Methods for analyzing chain mispairing in multispecific binding proteins

Abstract

To provide methods for monitoring production of a multispecific binding protein and one or more mispaired species by a cell line, as well as methods of production and screening related thereto.SOLUTION: In some embodiments, the methods comprise detecting an amount (e.g., a relative amount) of a multispecific binding protein and one or more mispaired species in a cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS). In some embodiments, the multispecific binding protein is a multispecific antibody, antibody fragment, or Fc fusion protein.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 926,313, filed October 25, 2019, and European Patent Application No. EP20315271.5, filed May 28, 2020, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Submission of sequence listing in ASCII TEXT FILE The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (Filename: 183952032840SEQLIST.TXT, Data Record: May 29, 2020, Size: 2KB).

[0003] Field The present disclosure relates to methods for monitoring the production of multispecific binding proteins and one or more mispaired species by host cells, and related production and screening methods.The present disclosure further relates to methods for monitoring the production of antibodies or antibody derivatives and one or more weight variant species by host cells, and related production and screening methods. [Background technology]

[0004] Multispecific antibodies that bind to two or more different epitopes on the same or different antigens have become an attractive therapeutic option in immuno-oncology in recent years (Non-Patent Document 1). Multitargeting has been used for various purposes, such as achieving enhanced drug specificity or mimicking natural ligands in signal transduction pathways, for example in hemophilia treatment through simultaneous binding to receptor pairs on the same cell surface (Non-Patent Document 2). A prominent application is the T cell engager (TCE) concept, where one arm of the molecule activates T cells via CD3 / CD28 receptor binding, and the other arm targets tumor antigens for tumor killing (Non-Patent Document 3; Non-Patent Document 4).

[0005] IgG-like trispecific antibodies (tsAbs) contain two different heavy chains and two different light chains and are generally expressed in a single host cell, followed by intracellular chain assembly. While this production method avoids the need to use two separate cell lines and purification steps, it can generate undesired mismatched species in addition to the desired tsAbs. In the absence of rational design and random association of subunits, the theoretical yield of correctly matched species is only 12.5% ​​(Figure 1B). Forced heterodimerization of heavy chains has been successfully achieved through protein engineering approaches such as knobs-into-holes design (Non-Patent Document 5). However, cognate pairing of light chains to the correct heavy chains remains a significant challenge in tsAb production.

[0006] Thus, there remains a need for multispecific binding proteins and methods for monitoring or analyzing the production of one or more mismatched species. In addition, there remains a need for methods for monitoring or analyzing the production of antibodies or antibody derivatives that contain multiple species of different molecular weights (e.g., with different chemical modifications, such as chemically modified cysteines or other residues, or with different glycoforms).

[0007] Includes patent applications, patent publications and UniProtKB / Swiss-Prot accession numbers All references cited in this specification are incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Baeuerle, PA & Reinhardt, C., "Cancer Res 2009, 69(12)", pp. 4941-4. [Non-Patent Document 2] Kitazawa, T. et al., Nat Med 2012, 18(10), pp. 1570-4 [Non-Patent Document 3] Krah, S. et al., N Biotechnol 2017, 39(Pt B), pp. 167-173 [Non-Patent Document 4] Correnti, C.E. et al., Leukemia 2018, 32(5), pp. 1239-1243 [Non-Patent Document 5] Ridgway, JB et al., Protein Eng 1996, 9(7), pp. 617-21 Summary of the Invention [Problem to be solved by the invention]

[0009] To address these and other needs, methods are provided herein for monitoring the production (e.g., by a cell line) of multispecific binding proteins and one or more mismatched species. These methods provide, inter alia, a high-throughput analytical platform based on denaturing SEC-LC-intact MS for the identification and relative quantification of strand mismatches and other IgG-related species. Advantageously, these methods allow intact MS analysis of mAb-related species (e.g., multispecific binding proteins, antibodies, Fc fusion proteins, antibody fragments, etc.) directly from clarified cell harvests, bypassing time-consuming and expensive purification (e.g., Protein A affinity chromatography) and buffer exchange steps. These methods can then be used to rapidly screen large numbers of clones for potential producing cell lines. [Means for solving the problem]

[0010] In some embodiments, provided herein are methods for monitoring the production of a multispecific binding protein and one or more mismatched species, comprising detecting the amount of the 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 by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS). 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 the one or more mismatched species comprises 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.

[0011] 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 Fc fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody, an antibody fragment, or an Fc fusion protein. In some embodiments, the one or more mismatched species are: an association of four polypeptide chains of a multispecific antibody comprising two first antibody heavy chains; an association of four polypeptide chains of a multispecific antibody comprising two second antibody heavy chains; an association of four polypeptide chains of a multispecific antibody comprising two first antibody light chains. and two second antibody light chains; and an association of four polypeptide chains of a multispecific antibody comprising two second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains that form three antigen binding sites, The first polypeptide chain of the binding protein has the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes a structure represented by The second polypeptide chain of the binding protein has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes a structure represented by The third polypeptide chain of the binding protein has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes a structure represented by The fourth polypeptide chain of the binding protein has the formula: V L3 -C L [IV] The structure includes a structure represented by During the ceremony: 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; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; Hinge is C H1 and C. H2 an immunoglobulin hinge region that connects the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, H1 and V L1 forms the first antigen-binding site, and V H2 and V L2 forms the second antigen-binding site, and V H3 and V L3forms a third antigen-binding site. In some embodiments, the one or more mispaired species comprises one or more of: an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula IV.

[0012] In some embodiments, detecting the amount of the multispecific binding protein and one or more mismatched species comprises deconvoluting one or more MS spectra obtained by MS. In some embodiments, the relative amount of the multispecific binding protein compared to the one or more mismatched species is detected. In some embodiments, the relative amount of the multispecific binding protein compared to the amount of one or more individual mismatched species is detected. In some embodiments, the relative amount of the multispecific binding protein compared to the total amount of mismatched species is detected. In some embodiments, the method further comprises providing or obtaining a cell culture medium (e.g., from a cell line producing the multispecific binding protein and one or more mismatched species) comprising the multispecific binding protein and one or more mismatched species prior to the detection. In some embodiments, the method further comprises isolating the cell culture medium from the cell line producing the multispecific binding protein and one or more mismatched species prior to the detection. In some embodiments, the cell culture medium is centrifuged prior to the detection. The multispecific binding protein and the one or more mismatched species are separated from the cell line by SE-UPLC. In some embodiments, the cell culture medium is subjected to SE-UPLC without a preceding chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without a preceding Protein A affinity chromatography. In some embodiments, the MS is an intact MS. In some embodiments, the MS is a quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is a denatured SE-UPLC. In some embodiments, the SE-UPLC is directly coupled to the MS. In some embodiments, the method further comprises contacting the multispecific binding protein and the one or more mismatched species with a protease prior to SE-UPLC-MS. In some embodiments, the protease is IdeS or IdeZ. In some embodiments, the SE-UPLC is performed at an initial flow rate of less than about 0.4 mL / min. In some embodiments, the SE-UPLC is performed at an initial flow rate of about 0.1 mL / min. In some embodiments, SE-UPLC is performed at a flow rate of about 0.1 mL / min for the first 25 minutes, followed by a flow rate of about 0.4 mL / min (e.g., 25-33 minutes). In some embodiments, SE-UPLC is performed using an isocratic elution with a mobile phase. In some embodiments, the mobile phase comprises a solution comprising 30:70 acetonitrile:water. In some embodiments, the mobile phase comprises formic acid and trifluoroacetic acid (TFA). In some embodiments, the mobile phase comprises about 0.05% formic acid and about 0.05% trifluoroacetic acid (TFA). In some embodiments, detecting the amount of the multispecific binding protein and the one or more mismatched species is accomplished within about 33 minutes. In some embodiments, the method is accomplished within about 33 minutes. In some embodiments, the MS is capable of resolving a mass difference of about 300 Da between the multispecific binding protein and one or more mismatched species or between two mismatched species. In some embodiments, MS is capable of resolving a mass difference of approximately 162 Da between a multispecific binding protein or mismatched species and one or more glycoforms.

[0013] 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, prior to detection, the cell line is cultured in a continuous cell culture, e.g., a stirred tank bioreactor, with cell culture medium. In some embodiments, prior to detection, the cell line is cultured in a batch cell culture with cell culture medium.

[0014] In some embodiments, provided herein is a method of producing a multispecific binding protein, comprising: (a) culturing 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) isolating the cell culture medium comprising the multispecific binding protein and one or more mismatched species from the cell line; (c) detecting the amount of the 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) removing at least a portion of the one or more 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 comprises two or more polypeptide chains that include at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

[0015] 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 or an Fc fusion protein. In some embodiments, the multispecific binding protein is a triplet antibody. The mispaired species may be one or more of: a multispecific antibody comprising two first antibody heavy chains; a multispecific antibody comprising two second antibody heavy chains; a multispecific antibody comprising two first antibody light chains; and a multispecific antibody comprising two second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains that form three antigen binding sites, The first polypeptide chain of the binding protein has the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes a structure represented by The second polypeptide chain of the binding protein has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes a structure represented by The third polypeptide chain of the binding protein has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes a structure represented by The fourth polypeptide chain of the binding protein has the formula: V L3 -C L [IV] The structure includes a structure represented by During the ceremony: 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; V H1is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; Hinge is C H1 and C. H2 an immunoglobulin hinge region that connects the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-light chain heavy chain pair, H1 and V L1 forms the first antigen-binding site, and V H2 and V L2 forms the second antigen-binding site, and V H3 and V L3 forms a third antigen-binding site. In some embodiments, the one or more mispaired species comprises one or more of: an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula IV.

[0016] In some embodiments, detecting the amount of the multispecific binding protein and one or more mismatched species comprises deconvoluting one or more MS spectra obtained by MS. In some embodiments, detecting the multispecific binding protein and one or more mismatched species comprises assessing the overall titer of the multispecific binding protein produced by the cell line. In some embodiments, detecting the multispecific binding protein and one or more mismatched species comprises assessing the overall titer of the mismatched species produced by the cell line. In some embodiments, the relative amount of the multispecific binding protein compared to the amount of one or more mismatched species is detected. In some embodiments, the relative amount of the multispecific binding protein compared to the amount of one or more individual mismatched species is detected. In some embodiments, the relative amount of the multispecific binding protein compared to the total amount of mismatched species is detected. In some embodiments, the cell culture medium is separated from the cell line by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC in (c) without a preceding chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC in (c) without a preceding Protein A affinity chromatography. In some embodiments, the MS is an intact MS. In some embodiments, the MS is a quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is a denatured SE-UPLC. In some embodiments, the SE-UPLC is directly coupled to the MS. In some embodiments, the method further comprises contacting the multispecific binding protein and the one or more mismatched species with a protease after culturing the cell line and prior to detecting. In some embodiments, the protease is IdeS or IdeZ. In some embodiments, the SE-UPLC is performed at an initial flow rate of less than about 0.4 mL / min. In some embodiments, SE-UPLC is performed at an initial flow rate of about 0.1 mL / min. In some embodiments, SE-UPLC is performed at a flow rate of about 0.1 mL / min for the first 25 minutes, followed by a flow rate of about 0.4 mL / min. In some embodiments, SE-UPLC is performed using an isocratic elution with a mobile phase. In some embodiments, the mobile phase comprises a solution comprising 30:70 acetonitrile:water. In some embodiments, the mobile phase comprises formic acid and trifluoroacetic acid (TFA). In some embodiments, the mobile phase comprises about 0.05% formic acid and about 0.05% trifluoroacetic acid (TFA). In some embodiments, detection is achieved within about 33 minutes. In some embodiments, the method is achieved within about 33 minutes. In some embodiments, the MS is capable of resolving a mass difference of about 300 Da between the multispecific binding protein and one or more mismatched species or between two mismatched species. In some embodiments, MS is capable of resolving a mass difference of approximately 162 Da between a multispecific binding protein or mismatched species and one or more glycoforms.

[0017] 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 cultured in continuous cell culture (e.g., stirred tank bioreactor). In some embodiments, the cell line is cultured in batch cell culture.

[0018] In some embodiments, provided herein is a method of screening a plurality of cell lines for production of a multispecific binding protein, comprising detecting an amount of multispecific binding protein produced by a first cell line of the plurality by a method of any one of the above embodiments; and detecting an amount of multispecific binding protein produced by a second cell line of the plurality by a method of any one of the above embodiments.

[0019] In some embodiments, the method further comprises: 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 selecting a cell line that produced a higher amount of multispecific binding protein based on the comparison. In some embodiments, the method further comprises: detecting the amount of one or more mismatched species produced by the first cell line by the method of any one of the above embodiments; and detecting the amount of one or more mismatched species produced by the second cell line by the method of any one of the above embodiments. In some embodiments, the method further comprises: after detecting the amount of one or more mismatched species produced by the first and second cell lines: comparing the amount of one or more mismatched species produced by the first cell line with the amount of one or more mismatched species produced by the second cell line; and selecting a cell line that produced a higher ratio of multispecific binding protein to one or more mismatched species based on the comparison. In some embodiments, cell lines are selected based on a high ratio of multispecific binding protein to the amount of one or more individual mismatch species. The antibody is selected based on a high ratio of multispecific binding protein to amount.

[0020] In some embodiments, provided herein is a method for monitoring production of an antibody or antibody derivative and one or more weight variant species, comprising detecting the amount of an antibody or antibody derivative and one or more weight variant species in a cell culture medium containing the antibody or antibody derivative and one or more weight variant species by size exclusion ultra performance liquid chromatography and mass spectrometry (SE-UPLC-MS), wherein the antibody or antibody derivative and the one or more weight variant species differ in molecular weight. In some embodiments, provided herein are methods of producing an antibody or antibody derivative, comprising: (a) culturing a cell line comprising one or more polynucleotides encoding the antibody or antibody derivative in a cell culture medium under conditions suitable for production of the antibody or antibody derivative and one or more weight variant species by the cell line; (b) isolating the cell culture medium comprising the antibody or antibody derivative and one or more weight variant species from the cell line; (c) detecting the 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) removing at least a portion of the one or more 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.

[0021] In some embodiments, provided herein is a method of screening a plurality of cell lines for the production of an antibody or antibody derivative, comprising: detecting the amount of an antibody or antibody derivative produced by a first cell line of the plurality by the method of any one of the above embodiments; and detecting the amount of an antibody or antibody derivative produced by a second cell line of the plurality by the method of any one of the above embodiments. In some embodiments, the method further comprises: comparing the amount of the antibody or antibody derivative produced by the first cell line with the amount of the antibody or antibody derivative produced by the second cell line; and selecting the cell line that produced a higher amount of the antibody or antibody derivative based on the comparison. In some embodiments, the method further comprises: detecting the amount of one or more weight variant species produced by the first cell line by the method of any one of the above embodiments; and detecting the amount of one or more weight variant species produced by the second cell line by the method of any one of the above embodiments. In some embodiments, the method further comprises, after detecting the amount of one or more weight variant species produced by the first and second cell lines: comparing the amount of one or more weight variant species produced by the first cell line with the amount of one or more weight variant species produced by the second cell line; and selecting cell lines that produced a higher ratio of antibody or antibody derivative to one or more weight variant species based on the comparison, or selecting cell lines that produced a higher proportion of a single weight variant species compared to the total amount of antibody or antibody derivative and one or more weight variant species produced. In some embodiments, the amount of antibody or antibody derivative and / or one or more weight variant species according to any embodiment herein refers to a relative amount (e.g., compared to one or more other species, or compared to the total amount of antibody / species produced).

[0022] 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 include species that are cysteinylated, N-acetylcysteinylated and glutathionylated and have a free cysteine ​​(e.g., that is not disulfide bonded to another cysteine ​​of the antibody or antibody derivative). In some embodiments, the antibody or antibody derivative is not N-glycosylated (e.g., in the antibody Fc region). In some embodiments, the antibody or antibody derivative is not N-glycosylated (e.g., in the antibody Fc region). The organism comprises a mutation in the Fc 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 antibody Fc region), and the method further comprises removing antibody N-glycosylation (e.g., prior to SE-UPLC-MS). In some embodiments, removing antibody N-glycosylation comprises treating the antibody with a peptide:N-glycosidase enzyme (e.g., PNGase F). In some embodiments, detecting the amount of the antibody or antibody derivative and one or more weight variant species comprises deconvoluting one or more MS spectra obtained by MS. In some embodiments, the method further comprises providing or obtaining a cell culture medium (e.g., from a cell line producing the antibody or antibody derivative and one or more weight variant species) that comprises the antibody or antibody derivative and one or more weight variant species prior to detection. In some embodiments, the method further comprises separating the cell culture medium from the cell line producing the antibody or antibody derivative and the one or more weight variant species prior to detection. In some embodiments, the cell culture medium is separated from the cell line by centrifugation prior to detection. In some embodiments, the cell culture medium is subjected to SE-UPLC without a preceding chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without a preceding Protein A affinity chromatography. In some embodiments, the MS is an intact MS. In some embodiments, the MS is a quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is a modified SE-UPLC. In some embodiments, the SE-UPLC is directly coupled to the MS. In some embodiments, the one or more weight variant species represent an antibody or antibody derivative species that includes a chemically modified cysteine ​​residue. In some embodiments, the antibody or antibody derivative and the one or more weight variant species differ in molecular weight by at least 119 Da.In some embodiments, MS can resolve cysteinylated (119 Da mass shift), N-acetylcysteinylated (161 Da mass shift) and glutathionylated (305 Da mass shift) species, mass differences in an antibody or antibody derivative and one or more weight variant species that differ in molecular weight by at least 162 Da. In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g., in the antibody Fc region) and one or more weight variant species represent glycoforms of the antibody or antibody derivative. In some embodiments, MS can resolve a mass difference of about 162 Da between the antibody or antibody derivative and one or more weight variant species that represent glycoforms of the antibody or antibody derivative. In some embodiments, the antibody or antibody derivative is a multispecific antibody.

[0023] It is understood that one, some or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. [Brief description of the drawings]

[0024] [Figure 1A] FIG. 1A provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites that bind to three target proteins CD28, CD3, and HER2. The first pair of polypeptides has dual variable domains with cross-orientation (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28 (CODV), and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes HER2 (Fab). The trispecific binding protein shown in FIG. 1A uses a constant region with a "knob-into-hole" mutation, where the knob is on the second pair of polypeptides that has a single variable domain. [Figure 1B]FIG. 1 illustrates species resulting from heavy (left) or light (right) chain mispairing. On the left side, species resulting from mispairing of two Fab heavy chains (top) or two CODV heavy chains (bottom). On the right side, species resulting from mispairing of two Fab light chains (bottom left), two CODV light chains (bottom right), or mispairing of a Fab and a CODV light chain to the wrong heavy chain (top right), as well as a correctly paired trispecific binding protein (top left). [Figure 2A] FIG. 2A shows deconvoluted mass spectrometry (MS) spectra of intact (FIG. 2A) and IdeS-digested F(ab')2 fragments (FIG. 2B) of anti-CD38 trispecific binding protein, with different species displayed. Represented species include the correctly paired trispecific binding protein (H1L1 / H2L2), a mispaired species of two Fab light chains (H1L1 / H2L1), and a mispaired species of two CODV light chains (H1L2 / H2L2). Annotation indicates experimental mass vs. theoretical mass (in brackets). [Figure 2B] FIG. 2A shows deconvoluted mass spectrometry (MS) spectra of intact (FIG. 2A) and IdeS-digested F(ab')2 fragments (FIG. 2B) of anti-CD38 trispecific binding protein, with different species displayed. Represented species include the correctly paired trispecific binding protein (H1L1 / H2L2), a mispaired species of two Fab light chains (H1L1 / H2L1), and a mispaired species of two CODV light chains (H1L2 / H2L2). Annotation indicates experimental mass vs. theoretical mass (in brackets). [Figure 3A] Figure 3: Generation of trispecific binding proteins from a series of clones Figure 3A shows the ranking of anti-CD38 trispecific binding protein producing clones by percentage of correct tsAb mass (purity). [Figure 3B] Figure 3B shows the production of trispecific binding proteins from a series of clones, and shows the productivity (normalized titer) of each clone in the same order (i.e., ranked by purity) as presented in Figure 3A. [Figure 3C]FIG. 1 shows pie charts depicting the contribution of mispaired and half-antibody impurities, and correctly paired species, in a randomly selected subset of anti-CD38 trispecific binding protein producing clones generated by batch culture. [Figure 4A] Figure 4: Analysis of trispecific binding proteins by SEC intact MS Figure 4A shows a representative base peak chromatogram of a denaturing SEC intact MS analysis of a tsAb clarified harvest fluid. [Figure 4B] Figure 4 shows the analysis of a trispecific binding protein by SEC intact MS: Figure 4B shows the composite spectrum under each chromatographic peak. [Figure 4C] Figure 4C shows the analysis of trispecific binding proteins by SEC intact MS. Figure 4C shows the expansion of the deconvoluted mass spectrum of a mixture of anti-CD38 trispecific binding protein and anti-HER2 trispecific binding protein degradation light chain mismatch species from two constructs with Δmass=302 Da. [Figure 5A] Figure 1 shows the relative quantification of correctly and mispaired species obtained for different column loads (µg protein), from 4.4 to 21.8 µg for anti-HER2 trispecific binding protein, and from 11.0 to 55.0 µg for anti-CD38 trispecific binding protein, demonstrating the robustness and reliability of this method for the analysis of low- and high-titer harvested samples. For anti-HER2 trispecific binding protein, the column loads are shown as 4.4 µg, 8.7 µg, 13.1 µg, 17.4 µg and 21.8 µg (from left to right). For anti-CD38 trispecific binding protein, the column loads are shown as 11.0 µg, 22.0 µg, 33.0 µg, 44.0 µg and 55.0 µg (from left to right). [Figure 5B]FIG. 5B shows raw and deconvoluted mass spectra obtained by SEC-LC-MS analysis of cleaned-up (FIG. 5B) and ProA-purified (FIG. 5C) anti-CD38 trispecific binding proteins, showing a comparison of the two methods. Annotations above the deconvoluted spectra indicate the experimental mass and % intensity of each species. [Figure 5C] FIG. 5B shows raw and deconvoluted mass spectra obtained by SEC-LC-MS analysis of cleaned-up (FIG. 5B) and ProA-purified (FIG. 5C) anti-CD38 trispecific binding proteins, showing a comparison of the two methods. Annotations above the deconvoluted spectra indicate the experimental mass and % intensity of each species. [Figure 5D] FIG. 5D shows the ranking of anti-HER2 trispecific binding protein producing clones based on the percentage of correct tsAb mass (purity) (FIG. 5D) and productivity measured by titer of the same clones (FIG. 5E). [Figure 5E] FIG. 5D shows the ranking of anti-HER2 trispecific binding protein producing clones based on the percentage of correct tsAb mass (purity) (FIG. 5D) and productivity measured by titer of the same clones (FIG. 5E). [Figure 6A] FIG. 6 shows the yield of the correct tsAb mass in different clones of anti-CD38 trispecific binding protein (FIG. 6A) and anti-HER2 trispecific binding protein (FIG. 6B) grown in different cell culture conditions (ambr or batch as indicated). [Figure 6B] FIG. 6 shows the yield of the correct tsAb mass in different clones of anti-CD38 trispecific binding protein (FIG. 6A) and anti-HER2 trispecific binding protein (FIG. 6B) grown in different cell culture conditions (ambr or batch as indicated). [Figure 6C] FIG. 6 shows the effect of different growth conditions on strand mispairing and half-antibody levels in anti-CD38 TCE clones grown under two different ambr conditions (FIGS. 6C and 6D) and batch culture conditions (FIG. 6E). [Figure 6D]FIG. 6 shows the effect of different growth conditions on chain mispairing and half-antibody levels in anti-CD38 TCE clones grown under two different ambr conditions (FIGS. 6C and 6D) and batch culture conditions (FIG. 6E). [Figure 6E] FIG. 6 shows the effect of different growth conditions on chain mispairing and half-antibody levels in anti-CD38 TCE clones grown under two different ambr conditions (FIGS. 6C and 6D) and batch culture conditions (FIG. 6E). [Figure 6F] FIG. 6 shows the effect of different growth conditions on strand mispairing levels in anti-HER2 TCE clones grown under ambr (FIG. 6F) or batch (FIG. 6G) culture conditions. [Figure 6G] FIG. 6 shows the effect of different growth conditions on strand mispairing levels in anti-HER2 TCE clones grown under ambr (FIG. 6F) or batch (FIG. 6G) culture conditions. [Figure 7] FIG. 1 illustrates an exemplary workflow for high-throughput intact MS according to some embodiments. In addition to high-throughput capabilities, further advantages include: (1) no need for Protein A purification, saving time and money; (2) more direct insight into cell line performance by providing information on potential wasted biomass on free chains, half antibodies and other variants that cannot pass Protein A purification; and (3) applicability to Fc-less modalities such as Fab, scFv and other antibody fragments. [Figure 8A] FIG. 1 shows deconvoluted intact mass spectra obtained for anti-HIV / CD28 / CD3 trispecific antibody producing cell clones with the highest and lowest levels of strand mispairing, respectively. [Figure 8B] FIG. 1 shows deconvoluted intact mass spectra obtained for anti-HIV / CD28 / CD3 trispecific antibody producing cell clones with the highest and lowest levels of strand mispairing, respectively. [Figure 9A]Figure 9A shows the productivity (surveyed by titer μg / mL; Figure 9A) and production (% correct mass; Figure 9B) of correctly paired products of 50 cell clones producing an anti-HIV / CD28 / CD3 trispecific grown under batch culture conditions. [Figure 9B] Figure 9A shows the productivity (surveyed by titer μg / mL; Figure 9A) and production (% correct mass; Figure 9B) of correctly paired products of 50 cell clones producing an anti-HIV / CD28 / CD3 trispecific grown under batch culture conditions. [Figure 10A] Detailed glycan profile obtained for reference mass (H1L1 / H2L2) for selected clones producing anti-HIV / CD28 / CD3 trispecific antibodies. Clones were cultivated under two different conditions: spin tubes (batch conditions; FIG. 10A) and ambr15 bioreactors (fed-batch conditions; FIG. 10B). [Figure 10B] Detailed glycan profile obtained for reference mass (H1L1 / H2L2) for selected clones producing anti-HIV / CD28 / CD3 trispecific antibodies. Clones were cultivated under two different conditions: spin tubes (batch conditions; FIG. 10A) and ambr15 bioreactors (fed-batch conditions; FIG. 10B). [Figure 11A] Figure 11 shows MS analysis of a collection of antibodies with an engineered cysteine ​​(Cys293) for cysteine ​​capping state. Figure 11A shows the identification of the Cys293 capping state of a species using direct intact MS analysis. This antibody contained an N300A mutation that removed N-glycosylation of the Fc region. Figure 11B shows the identification of the Cys293 capping state using direct intact MS analysis of an antibody that was treated with PNGase F to remove N-glycans prior to analysis. [Figure 11B]Figure 11 shows MS analysis of a collection of antibodies with an engineered cysteine ​​(Cys293) for cysteine ​​capping state. Figure 11A shows the identification of the Cys293 capping state of a species using direct intact MS analysis. This antibody contained an N300A mutation that removed N-glycosylation of the Fc region. Figure 11B shows the identification of the Cys293 capping state using direct intact MS analysis of an antibody that was treated with PNGase F to remove N-glycans prior to analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present disclosure provides, inter alia, methods for monitoring the production of multispecific binding proteins and one or more mismatched species, such as multispecific antibodies, antibody fragments, Fc fusion proteins, or other multispecific binding proteins 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 polypeptide chain, as well as 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 multispecific binding proteins, screening cell lines for the production of multispecific binding proteins, or the production of antibodies or Fc fusion proteins, and the production of antibodies or antibody derivatives (e.g., produced with one or more weight variant species) or screening cell lines for the production of antibodies or antibody derivatives (e.g., produced with one or more weight variant species). Advantageously, these methods allow intact MS analysis of mAb-related species directly in the clarified harvest, bypassing time-consuming and expensive purification (e.g., Protein A affinity chromatography) and buffer exchange steps, and can be used to rapidly screen large numbers of clones for potential production cell lines.

[0026] The description that follows sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but rather is provided as a description of example embodiments.

[0027] definition As utilized in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0028] It is understood that the aspects and embodiments of the disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. can be.

[0029] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides that comprise a polynucleotide can be ribonucleotides or deoxyribonucleic acids or modified forms of any type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate. The term "polynucleotide" expressly includes single-stranded and double-stranded forms of DNA.

[0030] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof: (1) the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature; (2) it is linked to a polynucleotide with which it is not linked in nature; or (3) it is not found in nature as part of a larger sequence.

[0031] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it is normally found; (2) is essentially free of other polypeptides from the same source, e.g., from the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is not associated (by covalent or non-covalent interactions) with portions of polypeptides with which it is naturally associated; (6) is operatively associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such isolated polypeptides may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, an isolated polypeptide is found in its natural environment and is substantially free of polypeptides or other contaminants that would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

[0032] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids that are typically involved in antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain that is involved in effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V H ) and three constant domains (C HI , C H2 and C H3 ), where V H The domain is located at the amino terminus of the polypeptide and is C H3 The domains are located at the carboxyl terminus, and full-length light chain immunoglobulin polypeptides contain a variable domain (V L ) and the constant domain (C L ), wherein the VL domain is at the amino terminus of the polypeptide and L The domain is at the carboxyl terminus.

[0033] Human light chains are typically classified as kappa and lambda light chains, while human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which are classified as IgM, IgD, and IgE, respectively. Antibody isotypes are defined as IgG, IgA and IgE. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids; see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), 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 typically exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by framework regions, allowing binding to a specific epitope. From the amino terminus to the carboxyl terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0034] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently by various 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 provided an unambiguous residue numbering system that can be applied to any variable region of an antibody, but also provided precise residue boundaries that define the three CDRs. These CDRs are referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) have described the Kabat CDRs as Certain portions within the CDRs were found to adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. These subportions were designated L1, L2, and L3 or H1, H2, and H3, with "L" and "H" designating the light and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, with boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in 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. Additionally, other CDR boundary definitions may not strictly adhere to one of the systems herein, and they may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues or even entire CDRs do not significantly affect antigen binding, but still fall within the scope of the Kabat method. The CDRs overlap. Certain embodiments use the CDRs defined by Kabat or Chothia, but the methods used herein can utilize CDRs defined by any of these systems. Identification of predicted CDRs using amino acid sequences is well known in the art, such as in Martin, AC “Protein sequence and structure analysis of antibody variable domains,” In Antibody Engineering, Vol. 2. Kontermann R., Dubel S., eds. Springer-Verlag, Berlin, pp. 33-51 (2010). The amino acid sequences of the heavy and / or light chain variable domains are also examined to identify the sequences of the CDRs by other conventional methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. The numbered sequences are approximated by eye. or by using an alignment program such as one of the CLUSTAL series of programs described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are conventionally used to accurately describe the framework and CDR regions, thus correcting the sequence-based assignments.

[0035] As used herein, the term "Fc" refers to a molecule that includes the sequence of a non-antigen-binding fragment resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may include a hinge region. The original immunoglobulin source of native Fc is preferably from human origin, and may be any immunoglobulin, although IgG1 and IgG2 are preferred. Fc molecules are composed of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. As used herein, the term "native Fc" is generic to the monomeric, dimeric and multimeric forms.

[0036] F(ab) fragments contain one light chain and one heavy chain V H and C H1 domain, where the V of the F(ab) fragment H -C H1 The heavy chain portion cannot form disulfide bonds with another heavy chain polypeptide. As used herein, a F(ab) fragment refers to one light chain containing two variable domains separated by an amino acid linker, and one light chain containing two variable domains and a C H1 It may also comprise one heavy chain containing the domains.

[0037] F(ab') fragments contain one light chain and a constant region (C) such that interchain disulfide bonds can form between two heavy chains to form an F(ab') molecule. H1 and C. H2 Typically, the portion of one heavy chain that contains more than one nucleotide (between the nucleotides and the nucleotides) is included.

[0038] As used herein, the term "binding protein" refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds 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 Fc 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 Fc regions fused to one, two, three or more antigen binding domains or other polypeptides (e.g., Fc fusion proteins). 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 a dual variable domain with cross over orientation, e.g., as described in WO2012135345. In some embodiments, the binding protein comprises four polypeptide chains that form four antigen binding sites, two of the polypeptide chains having the formula: V L1 -L1-V L2 -L2-C L [I] and having a structure represented by and the two polypeptide chains have the formula: V H2 -L3-V H1 -L4-C H1 -Fc [II] and having a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; VH2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2 , C H3 an immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-light heavy chain pair.

[0039] Trispecific binding proteins of the disclosure, unless otherwise specified, typically comprise four polypeptide chains that form at least three antigen binding sites, with the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] and having a structure represented by and the second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] and having a structure represented by and the third polypeptide chain has the formula: V H3 -C H1 [III] and having a structure represented by and the fourth polypeptide chain has the formula: V L3 -C L [IV] and having a structure represented by During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; Hinge is C H1 and C. H2 an immunoglobulin hinge region that connects the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-light heavy chain pair.

[0040] A "recombinant" molecule is one prepared, expressed, created, or isolated by recombinant means.

[0041] One embodiment of the disclosure provides a binding protein having biological and immunological specificity for between one and three target antigens. Another embodiment of the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Yet another embodiment of the disclosure provides a host cell that expresses such a binding protein (i.e., comprises a nucleic acid molecule or vector encoding a polypeptide chain forming such a binding protein). Provide.

[0042] As used herein, the term "swapability" refers to the interchangeability of variable domains within a binding protein format with retention of folding and ultimate binding affinity. "Full swappability" refers to the interchangeability of variable domains within a binding protein format with retention of folding and ultimate binding affinity, and the interchangeability of variable domains within a binding protein format with retention of binding affinity. H1 and V H2 Both orders of domains, hence V L1 and V L2 It refers to the ability to swap (i.e. reverse) the order of domains. H and V L It should be noted that this refers only to the location of the domain in a particular protein chain in the final format. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2 It may be derived from a domain and may be located at position V in a binding protein. H1 and V H2 Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 It may be derived from a domain and may be located at position V in a binding protein. H1 and V H2 This places the V H and V L The designation refers to the current location, not the original location in the parent antibody. H and V L Domains are therefore "interchangeable".

[0043] As used herein, the term "antigen" or "target antigen" or "antigen target" refers to a molecule or a portion of a molecule that can be bound by a binding protein and can be used in an animal to produce an antibody that can bind to the epitope of that antigen. A target antigen can have one or more epitopes. For each target antigen that is recognized by a binding protein, the binding protein can compete with the intact antibody that recognizes the target antigen.

[0044] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0045] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[0046] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

[0047] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0048] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

[0049] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In a specific embodiment, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0050] An "isolated" binding protein is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would prevent diagnostic or therapeutic uses for the binding protein, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified: (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver stain. Since at least one component of the binding protein's natural environment will not be present, an isolated binding protein includes the binding protein in situ within a recombinant cell.

[0051] As used herein, the term "substantially pure" or "substantially purified" refers 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 constitutes at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition constitutes greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminating species are not detectable in the composition by conventional detection methods) and the composition consists essentially of a single macromolecular species.

[0052] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and in certain embodiments 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 certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, a binding protein has an equilibrium dissociation constant of ≦10 -8 M, more preferably the equilibrium dissociation constant is ≦10 -9 M, most preferably the dissociation constant is ≦10 -10 When the nucleotide sequence is M, it is said to specifically bind to the antigen.

[0053] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, the term "KD" refers to a binding constant between a ligand (target antigen) and an analyte (binding protein) in solution. D " refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0054] As used herein, the term "specifically binds" means to bind to at least about 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11M, 1x10 -12 It refers to the ability of a binding protein or antigen-binding fragment thereof to bind to an antigen containing the epitope with a Kd of M or greater, and / or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.

[0055] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient flexibility to the light and heavy chain domains to fold into a crossed dual variable region immunoglobulin. The linker is inserted at the sequence level at the transition between the variable domains or between the variable and constant domains, respectively. The transition between domains can be identified because the approximate sizes of immunoglobulin domains are well understood. The exact location of the domain transition can be determined by locating peptide stretches that do not form secondary structure elements such as beta sheets or alpha helices, as demonstrated by experimental data, or can be inferred by modeling or secondary structure prediction techniques. The linkers described herein are Car is V L2 The C-terminus of L1 L1, located in the light chain between the N-terminus of the domain; and V L1 The C-terminus and C L The heavy chain linker is designated L2, which is located in the light chain between the N-terminus of the V domain. H1 The C-terminus of H2 L3, located between the N-terminus of the domain; and V H2 The C-terminus and C H1 Located between the N-terminus of the domain is known as L4.

[0056] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coded information into a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid linked to it. 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, which allows additional DNA segments to be inserted into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein, with the plasmid being the most commonly used form of vector. However, the 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 the same functions.

[0057] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Although such progeny may not in fact be identical to the parent cell, since certain modifications may occur in subsequent generations, either by mutation or environmental influences, 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 protein, including bacteria, yeast, baculovirus and mammalian expression systems (and phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the polypeptide chain of the binding protein, whereby the polypeptide chain is expressed in the host cell and preferably secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

[0058] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, modified to contain new DNA and the cell is transformed. For example, a cell is genetically modified from its native state and transformed. Following transformation, the transforming DNA may recombine the cell's DNA by physically integrating into the cell's chromosome, or may be transiently maintained as a non-replicating episomal element, or may replicate independently as a plasmid. A cell is considered stably transformed if the DNA replicates with the cell's division. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, where the exogenous DNA is introduced into the cell membrane and the cell is "transfected". Numerous transfection techniques are well known in the art. Such techniques involve the introduction of one or more exogenous DNA molecules into a suitable host cell. It is used to enter

[0059] As used herein and applied to an object, the term "naturally occurring" refers to an object that can be found in nature and is not manipulated by humans.For example, a polynucleotide or polypeptide that exists in an organism (including viruses), can be isolated from a natural source, and is not intentionally modified by humans, is naturally occurring.Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or that is structurally modified or synthesized by humans.

[0060] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs such as α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other atypical amino acids may also be suitable components for the polypeptide chain of the binding protein. Examples of atypical amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, 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 carboxyl terminal direction according to standard usage and convention.

[0061] Naturally occurring residues are divided into classes based on common side chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues influencing chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0062] Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

[0063] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptide chain of binding protein.For example, those skilled in the art can identify suitable regions of the polypeptide chain that can be modified without destroying activity by targeting regions that are not considered important for activity.Alternatively, those skilled in the art can identify residues and parts of molecules that are conserved in similar polypeptides.In addition, even regions that may be important for biological activity or for structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0064] method Certain aspects of the present disclosure relate to multispecific binding proteins and methods for monitoring the production of one or more mismatched species. In some embodiments, the methods include: and detecting the amount of product (e.g., antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mismatched species) by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS). In some embodiments, the product and one or more mismatched species are detected in the cell culture medium. Exemplary and non-limiting descriptions of multispecific binding proteins are provided herein.

[0065] Certain aspects of the present 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 method comprises detecting the amount of product (e.g., one or more weight variant species of an antibody or antibody derivative) by size exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS). In some embodiments, the antibody or antibody derivative and one or more weight variant species are detected in cell culture medium. The antibody or antibody derivative and one or more weight variant species can represent multiple species, for example, with different molecular weights (e.g., with different chemical modifications, e.g., with chemically modified cysteine ​​or other residues, or with different glycoforms). As demonstrated in the examples below, the methods described herein can be applied to the analysis of an antibody or antibody derivative and one or more weight variant species, for the analysis of an antibody, antibody fragment, Fc fusion protein, or multispecific binding protein, and one or more mismatched species.

[0066] In some embodiments, the cell culture medium is a clarified cell culture harvest. For example, the cell culture medium may be harvested from a cell culture and clarified by tangential flow filtration (TFF), depth filtration and / or centrifugation. In some embodiments, the method further comprises isolating or harvesting the cell culture medium from a cell line producing the product (e.g., an antibody, an antibody fragment, an Fc fusion protein or a multispecific binding protein and one or more mismatched species). In some embodiments, the cell culture medium is clarified by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior chromatographic separation. That is, in some embodiments, the cell culture medium has not been exposed to prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior Protein A affinity chromatography. That is, in some embodiments, the cell culture medium has not been contacted with Protein A.

[0067] In some embodiments, detecting the amount of the product (e.g., antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mismatched species) comprises deconvoluting one or more MS spectra obtained by MS. Methods for deconvoluting MS spectra are known in the art. Non-limiting examples of software useful for deconvolution are illustrated below.

[0068] In some embodiments, the relative amount of the multispecific binding protein is detected, e.g., relative to the amount of one or more mismatched species. For example, the amount of multispecific binding protein produced can be compared to the amount of one or more individual mismatched species and / or to the total amount of mismatched species (e.g., the total amount of all mismatched species or a particular subset thereof).

[0069] In some embodiments, the MS is an intact MS. In some embodiments, the MS is a quadrupole time-of-flight (QToF) MS. Various QToF MS methods are known in the art. Exemplary and non-limiting parameters of QToF MS are described in the examples below.

[0070] In some embodiments, the MS is about 1000 Da or less, about 900 Da or less, about 800 Da or less. MS can resolve mass differences of about 700 Da or less, about 600 Da or less, about 500 Da or less, about 400 Da or less, about 300 Da or less, or about 200 Da or less. In some embodiments, MS can resolve mass differences of about 300 Da. For example, in some embodiments, MS can resolve mass differences of about 300 Da between a multispecific binding protein and one or more mismatched species, or between two mismatched species. In some embodiments, MS can resolve mass differences of about 162 Da. For example, in some embodiments, MS can resolve mass differences of about 162 Da between glycoforms, for example between a multispecific binding protein or a mismatched species and one or more glycoforms.

[0071] In some embodiments, the SE-UPLC is modified SE-UPLC. In some embodiments, the SE-UPLC is directly connected to MS. Various SE-UPLC methods are known in the art. Exemplary and non-limiting conditions for SE-UPLC are described in the examples below.

[0072] In some embodiments, SE-UPLC is performed at a slower initial flow rate, followed by a faster flow rate. Without wishing to be bound by theory, it is believed that the slower initial flow rate allows for more efficient size-based separation of cell culture harvest components, while increasing the flow rate after this initial period enhances the overall speed of the method. In some embodiments, SE-UPLC is performed at an initial flow rate of less than about 0.4 mL / min, followed by a flow rate of about 0.4 mL / min or more. In some embodiments, the initial flow rate is less than about 0.4 mL / min, less than about 0.3 mL / min, less than about 0.2 mL / min, or about 0.1 mL / min. In some embodiments, the initial flow rate refers to the first 15, 20, or 25 minutes of SE-UPLC. In some embodiments, SE-UPLC is performed at a flow rate of less than about 0.4 mL / min for the first 25 minutes, followed by a flow rate of about 0.4 mL / min or more (e.g., from 25-33 minutes). In some embodiments, SE-UPLC is performed at a flow rate of about 0.1 mL / min for the first 25 minutes, followed by a flow rate of about 0.4 mL / min (e.g., from minutes 25-33).

[0073] In some embodiments, SE-UPLC is performed by diverting a portion(s) of the liquid chromatography (LC) flow without waste of the protein elution (e.g., 0-10 min and / or 24-33 min), thereby providing a more rapid analysis for screening and avoiding contamination of the MS source.

[0074] In some embodiments, SE-UPLC is performed using isocratic elution with a mobile phase. For example, in some embodiments, the mobile phase comprises a solution comprising 30:70 acetonitrile:water. In some embodiments, the mobile phase comprises formic acid (FA) and trifluoroacetic acid (TFA). Without wishing to be bound by theory, it is believed that while TFA is beneficial for liquid chromatography (LC) separation, it can suppress MS signals, and thus a mobile phase comprising a mixture of FA and TFA can enable effective liquid chromatography (LC) separation while maintaining a sufficient signal-to-noise ratio for MS analysis. In some embodiments, the mobile phase comprises about 0.05% formic acid and about 0.05% trifluoroacetic acid (TFA).

[0075] Advantageously, the methods provided herein allow for rapid analysis of products, thereby allowing for high throughput screening (e.g., of large numbers of potential producer cell lines). For example, in some embodiments, detection is achieved in about 30 minutes or less, about 33 minutes or less, about 35 minutes or less, about 40 minutes or less, about 45 minutes or less, or about 60 minutes or less.

[0076] In some embodiments, the method includes analyzing the product (e.g., an antibody, an antibody fragment, an Fc fusion protein, or a multispecific binding protein and one or more In some embodiments, the method further comprises contacting the fragments (mismatched or more mismatched species) with a protease. In some embodiments, the protease is IdeS or IdeZ, e.g., for analysis of antibody fragments.

[0077] 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 a species of cysteinylated, N-acetylcysteinylated or glutathionylated free cysteine ​​(e.g., not disulfide bonded to another cysteine ​​of the antibody or antibody derivative). In some embodiments, the antibody or antibody derivative is not N-glycosylated (e.g., in the antibody Fc region). In some embodiments, the antibody or antibody derivative comprises a mutation in the Fc 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 antibody Fc region) and the method further comprises removing the antibody N-glycosylation (e.g., prior to SE-UPLC-MS). In some embodiments, removing antibody N-glycosylation comprises treating the antibody with a peptide:N-glycosidase enzyme (e.g., PNGase F).

[0078] In some embodiments, one or more weight variant species represent antibody or antibody derivative species that contain chemically modified cysteine ​​residues. In some embodiments, MS is capable of resolving the 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 that contain free cysteine ​​residues (e.g., cysteines that are not disulfide bonded with other cysteine ​​residues of the antibody / antibody derivative) and / or antibodies and antibody derivatives that contain additional cysteine ​​residues incorporated in their Fc regions may be useful, for example, for conjugation of compounds (e.g., cytotoxic drugs to form antibody drug conjugates). This additional cysteine ​​is unpaired (free), i.e., not involved in disulfide bonds with other cysteines in the mAb molecule. Previous studies have shown that this additional cysteine ​​(Cys293) can form undesirable disulfide bonds with nearby cysteine ​​residues, otherwise known as "disulfide bond scrambling," potentially leading to structural instability of the antibody. Uncontrolled sulfhydryl chemical reactions can also pose significant manufacturing risks during the process of forming ADCs, leading to undesirable drug conjugation profiles. To avoid these risks, it is inevitable to have free cysteines capped with disulfide bond modifications such as cysteinylation and glutathionylation by adjusting cell culture conditions. These modifications can be selectively reduced to generate free cysteines prior to the drug conjugation step. The methods described herein can be used, for example, to analyze the generation of antibodies or antibody derivatives, including different weight variant species, for example species that contain chemically modified cysteines, such as cysteines capped with disulfide bond modifications such as cysteinylation and glutathionylation.

[0079] In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g., in the antibody Fc region) and one or more weight variant species represent glycoforms of the antibody or antibody derivative. In some embodiments, MS is capable of resolving a mass difference of about 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 embodiments, the antibody or antibody derivative is a multispecific antibody. In some embodiments, the antibody or antibody derivative is a monoclonal antibody.

[0080] Other aspects of the disclosure include antibodies, antibody fragments, Fc fusion proteins or multispecific A method for producing a binding protein is described. In some embodiments, the method includes culturing a cell line comprising one or more polynucleotides encoding a product (e.g., an antibody, an antibody fragment, an Fc fusion protein, or a multispecific binding protein, and one or more mismatched species) under conditions suitable for the cell line to produce the product; isolating a cell culture medium comprising the product (e.g., an antibody, an antibody fragment, an Fc fusion protein, or a multispecific binding protein, and one or more mismatched species) from the cell line; detecting the amount of the product (e.g., an antibody, an antibody fragment, an Fc 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 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 method includes culturing a cell line comprising one or more polynucleotides encoding a product (e.g., an antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mispaired species) under conditions suitable for production of the product by the cell line; separating a cell culture medium comprising the product (e.g., an antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mispaired species) from the cell line; detecting the amount of the product (e.g., an antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mispaired species) in the cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS); and determining the quality and / or purity of the multispecific binding protein produced by the cell line.In some embodiments, the method includes culturing a cell line comprising one or more polynucleotides encoding a product (e.g., an antibody, an antibody fragment, an Fc fusion protein or a multispecific binding protein and one or more mismatched species) under conditions suitable for production of the product by the cell line; isolating a cell culture medium comprising the product (e.g., an antibody, an antibody fragment, an Fc fusion protein or a multispecific binding protein and one or more mismatched species) from the cell line; detecting the amount of the product (e.g., an antibody, an antibody fragment, an Fc 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 determining one or both of the quality and purity of the multispecific binding protein produced by the cell line and removing 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 exemplary products described herein are useful in these and other methods.

[0081] Other aspects of the disclosure relate to methods for screening multiple cell lines for the production of a product (e.g., an antibody, an antibody fragment, an Fc fusion protein or a multispecific binding protein and one or more mismatched species). In some embodiments, the method includes detecting the amount of a product (e.g., an antibody, an antibody fragment, an Fc 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 the amount of a product (e.g., an antibody, an antibody fragment, an Fc fusion protein or a multispecific binding protein and one or more mismatched species) produced by a second cell line, e.g., different from the first cell line (e.g., using any of the methods described herein). Any of the exemplary products described herein are useful in these and other methods.

[0082] In some embodiments, the method comprises comparing the amount of a product (e.g., an antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mismatched species) produced by a first cell line with the amount of a product (e.g., an antibody, antibody fragment, Fc fusion protein or multispecific binding protein and one or more mismatched species) produced by a second cell line. and selecting the cell line that produced a higher amount of product based on this comparison. 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 screened. Advantageously, the methods described herein are rapid and amenable to high throughput screening.

[0083] In some embodiments, the method further comprises detecting the amount of one or more mismatched species produced by the first cell line and detecting the amount of one or more mismatched species produced by the second cell line. In some embodiments, the method further comprises, for example, after detecting the amount of one or more mismatched species produced by the first and second cell lines, comparing the amount of one or more mismatched species produced by the first cell line with the amount of one or more mismatched species produced by the second cell line. In some embodiments, based on this comparison, a cell line is selected that has provided a higher ratio of multispecific binding protein to one or more mismatched species. In some embodiments, a cell line is selected based on a higher ratio of multispecific binding protein to the amount of one or more individual mismatched species and / or a higher ratio of multispecific binding protein to the total amount of mismatched species.

[0084] In some embodiments, the selected cell line is selected as a production cell line.

[0085] Multispecific Binding Proteins Certain aspects of the present disclosure relate to multispecific binding proteins.

[0086] 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 Fc fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody or a trispecific Fc fusion protein. In some embodiments, one or more mispaired species comprises two or more polypeptide chains comprising at least one of the otherwise associated first and second polypeptide chains of the multispecific binding protein.

[0087] 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 mismatched species comprises one or more of the following: an association of four polypeptide chains of a multispecific antibody comprising two of the first antibody heavy chains; an association of four polypeptide chains of a multispecific antibody comprising two of the second antibody heavy chains; an association of four polypeptide chains of a multispecific antibody comprising two of the first antibody light chains; and an association of four polypeptide chains of a multispecific antibody comprising two of the second antibody light chains.

[0088] In some embodiments, a multispecific binding protein comprises four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein have dual variable domains with a crossed orientation and a second pair of polypeptides forming the binding protein have a single variable domain.

[0089] In some embodiments, a multispecific binding protein comprises one or two Fc regions fused to one, two, three or more antigen binding domains or other polypeptides (e.g., Fc fusion proteins). In some embodiments, a multispecific binding protein comprises a dual variable domain (DVD) immunoglobulin, e.g., as described in WO2012061558. In some embodiments, the multispecific binding protein comprises dual variable domains with cross-orientation, e.g., as described in WO2012135345. In some embodiments, the binding protein comprises four polypeptide chains that form four antigen-binding sites, where two polypeptide chains have a structure represented by the following formula: V L1 -L1-V L2 -L2-C L [I] The two polypeptide chains have a structure represented by the formula: V H2 -L3-V H1 -L4-C H1 -Fc[II] During the ceremony: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; Fc is the immunoglobulin hinge region and C H2 , C H3 an immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair.

[0090] In some embodiments, each of the three antigen-binding sites binds to a different target (e.g., a polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains that form the three antigen-binding sites, where a first polypeptide chain comprises a structure represented by the following formula: V L2 -L1-V L1 -L2-C L [I] The second polypeptide chain comprises a structure represented by the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The third polypeptide chain comprises a structure represented by the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The fourth polypeptide chain comprises a structure represented by the formula: V L3 -C L [IV] During the ceremony: 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; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; Hinge is C H1 and C H2 an immunoglobulin hinge region that connects the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair.

[0091] In some embodiments, the polypeptide of formula I and the polypeptide of formula II form a cross-light chain-heavy chain pair. H1 and V L1 forms the first antigen-binding site, and V H2 and V L2 forms the second antigen-binding site, and V H3 and V L3 forms the third antigen-binding site.

[0092] In some embodiments, the one or more mismatched species comprises one or more of the following: an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula I; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula II; an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula III; and an association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula IV.

[0093] In some embodiments, the multispecific binding protein is trispecific and / or trivalent. In some embodiments, the multispecific binding protein is bispecific and / or bivalent.

[0094] Any of the antigen-binding sites described herein may be useful in the trispecific binding protein of the present disclosure, including, for example, four polypeptide chains having the structure described above.For example, in some embodiments, the trispecific binding protein of the present disclosure includes a VH1 and VL1 domain pair that forms a first antigen-binding site, a VH2 and VL2 domain pair that forms a second antigen-binding site, and a VH3 and VL3 domain pair that forms a third antigen-binding site.In some embodiments, the trispecific binding protein of the present disclosure includes a VH1 and VL1 domain pair that forms a first antigen-binding site, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site. In some embodiments, the trispecific binding protein of the present disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site. In some embodiments, the trispecific binding protein of the present disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds to a tumor target protein. In some embodiments, the trispecific binding protein of the present disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds to an HIV target protein. In some embodiments, a trispecific binding protein of the disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds to an HIV target protein, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to an HIV target protein, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds to an HIV target protein.In some embodiments, the trispecific binding protein of the present disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds to a tumor targeting protein. In some embodiments, the trispecific binding protein of the present disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds to a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds to a CD38 polypeptide. In some embodiments, a trispecific binding protein of the disclosure comprises a VH1 and VL1 domain pair that forms a first antigen-binding site that binds a CD28 polypeptide, a VH2 and VL2 domain pair that forms a second antigen-binding site that binds a CD3 polypeptide, and a VH3 and VL3 domain pair that forms a third antigen-binding site that binds a HER2 polypeptide.

[0095] In some embodiments, the binding protein of the present disclosure binds to one or more tumor targeting proteins and one or more T cell targeting proteins. In some embodiments, the binding protein is capable of specifically binding to one tumor targeting protein and two different epitopes on a single T cell targeting protein. In some embodiments, the binding protein is capable of specifically binding to one tumor targeting protein and two different T cell targeting 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 the two T cell targeting proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds to the tumor targeting protein. In some embodiments, the targeting protein is CD38 or HER2. Additional tumor targeting proteins are provided below. In some embodiments, the one or more T cell targeting proteins are one or more of CD3 and CD28.

[0096] Exemplary multispecific binding proteins that can be used in the methods of the disclosure also include, but are not limited to, those described in International Publication Nos. WO2017074878, WO2017180913, WO2018151841, and WO2019074973.

[0097] 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 CD38 polypeptide, 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 a polypeptide of formula I and a polypeptide of formula II form a cross-light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above anti-CD38 antigen-binding sites are VH and VL domains of any of the above anti-CD38 antigen-binding sites. H3 and V L3and forms a third antigen-binding site that binds to the CD38 polypeptide. H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site which binds to the CD38 polypeptide.

[0098] 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 the three antigen-binding sites described above, wherein V H3 and V L3 The pair of domains forms a third antigen-binding site that binds to the HER2 polypeptide.

[0099] In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site that binds to a tumor targeting protein. In some embodiments, the tumor targeting protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor targeting protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, tumor targeting proteins of the disclosure include, but are not limited to, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (aka VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (aka MCP-1), CCL3 (aka MIP-1a), CCL4 (aka MIP-1b), CCL5 (aka RANTES), CCL7 (aka MCP-3), CCL8 (aka mcp-2), CCL11 (aka eotaxin), CCL15 (aka MIP-1d), CCL17 (aka TARC), CCL19 (aka MIP-3b), CCL20 (aka MIP-3a), CCL21 (aka MIP-2), CCL24 (aka MIP-4), CCL25 (aka MIP-5), CCL26 (aka MIP-6), CCL27 (aka MIP-7), CCL28 (aka MIP-8), CCL29 (aka MIP-9), CCL30 (aka MIP-10), CCL31 (aka MIP-11), CCL32 (aka MIP-12), CCL33 (aka MIP-13), CCL34 (aka MIP-14), CCL35 (aka MIP-15), CCL36 (aka MIP-16), CCL37 (aka MIP-17), CCL38 (aka MIP-19), CCL39 (aka MIP-20), CCL39 (aka MIP-21), CCL39 (aka MIP-22), CCL39 (aka MIP-23), CCL39 (aka MIP-24), CCL39 (aka MIP-25), CCL39 (aka MIP-26), CCL39 (aka MIP-27), CCL39 (aka PIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, IgE receptor), 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 PDL 1), 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 SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP , FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2R beta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the IL25 receptor), IL18, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36, IL37, IL38, IL39, IL40, IL41, IL42, IL43, IL44, IL45, IL46, IL47, IL48, IL49, IL50, IL51, IL42, IL43, IL44, IL45, IL46, IL47, IL48, IL49, IL49, IL52, IL41, IL42, IL43, IL44, IL45, IL45, IL46, IL47, IL48, IL49 ... , IL27, IL33, IL35, ITGB4 (aka b4 integrin), 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 the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa,These include TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0100] In some embodiments, the binding protein of the present disclosure comprises an antigen binding site that binds to an HIV target protein. In some embodiments, the binding protein of the present disclosure comprises three antigen binding sites that each bind to an HIV target protein. In some embodiments, the binding protein specifically binds to one or more HIV target proteins (e.g., as described below) and one or more target proteins on a T cell that includes a T cell receptor complex. Examples of target proteins on a T cell include, but are not limited to, CD3 and CD28, among others. In some embodiments, the HIV target protein is glycoprotein 120, glycoprotein 41, or glycoprotein 160. In some embodiments, the binding protein binds to one or more of the following: glycoprotein 120, glycoprotein 41, and glycoprotein 160. Exemplary HIV target proteins include, but are not limited to, the MPER of the HIV-1 gp 41 protein, the CD4 binding site of the HIV-1 gp120 protein, a glycan in the V3 loop of the HIV-1 gp120 protein, or the trimer apex of the HIV-1 gp120 protein or gp160. For example, in some embodiments, the binding proteins of the present disclosure comprise an antigen-binding site that binds to the CD4 binding site of the HIV-1 gp120 protein. Exemplary antigen-binding sites that bind to HIV target proteins contemplated for use herein include, but are not limited to, those described in International Publication No. WO2017 / 074878, such as those from antibodies CD4BS "a", CD4BS "b", MPER, MPER_100W, V1 / V2 "a", V1 / V2 "b" or V3.

[0101] In some embodiments, the 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, the 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, the 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, the 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 CD38 polypeptide. In some embodiments, the 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 HER2 polypeptide. In some embodiments, the 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 targeting protein.

[0102] 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 but not limited to 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 a polypeptide of formula I and a polypeptide of formula II form a cross-light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above anti-CD28 antigen-binding sites are VH and VL domains. H1 and V L1 and forms a first antigen-binding site that binds to the CD28 polypeptide. H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site that binds to a tumor targeting protein (including, but not limited to, CD38 or HER2).

[0103] In some embodiments, the 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, the 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 and one of which binds to a CD3 polypeptide. In some embodiments, the 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 CD38 polypeptide. In some embodiments, the 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, the 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 targeting protein.

[0104] 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 but not limited to 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-light chain-heavy chain pair (e.g., as described herein). In some embodiments, the V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide. H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site that binds to a tumor targeting protein (including, but not limited to, CD38 or HER2).

[0105] Linker In some embodiments, the linkers L1, L2, L3 and L4 are zero amino acids (length=0) to about 100 amino acids in length, or less than 100, 50, 40, 30, 20 or 15 amino acids in length. The linkers may be 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid in length. The L1, L2, L3 and L4 in one binding protein can all have the same amino acid sequence or can all have different amino acid sequences.

[0106] Examples of suitable linkers include, for example, 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 Publication Nos. WO2017 / 074878 and WO2017 / 180913. The examples listed above are in no way intended to limit the scope of the present disclosure, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine and proline have been shown to be suitable for binding proteins.

[0107] The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure elements that need to be achieved in the linker.For example, glycine, serine and alanine are best for linkers with maximum flexibility.If a stronger and more extended linker is required, some combinations of glycine, proline, threonine and serine are beneficial.Any amino acid residue can be considered as a linker in combination with other amino acid residues to build larger peptide linkers as needed according to desired properties.

[0108] In some embodiments, L1 is at least twice as long as L3. In some embodiments, L2 is at least twice as long as L4. In some embodiments, L1 is at least twice as long as L3 and L2 is at least twice as long as L4. In some embodiments, L1 is 3-12 amino acid residues in length, L2 is 3-14 amino acid residues in length, L3 is 1-8 amino acid residues in length and L4 is 1-2 amino acid residues in length. In some embodiments, L1 is 5-10 amino acid residues in length, L2 is 5-8 amino acid residues in length, L3 is 1-5 amino acid residues in length, and L4 is 1-2 amino acid residues in length. In some embodiments, 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.

[0109] In some embodiments, L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 1), GGGGSGGGGSGGGGS (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, L2, L3, and L4 are each independently comprised of a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 1), GGGGSGGGGSGGGGS (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: 4), L2 comprises the sequence TKGPS (SEQ ID NO: 3), L3 comprises the sequence S, and L4 comprises the sequence RT.

[0110] In some embodiments, at least one of L1, L2, L3, or L4 comprises the sequence DKTHT (SEQ ID NO: 6). In some embodiments, L1, L2, L3, and L4 comprise the sequence DKTHT (SEQ ID NO: 6).

[0111] Fc Region and Constant Domains In some embodiments, the binding protein of the present disclosure is C H1 an Fc region linked to an immunoglobulin hinge region and H2 and C H3 In some embodiments, the binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region comprising an immunoglobulin heavy chain constant domain. H1 an Fc region linked to an immunoglobulin hinge region andH2 and C H3 In some embodiments, the binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region comprising an immunoglobulin heavy chain constant domain. H1 an Fc region linked to an immunoglobulin hinge region and H2 and C H3 a second polypeptide chain further comprising an Fc region comprising an immunoglobulin heavy chain constant domain; and H1 an Fc region linked to an immunoglobulin hinge region and H2 and C H3 and a third polypeptide chain further comprising an Fc region comprising an immunoglobulin heavy chain constant domain.

[0112] In some embodiments, the binding protein of the present disclosure comprises a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, such as an Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region comprises an antibody hinge, C H1 , C H2 , C H3 and possibly C H4 In some embodiments, the Fc region comprises a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the mutations described below. In some embodiments, the Fc region is an Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the heavy chain constant region is an Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the light chain constant region is an Fc region of one of the light chain polypeptides (e.g., polypeptide 1 or 4) of the binding proteins shown in Table 4.

[0113] In some embodiments, the binding proteins of the disclosure comprise one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor) or refers to the sequence. Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can include molecules or sequences that are humanized from a non-human native Fc. In addition, native Fc includes regions that may be removed because they provide structural functions or biological activities that are not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or that have been modified in one or more Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity due to expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0114] In some embodiments, a binding protein of the disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, a binding protein of the disclosure comprises a "knob" mutation on the third polypeptide chain and a "hole" mutation on the second polypeptide chain. In some embodiments, the "knob" mutation comprises a 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 "knob" mutation comprises a substitution 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 "hole" mutation comprises a substitution(s) at positions corresponding to positions 407, and optionally 349, 366 and / or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y407V or Y407T, and optionally Y349C, T366S and / or L368A. In some embodiments, the "hole" mutation comprises a substitution at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A and Y407V.

[0115] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising an amino acid substitution(s) at a position corresponding to position 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are T366W or T366Y and optionally S354C; wherein the second Fc region comprises an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, and wherein the second Fc region comprises an amino acid substitution(s) at positions corresponding to positions 407, and optionally 349, 366 and / or 368 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y407V or Y407T, and optionally Y349C, T366S and / or L368A.

[0116] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises an amino acid substitution(s) at positions corresponding to positions 407, and optionally 349, 366 and / or 368 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y407V or Y407T, and optionally Y349C, T366S and / or Y366C. and L368A; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution(s) at a position corresponding to position 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W or T366Y and optionally S354C.

[0117] In some embodiments, the second polypeptide chain comprises a first Fc region linked to CH1, the first Fc region further comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution(s) at a position corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366S, L368A and / or Y407V.

[0118] In some embodiments, the second polypeptide chain comprises a first Fc region linked to CH1, further comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution(s) at a position corresponding to position 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366S, L368A and / or Y407V; and the third polypeptide chain comprises a second Fc region linked to CH1, further comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitution is T366W.

[0119] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are Y349C, T366S, L368A and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc The region is a human IgG4 Fc region.

[0120] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, S354C, T366W and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. The second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. The second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, S354C, T366W and R409K.

[0121] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. The second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. The second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354 and 366 of human IgG4 according to the EU index, where the amino acid substitutions are F234A, L235A, S354C and T366W.

[0122] In some embodiments, the binding proteins of the disclosure have effector functions, such as Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and and / or one or more mutations to reduce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain comprises H1 a first Fc region linked to an immunoglobulin hinge region and H2 and C H3 The first polypeptide chain further comprises an Fc region comprising an immunoglobulin heavy chain constant domain; H1 a second Fc region linked to an immunoglobulin hinge region and H2 and C H3 and further comprising a second Fc region comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions each comprise an amino acid substitution at a position corresponding to positions 234 and 235 of human IgG1 according to the EU index, where the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and these Fc regions each comprise an amino acid substitution at a position corresponding to positions 234 and 235 of human IgG1 according to the EU index, where the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain is C H1 a first Fc region linked to an immunoglobulin hinge region and H2 and C H3 The first polypeptide chain further comprises an Fc region comprising an immunoglobulin heavy chain constant domain; H1 a second Fc region linked to an immunoglobulin hinge region and H2 and C H3and further comprising a second Fc region comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and these Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, where the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, which comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, where the amino acid substitutions are F234A and L235A. H1 a first Fc region linked to an immunoglobulin hinge region and H2 and C H3 a second polypeptide chain further comprising a first Fc region comprising an immunoglobulin heavy chain constant domain; H1 a second Fc region linked to an immunoglobulin hinge region and H2 and C H3 a third polypeptide chain further comprising a second Fc region comprising an immunoglobulin heavy chain constant domain, wherein the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A.

[0123] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain. and a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; the third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain. The second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4 according to the EU index, where the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K.

[0124] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 228, 234, 235 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at position 236; and / or R409K.

[0125] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or an effector function of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC)).

[0126] In some embodiments, the Fc region is a human IgG1 Fc 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 Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299 and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A and / or Y300S.

[0127] In some embodiments, the binding proteins of the disclosure comprise one or more mutations that improve stability, for example, of the hinge region and / or dimer interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations improve stability of the IgG4 hinge region and / or dimer interface according to the EU index. In some embodiments, the binding protein comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4, where the amino acid substitutions are S228P and R409K. H1 a first Fc region linked to an immunoglobulin hinge region and H2 and C H3 a second polypeptide chain further comprising a first Fc region comprising an immunoglobulin heavy chain constant domain; and H1 a second Fc region linked to an immunoglobulin hinge region and H2 and C H3and a third polypeptide chain further comprising a second Fc region comprising an immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise an amino acid substitution at a position corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to improve stability. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0128] In some embodiments, the Fc region is a human IgG1 Fc 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 Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299 and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A and / or Y300S.

[0129] Nucleic acids and vectors Standard recombinant DNA methods are used to construct polynucleotides that code for polypeptides that form binding proteins, to incorporate these polynucleotides into recombinant expression vectors, and to introduce such vectors into host cells.See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd Edition).Enzymatic reactions and purification techniques can be carried out according to manufacturer's specifications as commonly accomplished in the art, or as described herein.Unless specific definitions are provided, the nomenclature used in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, and the test methods and techniques thereof, are well known and commonly used in the art.Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical manufacturing, formulation, delivery, and patient treatment.

[0130] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to induce transcription of the nucleic acid sequence encoding the binding protein.Promoter can refer to a nucleic acid control sequence that induces transcription of a nucleic acid.When the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence, the first nucleotide sequence is operably linked to the second nucleic acid sequence.For example, a promoter is operably linked to the binding protein coding sequence if the promoter affects the transcription or expression of the coding sequence. Examples of promoters include, but are not limited to, promoters obtained from the genome of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (such as actin promoter, immunoglobulin promoter, heat shock promoter, etc.), CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK) promoter, tetracycline-inducible promoter (Masui et al., Nucleic acid Res. 33:e43, 2005), lac system, tr Examples of promoters that may be included include the p system, the tac system, the trc system, the major operator and promoter regions of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast alpha-mating factor. A polynucleotide encoding a binding protein of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or that will be readily recognized by one of skill in the art.

[0131] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. An expression vector can include one or more regulatory sequences operably 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 include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic cell viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors can include, for example, plasmids, cosmids, episomes, transposons and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus 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 and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

[0132] host cell Other aspects of the disclosure relate to host cells (e.g., isolated host cells) that contain one or more of the isolated polynucleotides, vectors and / or vector systems described herein. For example, the host cells or cell lines can be used to produce the products of the disclosure.

[0133] In some embodiments, the isolated host cell of the present disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In an embodiment, the host cell is an insect cell. Examples of insect host cells include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni) cells (e.g., High Five™ cells), and Spodoptera frugiperda. The present invention relates to a method for the preparation of a medaka-based ... In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293™ cells, Chinese hamster ovary (CHO) cells, baby hamster kidney cells (e.g., BHK, ATCC CCL10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL2), canine kidney cells (e.g., MDCK, ATCC CCL34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL1442), human lung cells (e.g., W138, ATCC CCL75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2) and bone marrow The present invention may include tumor cells (e.g., NS0 and Sp2 / 0 cells).

[0134] In some embodiments, the host cells or cell lines of the present disclosure are cultured in continuous cell culture, for example in stirred tank bioreactors. In some embodiments, the host cells or cell lines of the present disclosure are cultured in batch cell culture. Suitable stirred tank and batch cell culture equipment and techniques are known in the art; exemplary, non-limiting descriptions are provided in the Examples below. Exemplary conditions and techniques suitable for the production of any of the products described herein using the host cells or cell lines described herein (e.g., above) are known in the art.

[0135] Another aspect of the present disclosure relates 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) containing 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 a host cell under conditions for expressing a protein are well known to those of skill in the art. Methods for isolating a protein from a cultured host cell are well known to those of skill in the art, including, for example, by affinity chromatography (e.g., two-step affinity chromatography including protein A affinity chromatography followed by size exclusion chromatography). EXAMPLES

[0136] The following examples illustrate specific embodiments of the disclosure and various uses thereof, they are presented for illustrative purposes only and should not be considered as limiting the scope of the invention in any way. EXAMPLES

[0137] Identification of strand mispairing and clonal variants for asymmetric trispecific binding proteins IgG-like multispecific antibodies with asymmetric constructs have become a widely used format for therapeutic applications in recent years. Correct assembly of subunits in this class of therapeutics is a critical quality attribute (CQA) that has a direct impact on biological activity. Therefore, early development efforts (e.g., clonal selection) must be guided by information about potential strand mispairing to enable timely decision-making and risk mitigation.

[0138] Mismatched antibodies may have suboptimal levels of expected biological activity and are considered product-related impurities. Removal of such mAb-related impurities requires additional purification steps after the Protein A (ProA) column, increasing the cost and timeline of process development. Therefore, any potential strand mismatches must be identified and monitored during early development, starting with cell line development. This calls for a high-throughput analytical platform that allows a large number of samples to be screened for mismatch levels with a fast turnaround time.

[0139] For identification and relative quantification of mispaired species in bispecific IgG, intact protein mass spectrometry was used. Several different intact protein MS approaches based on reversed-phase separation or size-exclusion liquid chromatography for denaturing and native MS analysis have been reported (Wang, C. et al. MAbs 2018, 10(8), pp. 1226-1235; Schaefer, W. et al. MAbs 2016, 8(1), pp. 49-55; Schachner, L. et al. Anal Chem 2016, 88(24), pp. 12122-12127; Yin, Y. et al. MAbs 2016, 8(8), pp. 1467-1476). These approaches require that antibodies be purified, typically through Protein A affinity, prior to LC-MS analysis.

[0140] The following example describes 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 FIG. 1A. This intact mass method can be performed directly on clarified harvest fluids without prior purification or sample preparation. This method can be performed directly on cell culture harvest fluids prior to Protein A purification to provide direct and unbiased information on the type and distribution of mAb-related species produced by the cells, giving insight into the performance and characteristics of the clones as well as the growth conditions. This analytical platform enabled the screening of a large number of CHO (Chinese Hamster Ovary) cell clones expressing different tsAb constructs for mismatch and half-antibody levels. Results from this intact mass analysis method, especially in early development, facilitate the selection of CHO clones that produce tsAbs of suitable product quality.

[0141] material and method Antibody expression and purification Trispecific antibodies (tsAbs) were produced by co-expression of all four subunits in a single CHO host cell line using batch culture in centrifuge tubes or fed-batch process in ambr® bioreactor system. Titers were determined in harvest fluids using an Octet with a ProA biosensor. Clarified cell culture harvest samples were stored frozen at -80°C prior to mass spectrometry. Purification of tsAbs from clarified cell culture fluids was performed using Protein A centrifuge columns in a 96-well plate format using a Tecan liquid handling system followed by concentration measurement using an Octet.

[0142] IdeS digestion of antibodies To a 10 μL solution of tsAb at a concentration of approximately 2 mg / mL, 20 μL of 25 mM Tris buffer pH 7.2 was added. The sample was mixed with 1.5 μL of IdeS enzyme solution (66.6 units / μL) from Genovis Inc. (Cambridge, MA, USA), followed by incubation at 37° C. for 2 h.

[0143] Size Exclusion Chromatography (SEC) for Mass Spectrometry (MS) Size-exclusion liquid chromatography (LC) separation was performed using a UPLC BEH SEC column, 1.7 μm, 200 Å, 4.6×300 mm, controlled by Acquity UPLC H-class (Waters Corp., Milford, MA, United States). For the ProA purified antibody samples, 0.1% trifluoroacetic acid (TFA) in 30:70 acetonitrile:water at a flow rate of 0.2 mL / min for 15 min, and for the clarified harvested samples, 0.05% formic acid (FA) and 0.05% TFA in 30:70 acetonitrile:water at flow rates of 0.1 mL / min (0 to 25 min) and 0.4 ml / min (25 to 33 min) in isocratic mode were used as the mobile phase. Both FA and TFA were LC-MS grade from Sigma (St. Louis, MO, United States). LC-MS water and acetonitrile were from Thermo Fisher Scientific (Waltham, MA, United States).

[0144] mass spectrometry Liquid chromatography (LC) was coupled to a Xevo G2 QToF mass analyzer (Waters Corp., Milford, MA, United States) for online intact MS data acquisition in the m / z range of 500–4000 using sensitivity mode and the following source parameters: capillary voltage of 3 kV, sample voltage of 40 V, and sample temperature of 100°C. pulling cone voltage, extraction cone voltage of 2.5V, source temperature of 150°C, desolvation temperature of 500°C, cone gas flow of 50L / hr, desolvation gas flow of 800L / hr, collision energy of 6V.

[0145] Data analysis For deconvolution and relative quantification, acquired LC-MS raw files were batch processed using Byos® (Protein Metrics Inc.).

[0146] result Mispairing of the heavy and light chains in a trispecific antibody results in a mass shift from the expected mass of the correctly paired species, which is easily detectable by intact protein mass spectrometry. The only exception is a potential light chain-scrambled molecule that has the same mass as the correctly paired structure and is therefore indistinguishable by mass spectrometry (Figure 1B).

[0147] Intact MS analysis of cell culture harvest fluids is a challenge due to the complex matrix, demanding effective chromatographic separation with MS detection on-line. 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 harvest, as well as their inherently low ionization efficiency due to their large size (up to 210 kDa for some mismatched species). Therefore, to obtain high-quality MS data from direct injection of harvest fluids without sample preparation, we undertook optimization of chromatographic parameters including flow rate, mobile phase modifiers and their organic content, SEC column pore size as well as protein loading on the column, as well as MS source parameters during the development of the SE-UPLC-intact MS method.

[0148] The initial workflow involved automated ProA purification of tsAbs from clarified harvest, followed by denaturing SEC-LC coupled to a QToF MS for intact MS data acquisition. Denaturing SEC allows for rapid (15 min) and efficient desalting of tsAbs as well as separation from overexpressed light chains that are often co-purified with mAbs.

[0149] Figure 2A shows the deconvoluted mass spectrum of CD38TCE tsAb, where the mass of 174,188 Da corresponds to the desired assembly, H1L1 / H2L2. Two other masses observed at 160,683 Da and 187,692 Da match the theoretical masses of the light chain mispaired species H1L1 / H2L1 and H1L2 / H2L2, respectively. To further verify the structural assignment of the species, we digested the tsAb sample with the cysteine ​​protease IdeS, which digests antibodies at a specific site below the hinge, generating pools of F(ab')2 and Fc / 2 fragments. As seen in Figure 2B, three distinct Fab fragments with the same mass shift pattern observed in the intact molecule appeared after IdeS digestion, further confirming the light chain mispaired assignment.

[0150] The development of cell lines for protein therapeutics, including antibodies, is the foundation of early development. The primary goal is to generate stable monoclonal cell lines (commonly CHO cells) that can consistently express the target recombinant protein in large amounts and with the desired product quality attributes such as glycosylation, charge heterogeneity, etc. (Chusainow, J. et al. Biotechnol. Bioeng 2009, 102(4), pp. 1182-96). In the context of multispecific antibodies, potential mispairing of subunits is a critical quality attribute (CQA) that needs to be monitored during the selection of pools and final production clones.

[0151] A denaturing SEC-intact MS method was used to determine the types and relative levels of product-associated impurities, including mismatched strands and half-antibodies, that co-purify with the desired tsAb forms. Intact MS analysis of ProA purified material from 38 clones of CHO cells expressing anti-CD38 TCE was undertaken. Relative levels of different species were calculated based on mass intensity after deconvolution of combined MS spectra acquired across tsAb chromatographic peaks. Although different IgG-related species can vary widely in mass compared to the full tsAb structure, especially for half antibodies, and thus in their ionization efficiency, MS-based relative quantification of species is a useful tool to compare and rank clones based on their mismatch and half antibody levels.

[0152] Figure 3A shows the % purity (i.e., percentage of correct mass) in different clones of anti-CD38 TCE constructs grown using batch CHO cell culture. A high degree of variability in purity was observed between clones, ranging from 90% to 10%. Furthermore, as shown in Figure 3C, the relative levels of mispaired species and half antibodies differed significantly between clones. Furthermore, aligning the purity results with the clonal productivity values ​​measured by titer (Figure 3B) demonstrates the absence of correlation between the two, implying that highly productive clones could potentially give rise to high degrees of mispaired, incompletely assembled mAb species.

[0153] These results demonstrate the importance of screening tsAb clones for mispairing levels and using that information in conjunction with productivity and other cell culture parameters for cell line development and selection of the best producing clones. EXAMPLES

[0154] Direct, intact MS analysis of clarified harvest material To reduce costs and lead times for clone screening and streamline workflow, denaturing SEC-MS method parameters were optimized to enable intact MS measurements directly in clarified cell culture fluids of mAb species generated during cell line development by bypassing any prior purification or sample manipulation.

[0155] Intact MS analysis of cell culture fluids is challenged by the complex matrix, especially the presence of non-ionic detergents such as Pluronic F-68 that are commonly added to the medium to protect cells from hydrodynamic injury, which can lead to coelution 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 harvest, as well as the inherently low ionization efficiency of tsAbs due to their large size (up to 210 kDa for some mispaired species). Chromatographic separation of proteins from detergents on reversed-phase (RP) stationary phases commonly used for intact protein MS can be quite difficult, if not impossible, due to the hydrophobicity of both types of species. Moreover, a given RP-LC method shows different selectivity behavior for different tsAbs depending on the amino acid sequence, and cannot be used as a platform method for multiple constructs of trispecific antibodies.

[0156] In this example, SEC-LC under denaturing conditions was successfully used for size-based separation of antibody species from small molecules and detergents 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 acquisition of high quality MS data for tsAb species in harvests equivalent to ProA purified material. As seen in the base peak chromatograms in Figures 4A and 4B, the optimized denaturing SEC method efficiently separated mAb-related species including tsAb and overexpressed CODV light chains and Fab light chains from each other and from detergent species.

[0157] The final detergent elution in the chromatogram and flow was discarded to avoid MS source contamination (Figure 4A). This SEC-MS method is a non-product specific platform, allowing for rapid, high-throughput screening of a large number of clones across different tsAb constructs. Furthermore, the developed method can be applied to other mAb-related modalities, including Fc-free antibody formats such as scFv2 and Fab fusion proteins (Brinkmann, U. and Kontermann, REMAb 2017, 9(2), pp. 182-212), which cannot be purified using the automated ProA purification system.

[0158] The minimum mass difference between the two mismatched species in the anti-CD38 TCE construct is 810 Da and 1095 Da in the anti-HER2 TCE construct, well below the mass resolution of the method. To demonstrate the ability of the method to more precisely resolve mismatched species by mass, cleaned harvest samples from both constructs were mixed in a 1:1 ratio to create a more complex mixture of mismatched species. Both samples contained H1L1 / H2L1 species with a theoretical mass value difference of approximately 302 Da for CD38 TCE and anti-HER2 TCE. As can be seen in Figure 4C, online SEC-MS analysis of the harvest completely resolved these two species and their glycoforms, marked by a mass shift of +162 Da.

[0159] Next, the repeatability, reliability and robustness of the MS signal was assessed using clarified harvest fluids of two different tsAb constructs: anti-HER2 TCE anti-CD38 TCE with titers of 435 and 1100 μg / mL, respectively. Figure 5A shows that the relative levels of the three different tsAb assemblies are consistent across different column loads achieved by increasing the injection volume from 10 to 50 μL. These results demonstrate the robustness and reliability of MS quantification across column loads ranging from 4 to 55 μg. Thus, online SEC-MS with a fixed injection volume of clarified harvest can be used for clones of low and high titers, generally in the range of 100 to 1100 μg / mL. The repeatability of the method, assessed by triplicate LC-MS analysis at each protein load level across the two tsAb constructs in Figures 4A-4C, returned %RSD values ​​of less than 10% (Table A).

[0160] [Table 1]

[0161] Furthermore, comparison of mismatch quantification results obtained by SEC-LC-intact MS analysis of clarified harvest and ProA purified material showed close agreement between the two methods, further confirming the suitability of direct intact MS analysis of harvest for mismatch determination (Figures 5B and 5C).

[0162] The harvest SEC-MS method was utilized 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, whereas the light chain mismatched species H1L1 / H2L1 (162,187 Da) and H1L2 / H2L2 (188,965 Da) were detected at various levels in all tested samples. Zero or very low levels of half antibodies were detected in the tested clones. As with anti-CD38 TCE, no correlation was observed between mismatch levels and clonal productivity (Figures 5D and 5E). EXAMPLES

[0163] Effect of cell culture growth conditions on strand mispairing To investigate the effect of cell growth conditions on strand mispairing and half-antibody formation in tsAbs, two different sets of experiments were performed.

[0164] In the first study, anti-CD38TCE clones were a) grown in batch culture and harvested on day 10, b) and c) grown in ambr® microbioreactors under two different growth conditions. SEC-intact MS analysis (ProA purified material) showed variation in the distribution of mismatches and half antibodies in each clone grown under the three different conditions (Figures 6C-6E). However, as seen in Figure 6C, clone ranking based on total levels of impurities remained generally unchanged regardless of growth condition.

[0165] Similar findings were found in testing of anti-HER2 TCE, where clarified harvest fluids from selected clones grown under batch culture conditions (harvested on day 10) and in ambr® microbioreactors were analyzed by SEC-LC intact MS for their relative mismatch levels. Although the distribution of mismatch species in each given clone differed between batch culture and ambr conditions (Figures 6F and 6G), clone rankings based on total mismatches were generally similar between the two growth conditions (Figure 6B). These results vividly demonstrate the importance and validity of early clone selection based on mismatch data, despite inevitable changes in process conditions later in drug development.

[0166] It is noteworthy that in both studies there was a greater variation in yield of correctly paired tsAb among clones grown in ambr® compared to batch culture; this is indicated by the % purity range 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 top clones appeared to perform better in the ambr bioreactor than in batch culture, as suggested by the 10-20% higher purity.

[0167] conclusion Misassembled species are product-related impurities that commonly occur during the manufacturing of multispecific antibodies, leading to reduced production yields and requiring costly purification steps as well as robust analytical methods for early monitoring. In the above examples, a high-throughput analytical platform based on denaturing SEC-LC-intact MS for the identification and relative quantification of chain mispairing and other IgG-related species was described. The developed method allows for direct intact MS analysis of mAb-related species in the clarified harvest, avoiding time-consuming and expensive ProA purification and buffer exchange steps (Figure 7). Intact MS analysis of two different tsAb constructs; anti-CD38 TCE and anti-CD38 TCE from different clones of CHO cells. and anti-HER2 TCE showed variation in the levels of correctly matched tsAb yields among clones, but there was no clear correlation between clone productivity (titer) and mismatches. Furthermore, different growth conditions may affect the type and distribution of impurities, but did not affect clone quality ranking, validating the validity of early clone selection based on mismatch data despite inevitable changes in process conditions later in drug development. EXAMPLES

[0168] Intact mass analysis of harvested samples for trispecific binding proteins Intact protein mass spectrometry has been used by others in the past for the identification and relative quantification of biotherapeutics, their variants and product-related impurities. Several different intact protein MS approaches based on reversed-phase separation or size-exclusion LC for denaturing and native MS analysis have been reported (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 require that antibodies be purified, typically through Protein A affinity, prior to LC-MS analysis.

[0169] The above example describes a high-throughput analytical platform consisting of denaturing size-exclusion liquid chromatography (SEC) coupled to QToF mass spectrometry for intact MS analysis of strand mispairing in trispecific antibodies (tsAbs). See also Tousi, F. et al. (2020) Anal. Chem. 92:2764-2769. This intact mass method can be performed directly on clarified harvest fluids without prior purification or sample preparation. This analytical platform enabled the screening of a large number of CHO (Chinese Hamster Ovary) cell clones expressing different tsAb constructs for their levels of mispaired species and half-antibodies. Results from this intact mass spectrometry method, especially in early development, facilitate the selection of CHO clones producing tsAbs of acceptable product quality.

[0170] Examples 4 and 5 describe the application of the same analytical method to clonal selection for different mAb-related constructs: an anti-HIV / CD28 / CD3 trispecific antibody (Example 4) and a cysteine ​​engineered monoclonal antibody (mAb) (Example 5).

[0171] material and method Size Exclusion Chromatography (SEC) Size-exclusion LC separation was performed using a UPLC BEH SEC column, 1.7 μm, 200 Å, 4.6 × 300 mm, controlled by an Acquity UPLC H-class (Waters Corp., Milford, MA, United States). A solution of 0.05% formic acid (FA) and 0.05% TFA in 30:70 acetonitrile:water with a flow rate of 0.1 mL / min (0 to 25 min) and 0.4 ml / min (25 to 33 min) in isocratic mode was used as the mobile phase. A 50 μL volume of the clarified harvest was directly injected into the LC-MS system using an Acquity autosampler set at 4 °C. FA and TFA were both LC-MS grade from Sigma (St. Louis, MO, United States). Water and acetonitrile for LC-MS were from Thermo Fisher Scientific (Waltham, MA, United States).

[0172] Mass spectrometry (MS) The LC was monitored using a Xevo G2 (or G2XS) QToF mass analyzer (Wa The Xevo G2 instrument was connected to a 300 sq. m. FT-IR spectrometer (Microelectron Microscopy, Inc., Milford, MA, United States). 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 of 50 L / hr, desolvation gas flow of 800 L / hr, and collision energy of 6 V. The ESI parameters for the Xevo G2XS were: capillary voltage of 3 kV, sampling cone voltage of 200 V, source temperature of 150° C., source offset of 150, desolvation temperature of 250° C., cone gas flow of 50 L / hr, desolvation gas flow of 800 L / hr, and collision energy of 6 V.

[0173] Data analysis Acquired LC-MS raw files were batch processed using Byos® (Protein Metrics Inc.) for deconvolution, annotation and relative quantification.

[0174] result Cleaned cell culture harvest samples from 50 clones of CHO cells expressing anti-HIV / CD28 / CD3 tsAb were analyzed using the denaturing SEC-intact MS method described above. Analysis of the acquired MS raw files revealed a high variability in the level (%) of mismatches between the clones tested, ranging from as low as 1% to 100% mismatches. H1L1 / H2L1 (165-167 kDa) was the predominant mismatch species. A small number of clones contained the H1L2 / H2L2 mismatch form. H2L2 / H2L2 homodimers were detected in a few clones. Half antibodies (H1L1 and H2L2) were present in some clones, albeit at low levels. Unlike the previously tested trispecific antibodies, no significant amount of H1L2 / H2L2 type mismatches was detected in the HIV TCE clones. 8A and 8B show deconvoluted intact mass spectra for anti-HIV / CD28 / CD3 clones with the highest and lowest levels of strand mismatching, respectively.

[0175] Figures 9A and 9B show clonal productivity as measured by Protein A titer and relative levels of correct mass for 50 cell clones producing anti-HIV / CD28 / CD3 trispecifics grown under batch culture conditions during the clonal selection step as part of cell line development. The results of the mismatch analysis combined with the productivity data allowed the selection of clones with the highest titers as well as the lowest levels of strand mismatching. Interestingly, some clones with relatively high productivity showed 0% correctly matched tsAb as can be seen at the far right of the column graph in Figure 9B.

[0176] The excellent chromatographic separation combined with the availability of a highly sensitive mass spectrometer allowed the elucidation of a highly heterogeneous glycan profile at the intact level due to the presence of sialylated N-glycans on the Fab LC in addition to the usual heavy chain Fc N-glycans. This allows the rapid monitoring of the N-glycosylation profile of clones without the need to perform more thorough glycan analysis using typically labor-intensive and time-consuming methods. As an example, Figures 10A and 10B show the detailed glycan profile of a reference mass (H1L1 / H2L2) in a selected clone producing anti-HIV / CD28 / CD3 cultured in two different conditions: 1-centrifuge tube (batch condition) and 2-ambr15 bioreactors (fed-batch condition), respectively, showing a shift towards more galactosylated and sialylated N-glycans as evident from the shift towards higher mass values. Due to the extreme glycan heterogeneity added by light chain glycosylation, the molecular weights of the reference form (H1L1 / H2L2) and the H1L1 / H2L1 mismatched form range from 1000 Da to 2000 Da, respectively. Glycotype assignments for the intact anti-HIV / CD28 / CD3 reference form are detailed in Table 1.

[0177] [Table 2] EXAMPLES

[0178] Intact mass analysis of a collection of cysteine ​​engineered antibodies This product is a thiomAb, i.e., an antibody that includes an additional cysteine ​​residue incorporated into its Fc region that is beneficial for the conjugation of compounds, e.g., cytotoxic drugs to form antibody-drug conjugates. This additional cysteine ​​is unpaired (free), i.e., not involved in disulfide bonds with other cysteines in the mAb molecule. Previous studies have shown that this additional cysteine ​​(Cys293) can form undesired disulfide bonds, otherwise known as "disulfide bond scrambling," with nearby cysteine ​​residues, potentially leading to structural instability of the antibody. Uncontrolled sulfhydryl chemistry can also pose significant manufacturing risks during the process of forming ADCs, leading to undesirable drug conjugation profiles. To avoid these risks, it is inevitable to have free cysteines capped with disulfide bond modifications, such as cysteinylation and glutathionylation, by adjusting cell culture conditions. These modifications can be selectively reduced to generate free cysteines prior to the drug conjugation step.

[0179] In this example, the developed denaturing SEC-intact MS of cell culture harvests is used for rapid screening of clones for their Cys293 capping status during cell line development. The first thiomAb candidate tested contained a cytosine residue in the Fc region. It had an N300A mutation that eliminated N-glycosylation. The absence of glycans allowed identification of the Cys293 capping by direct intact MS analysis of the harvest without the need for any sample manipulation (Figure 11A).

[0180] However, the second thiomAb candidate with a typical Fc N-glycosylation profile showed a highly complex pattern in the intact MS data due to the presence of multiple capping species including N-acetylcysteinylation (+161 Da) that interfered with the mass shift due to the addition of a hexose monosaccharide residue to the N-glycan (+162 Da). To circumvent this issue and facilitate data interpretation, the cleaned harvest from the second candidate was treated with PNGase F to remove the N-glycans under native conditions. The deglycosylated harvest was subsequently subjected to SEC-intact MS as before. Figure 11B shows the deconvoluted intact mass spectrum obtained for the second candidate after deglycosylation, where three types of capping modifications are identified: cysteinylation with a mass shift of +119 Da; (Cys), N-acetylcysteinylation (N-acetylCys) with a mass shift of +161 Da, and glutathionylation (Glu) with a mass shift of +305 Da. Screening of 22 clones of CHO cells expressing thiomAb by this method showed 100% capping of the engineered cysteines in all tested clones.

Claims

1. 1. A method for monitoring the production of a multispecific binding protein and one or more mismatched species, comprising: Detecting the amount of the multispecific binding protein and the one or more mismatched species in a cell culture medium containing the multispecific binding protein and the one or more mismatched species by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), thereby monitoring the production of the multispecific binding protein and the one or more mismatched species. Including, A multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain that is different from the first polypeptide chain; The method, wherein the one or more mismatched species comprises 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. 2. The method of 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 that is different from the first antibody heavy chain, and a second antibody light chain that is different from the first antibody light chain.

3. One or more mismatch species: (i) the assembly of four polypeptide chains of a multispecific antibody comprising two first antibody heavy chains; (ii) the assembly of four polypeptide chains of a multispecific antibody comprising two second antibody heavy chains; (iii) the assembly of four polypeptide chains of a multispecific antibody comprising two first antibody light chains; and (iv) Assembly of four polypeptide chains of a multispecific antibody comprising two second antibody light chains. The method of claim 2 , comprising one or more of the following:

4. A multispecific binding protein comprises four polypeptide chains that form three antigen-binding sites, The first polypeptide chain of the binding protein has the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes a structure represented by The second polypeptide chain of the binding protein has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes a structure represented by The third polypeptide chain of the binding protein has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes a structure represented by The fourth polypeptide chain of the binding protein has the formula: V Ｌ３ -C Ｌ [IV] The structure includes a structure represented by During the ceremony: 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; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; Hinge is C Ｈ１ and C. Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; The polypeptide of formula I and the polypeptide of formula II form a cross-light chain heavy chain pair, Ｈ１ and V Ｌ１ forms the first antigen-binding site, and V Ｈ２ and V Ｌ２ forms the second antigen-binding site, and V Ｈ３ and V Ｌ３ forms a third antigen-binding site, The method of claim 1.

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

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

7. The method of any one of claims 1 to 6, wherein the relative amount of a multispecific binding protein compared to one or more mismatched species is detected.

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

9. The method of claim 7, wherein the relative amount of the multispecific binding protein compared to the total amount of mismatched species is detected.

10. The method of any one of claims 1 to 9, wherein the cell culture medium is a cell culture harvest.

11. 11. The method of claim 10, further comprising isolating the cell culture medium from the cell line producing the multispecific binding protein and the one or more mismatched species prior to detection.

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

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

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

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

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

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

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

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

20. 20. The method of claim 19, wherein the SE-UPLC is performed at an initial flow rate of about 0.1 mL / min.

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

22. The method of any one of claims 1 to 21, wherein SE-UPLC is performed using isocratic elution with the mobile phase.

23. 23. The method of claim 22, wherein the mobile phase comprises a solution comprising 30:70 acetonitrile:water.

24. 24. The method of claim 22 or claim 23, wherein the mobile phase comprises formic acid and trifluoroacetic acid (TFA).

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

26. The method of any one of claims 1 to 25, wherein detection is achieved within about 33 minutes.

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

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

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

30. 30. The method of claim 29, wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line.

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

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

33. 1. A method for producing a multispecific binding protein, comprising: (a) culturing a cell line comprising one or more polynucleotides encoding a multispecific binding protein in a cell culture medium under conditions suitable for production of the multispecific binding protein and one or more mismatched species by the cell line; (b) isolating from the cell line the cell culture medium containing the multispecific binding protein and one or more mismatched species; (c) detecting the amount of the 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) removing at least a portion of one or more mismatched species from the multispecific binding protein produced by the cell line, or determining the quality and / or purity of the multispecific binding protein produced by the cell line. Including, A multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain that is different from the first polypeptide chain; The method, wherein the one or more mismatched species comprises 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.

34. 34. The method of 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 that is different from the first antibody heavy chain, and a second antibody light chain that is different from the first antibody light chain.

35. One or more mismatch species: (i) the assembly of four polypeptide chains of a multispecific antibody comprising two first antibody heavy chains; (ii) the assembly of four polypeptide chains of a multispecific antibody comprising two second antibody heavy chains; (iii) the assembly of four polypeptide chains of a multispecific antibody comprising two first antibody light chains; and (iv) Assembly of four polypeptide chains of a multispecific antibody comprising two second antibody light chains.

35. The method of claim 34, comprising one or more of the following:

36. A multispecific binding protein comprises four polypeptide chains that form three antigen-binding sites, The first polypeptide chain of the binding protein has the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes a structure represented by The second polypeptide chain of the binding protein has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes a structure represented by The third polypeptide chain of the binding protein has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes a structure represented by The fourth polypeptide chain of the binding protein has the formula: V Ｌ３ -C Ｌ [IV] The structure includes a structure represented by During the ceremony: 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; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; Hinge is C Ｈ１ and C. Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; The polypeptide of formula I and the polypeptide of formula II form a cross-light chain heavy chain pair, Ｈ１ and V Ｌ１ forms the first antigen-binding site, and V Ｈ２ and V Ｌ２ forms the second antigen-binding site, and V Ｈ３ and V Ｌ３ forms a third antigen-binding site, 34. The method of claim 33.

37. One or more mismatch species: (i) association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula I; (ii) association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula II; (iii) association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula III; and (iv) Association of four polypeptide chains of a binding protein comprising two polypeptide chains according to formula IV 37. The method of claim 36, comprising one or more of the following:

38. 38. The method of any one of claims 33 to 37, wherein detecting the amount of multispecific binding protein and one or more mismatched species comprises deconvoluting one or more MS spectra obtained by MS.

39. 39. The method of any one of claims 33 to 38, wherein detecting the multispecific binding protein and one or more mismatched species comprises assessing the overall titer of multispecific binding protein produced by the cell line.

40. 40. The method of any one of claims 33 to 39, wherein detecting the multispecific binding protein and one or more mismatched species comprises assessing the overall titer of mismatched species produced by the cell line.

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

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

43. 42. The method of claim 41, wherein the relative amount of the multispecific binding protein compared to the total amount of mismatched species is detected.

44. The method of any one of claims 33 to 43, wherein the cell culture medium is separated from the cell line by centrifugation.

45. The cell culture medium was subjected to SE-U purification in (c) without prior chromatographic separation. The method of any one of claims 33 to 44, which is subjected to PLC.

46. 45. The method of any one of claims 33 to 44, wherein the cell culture medium is subjected to SE-UPLC in (c) without preceding Protein A affinity chromatography.

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

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

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

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

51. 51. The method of any one of claims 33 to 50, wherein the SE-UPLC is performed at an initial flow rate of less than about 0.4 mL / min.

52. 52. The method of claim 51, wherein the SE-UPLC is performed at an initial flow rate of about 0.1 mL / min.

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

54. The method of any one of claims 33 to 53, wherein SE-UPLC is performed using isocratic elution with the mobile phase.

55. 55. The method of claim 54, wherein the mobile phase comprises a solution comprising 30:70 acetonitrile:water.

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

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

58. 58. The method of any one of claims 33 to 57, wherein (c) is accomplished within about 33 minutes.

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

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

61. The method of any one of claims 33 to 60, wherein the cell line is a mammalian cell line.

62. 62. The method of claim 61, wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line. The method described.

63. 63. The method of any one of claims 33 to 62, wherein the cell line is cultured in (a) in a continuous, stirred tank bioreactor culture.

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

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

66. 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) selecting a cell line that produced a higher amount of the multispecific binding protein based on the comparison.

66. The method of claim 65, further comprising:

67. 67. The method of claim 65 or claim 66, further comprising detecting the amount of one or more mismatched species produced by the first cell line by the method of any one of claims 33 to 64; and detecting the amount of one or more mismatched species produced by the second cell line by the method of any one of claims 33 to 64.

68. 68. The method of claim 67, further comprising, after detecting the amount of the one or more mismatched species produced by the first and second cell lines: comparing the amount of the one or more mismatched species produced by the first cell line with the amount of the one or more mismatched species produced by the second cell line; and selecting a cell line that produced a higher ratio of multispecific binding protein to the one or more mismatched species based on the comparison.

69. 69. The method of claim 68, wherein the cell line is selected based on a high ratio of multispecific binding protein to the amount of one or more individual mismatched species.

70. 69. The method of claim 68, wherein the cell line is selected based on a high ratio of multispecific binding protein to the total amount of mismatched species.

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

72. 1. A method for producing a multispecific binding protein, comprising: (a) culturing a cell line comprising one or more polynucleotides encoding an antibody or antibody derivative in a cell culture medium under conditions suitable for production of the antibody or antibody derivative and one or more heavy variant species by the cell line; (b) isolating from the cell line a cell culture medium comprising the antibody or antibody derivative and one or more weight variant species; (c) detecting the 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) removing at least a portion of one or more weight variant species from the antibody or antibody derivative produced by the cell line, or determining the quality and / or purity of the antibody or antibody derivative produced by the cell line. Including, The method, wherein the antibody or antibody derivative and the one or more weight variant species differ in molecular weight.

73. 73. The method of claim 71 or claim 72, wherein the antibody or antibody derivative and one or more weight variant species include cysteinylated, N-acetylcysteinylated and glutathionylated species having a free cysteine.

74. 73. The method of claim 71 or claim 72, wherein the antibody or antibody derivative and one or more weight variant species include species that contain chemically modified cysteine ​​residues.

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

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

77. 77. The method of any one of claims 71 to 76, wherein the antibody or antibody derivative comprises mutations in the Fc region, excluding N-glycosylation in the Fc region.

78. 78. The method of claim 77, wherein the antibody or antibody derivative comprises an N300A mutation as numbered by the EU index.

79. 77. The method of any one of claims 71 to 76, further comprising removing N-glycosylation in the Fc region prior to detection via SE-UPLC-MS.

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

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

82. 82. A method according to any one of claims 71 to 81, capable of resolving mass differences in cysteinylated, N-acetylcysteinylated and glutathionylated species.

83. 73. The method of claim 71 or claim 72, wherein the antibody or antibody derivative and the one or more weight variant species comprises a glycoform of the antibody or antibody derivative.

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

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

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

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

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

89. 89. The method of any one of claims 71 to 88, wherein the relative amount of the antibody or antibody derivative compared to one or more weight variant species is detected.

90. 90. The method of claim 89, wherein the relative amount of antibody or antibody derivative compared to the amount of one or more individual weight variants is detected.

91. 90. The method of claim 89, wherein the relative amount of antibody or antibody derivative compared to the total amount of weight variant species is detected.

92. 72. The method of claim 71, wherein the cell culture medium is a cell culture harvest.

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

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

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

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

97. 96. The method of any one of claims 71 to 95, wherein the cell culture medium is subjected to SE-UPLC without preceding Protein A affinity chromatography.

98. The method of any one of claims 71 to 97, wherein the MS is an intact MS.

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

100. The method according to any one of claims 71 to 99, wherein the SE-UPLC is a modified SE-UPLC. How to.

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

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

103. 103. The method of claim 102, wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line.

104. The method of any one of claims 72 to 103, wherein the cell line is cultured in a continuous, stirred tank bioreactor culture.

105. The method of any one of claims 72 to 103, wherein the cell line is cultivated in batch cell culture.

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

107. After (a) and (b): (c) comparing the amount of the antibody or antibody derivative produced by the first cell line with the amount of the antibody or antibody derivative produced by the second cell line; and (d) selecting a cell line that produced a higher amount of antibody or antibody derivative based on the comparison.

107. The method of claim 106, further comprising:

108. 108. The method of claim 106 or claim 107, further comprising detecting the amount of one or more weight variant species produced by a first cell line by the method of any one of claims 72 to 105; and detecting the amount of one or more weight variant species produced by a second cell line by the method of any one of claims 72 to 105.

109. 109. The method of claim 108, further comprising, after detecting the amount of one or more weight variant species produced by the first and second cell lines: comparing the amount of the one or more weight variant species produced by the first cell line with the amount of the one or more weight variant species produced by the second cell line; and selecting a cell line that produced an antibody or antibody derivative in a higher ratio to the one or more weight variant species based on the comparison.

110. 110. The method of claim 109, wherein the cell line is selected based on having a high ratio of antibody or antibody derivative to the amount of one or more individual weight variant species.

111. 110. The method of claim 109, wherein the cell line is selected based on a high ratio of antibody or antibody derivative to total amount of heavy variant species.

Images