprising, after detecting the quantity of one or more weight variant species produced by the first and second cell lines: comparing the quantity of one or more weight variant species produced by the first cell line with the quantity of one or more weight variant species produced by the second cell line; and based on the comparison, selecting the cell line that produced the highest proportion of antibodies or antibody derivatives to one or more weight variant species.

110. The method according to claim 109, wherein the cell line is selected based on a higher proportion of antibodies or antibody derivatives with respect to the amount of one or more individual weight variant species.

111. The method according to claim 109, wherein the cell line is selected based on a higher proportion of antibodies or antibody derivatives with respect to the total amount of weight variant species.