METHODS FOR ANALYZING CHAIN MISP AIRING IN MULTISPECIFIC BINDING

PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/926,313, filed October 25, 2019, and EP Application No. EP20315271.5, filed May 28, 2020, the disclosures of each of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952032840SEQLIST.TXT, date recorded: May 29, 2020, size: 2 KB).

FIELD

[0003] The disclosure relates to methods for monitoring production of a multispecific binding protein and one or more mispaired species by a host cell, as well as methods of production and screening related thereto. The disclosure further relates to methods for monitoring production of an antibody or antibody derivative and one or more weight variant species by a host cell, as well as methods of production and screening related thereto.

BACKGROUND

[0004] Multi-specific antibodies that bind two or more different epitopes on the same or different antigens have become attractive therapeutic options in immune-oncology in recent years (Baeuerle, P. A. & Reinhardt, C., Cancer Res 2009, 69 (12), 4941-4). Multi-targeting has been used for different purposes, such as achieving enhanced drug specificity or mimicking natural ligands in signaling pathways, for example in hemophilia treatments through simultaneous binding to receptor pairs on the surface of the same cell (Kitazawa, T. et al. Nat Med 2012, 18 (10), 1570-4). A prominent application is the T-cell engager (TCE) concept where one arm of the molecule activates T-cells via CD3/CD28 receptor binding and the other arm targets a tumor antigen for tumor killing (Krah, S. et al. N Biotechnol 2017, 39 (Pt B), 167-173; Correnti, C. E. et al. Leukemia 2018, 32 (5), 1239-1243).

[0005] IgG-like tri-specific antibodies (tsAb) are comprised of two different heavy chains and two different light chains, commonly expressed in a single host cell followed by intracellular chain assembly. While this method of production eliminates the need to have two separate cell lines and purification processes, it can generate unwanted mispaired species in addition to the desired tsAb. In the absence of rational design and with random association of the subunits, the theoretical yield of the correctly paired species is only 12.5% (FIG. IB). Forced heterodimerization of the heavy chains has been successfully accomplished through protein engineering approaches such as the knobs-into-holes design (Ridgway, J. B . et al. Protein Eng 1996, 9 (7), 617-21). However, cognate pairing of the light chains to the correct heavy chains remains a significant challenge in tsAb production.

[0006] As such, there remains a need for methods for monitoring or analyzing production of a multispecific binding protein and one or more mispaired species. In addition, there remains a need for methods for monitoring or analyzing production of antibody or antibody derivatives that comprise multiple species varying in molecular weight ( e.g. , with different chemical modifications, such as chemically modified cysteine or other residues, or with different glycoforms).

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

[0008] To meet these and other needs, provided herein are methods for monitoring production of a multispecific binding protein and one or more mispaired species (e.g, by a cell line). These methods provide, inter alia , a high throughput analytical platform based on denaturing SEC-LC-Intact MS for identification and relative quantitation of chain mispairing and other IgG-related species. Advantageously, these methods allow for intact MS analysis of mAb-related species (e.g, multispecific binding proteins, antibodies, Fc fusion proteins, antibody fragments, and so forth) directly from a clarified cell harvest, bypassing time consuming and expensive purification (e.g, protein A affinity chromatography) and buffer exchange steps. As such, these methods can be used to rapidly screen a large number of clones for potential production cell lines.

[0009] In some embodiments, provided herein are methods for monitoring production of a multispecific binding protein and one or more mispaired species, the methods

comprising: detecting, by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of a multispecific binding protein and one or more mispaired species in a cell culture medium comprising the multispecific binding protein and the one or more mispaired species. In some embodiments, the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, and the one or more mispaired species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

[0010] 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, antibody fragment, or Fc fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody, antibody fragment, or Fc fusion protein. In some embodiments, the one or more mispaired species comprises one or more of: an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody heavy chains; an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody heavy chains; an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody light chains; and an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains that form the three antigen binding sites,

wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

VL2-LI-VLI-L2-CL [I]

and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II]

and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

Vro-Cm-hinge-Cm-Cro [III]

and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin Cm heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, wherein VHI and VLI form a first antigen binding site, wherein Vm and VL2 form a second antigen binding site, and wherein V and VL3 form a third antigen binding site. In some embodiments, the one or more mispaired species comprise one or more of: an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula I; an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula II; an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula III; and an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula IV.

[0011] In some embodiments, detecting the amount of the multispecific binding protein and the one or more mispaired species comprises deconvoluting one or more MS spectra obtained by the MS. In some embodiments, a relative amount of the multispecific binding protein as compared to the one or more mispaired species is detected. In some embodiments, a relative amount of the multispecific binding protein as compared to an amount of one or more individual mispaired species is detected. In some embodiments, a relative amount of the multi specific binding protein as compared to an overall amount of mispaired species is detected. In some embodiments, the methods further comprise, prior to detection, providing or obtaining ( e.g ., from a cell line that produces the multispecific binding protein and one or more mispaired species) a cell culture medium comprising the multispecific binding protein and one or more mispaired species. In some embodiments, the methods further comprise, prior to detection, separating the cell culture medium from a cell line that produces the multispecific binding protein and one or more mispaired species. In some embodiments, prior to detection, the cell culture medium is separated from the cell line by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC without a prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography. In some embodiments, the MS is intact MS. In some embodiments, the MS is quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is denaturing SE-UPLC.

In some embodiments, the SE-UPLC is directly coupled to the MS. In some embodiments, the methods further comprise, prior to SE-UPLC-MS, contacting the multispecific binding protein and one or more mispaired species with a protease. In some embodiments, the protease is IdeS or IdeZ. In some embodiments, SE-UPLC is performed with an initial flow rate of less than about 0.4mL/min. In some embodiments, SE-UPLC is performed with an initial flow rate of about 0. lmL/min. In some embodiments, SE-UPLC is performed with a flow rate of about O.lmL/min for the first 25 minutes, followed by a flow rate of about 0.4mL/min (e.g., for minutes 25-33). In some embodiments, SE-UPLC is performed using 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 one or more mispaired species is accomplished in about 33 minutes or less. In some embodiments, In some embodiments, the method is accomplished in about 33 minutes or less. In some embodiments, the MS is capable of resolving a mass difference of about 300 Da between the multispecific binding protein and the one or more mispaired species, or between two mispaired species. In some embodiments, the MS is capable of resolving a mass difference of about 162 Da between the multispecific binding protein or mispaired species and one of more glycoforms.

[0012] 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 with the cell culture medium in a continuous cell culture, e.g ., in a stirred tank bioreactor. In some embodiments, prior to detection, the cell line is cultured with the cell culture medium in a batch cell culture.

[0013] In some embodiments, provided herein are methods for producing a multispecific binding protein, the methods 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 production of the multispecific binding protein and one or more mispaired species by the cell line; (b) separating, from the cell line, the cell culture medium comprising the multispecific binding protein and one or more mispaired species; (c) detecting an amount of the 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 (d) removing at least a portion of one or more of the mispaired species from the multispecific binding protein produced by the cell line, or determining one or both of 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 mispaired species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

[0014] 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 Fc fusion protein. In some embodiments, the multispecific binding protein is a trispecific antibody or Fc fusion protein. In some embodiments, the one or more mispaired species comprises one or more of: an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody heavy chains; an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody heavy chains; an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody light chains; and an association of four polypeptide chains of the

multispecific antibody that comprises two of the second antibody light chains. In some embodiments, the multispecific binding protein comprises four polypeptide chains that form the three antigen binding sites,

wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

VL2-LI-VLI-L2-CL [I]

and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II]

and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

Vm-Cm-hinge-Cm-Cm [III]

and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

Cm is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin Cm heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, wherein VHI and VLI form a first antigen binding site, wherein Vm and VL2 form a second antigen binding site, and wherein V and VL3 form a third antigen binding site. In some embodiments, the one or more mispaired species comprise

one or more of: an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula I; an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula II; an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula III; and an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula IV.

[0015] In some embodiments, detecting the amount of the multispecific binding protein and one or more mispaired species comprises deconvoluting one or more MS spectra obtained by MS. In some embodiments, detecting the multispecific binding protein and one or more mispaired species comprises assessing overall titer of the multispecific binding protein produced by the cell line. In some embodiments, detecting the multispecific binding protein and one or more mispaired species comprises assessing overall titer of the mispaired species produced by the cell line. In some embodiments, a relative amount of the multispecific binding protein as compared to the one or more mispaired species is detected. In some embodiments, a relative amount of the multispecific binding protein as compared to an amount of one or more individual mispaired species is detected. In some embodiments, a relative amount of the multispecific binding protein as compared to an overall amount of mispaired 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 prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC in (c) without prior protein A affinity chromatography. In some embodiments, the MS is intact MS. In some embodiments, the MS is quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is denaturing SE-UPLC. In some embodiments, the SE-UPLC is directly coupled to the MS. In some embodiments, the methods further comprise, after culturing the cell line and prior to detection, contacting the multispecific binding protein and one or more mispaired species with a protease. In some embodiments, the protease is IdeS or IdeZ. In some embodiments, SE-UPLC is performed with an initial flow rate of less than about 0.4mL/min. In some embodiments, SE-UPLC is performed with an initial flow rate of about 0. lmL/min. In some embodiments, SE-UPLC is performed with a flow rate of about 0. lmL/min for the first 25 minutes, followed by a flow rate of about 0.4mL/min. In some embodiments, SE-UPLC is performed using isocratic elution with a mobile phase. In some embodiments, the mobile phase comprises a solution comprising

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 accomplished in about 33 minutes or less. In some embodiments, the method is accomplished in about 33 minutes or less. In some embodiments, the MS is capable of resolving a mass difference of about 300 Da between the multispecific binding protein and the one or more mispaired species, or between two mispaired species. In some embodiments, the MS is capable of resolving a mass difference of about 162 Da between the multispecific binding protein or mispaired species and one of more glycoforms.

[0016] 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 a continuous cell culture ( e.g ., in a stirred tank bioreactor). In some embodiments, the cell line is cultured in a batch cell culture.

[0017] In some embodiments, provided herein are methods for screening a plurality of cell lines for production of a multispecific binding protein, the methods comprising: detecting an amount of a multispecific binding protein produced by a first cell line of the plurality according to the method of any one of the above embodiments; and detecting an amount of the multispecific binding protein produced by a second cell line of the plurality according to the method of any one of the above embodiments.

[0018] In some embodiments, the methods further comprise: comparing the amount of multispecific binding protein produced by the first cell line with the amount of multispecific binding protein produced by the second cell line; and based on the comparison, selecting the cell line that produced the higher amount of the multispecific binding protein. In some embodiments, the methods further comprise: detecting an amount of one or more mispaired species produced by the first cell line according to the method of any one of the above embodiments; and detecting an amount of one or more mispaired species produced by the second cell line according to the method of any one of the above embodiments. In some embodiments, the methods further comprise: after detecting the amount of one or more mispaired species produced by the first and second cell lines: comparing the amount of the one or more mispaired species produced by the first cell line with the amount of the one or more mispaired species produced by the second cell line; and based on the comparison, selecting the cell line that produced the higher ratio of multispecific binding protein to one or more mispaired species. In some embodiments, the cell line is selected based on a higher ratio of multispecific binding protein to the amount of one or more individual mispaired species. In some embodiments, the cell line is selected based on a higher ratio of multispecific binding protein to the overall amount of mispaired species.

[0019] In some embodiments, provided herein are methods for monitoring production of an antibody or antibody derivative and one or more weight variant species, the methods comprising: detecting, by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of an antibody or antibody derivative and one or more weight variant species in a cell culture medium comprising the antibody or antibody derivative and one or more weight variant species, wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight. In some embodiments, provided herein are methods for producing an antibody or antibody derivative, the methods comprising: (a) culturing a cell line comprising one or more polynucleotides encoding the 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 weight variant species by the cell line; (b) separating, from the cell line, the cell culture medium comprising the antibody or antibody derivative and one or more weight variant species; (c) detecting an amount of the antibody or antibody derivative and one or more weight variant species in the cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS); and (d) removing at least a portion of one or more of the weight variant species from the antibody or antibody derivative produced by the cell line, or determining one or both of quality and purity of the antibody or antibody derivative produced by the cell line.

[0020] In some embodiments, provided herein are methods for screening a plurality of cell lines for production of an antibody or antibody derivative, the methods comprising: detecting an amount of an antibody or antibody derivative produced by a first cell line of the plurality according to the method of any one of the above embodiments; and detecting an amount of the antibody or antibody derivative produced by a second cell line of the plurality according to the method of any one of the above embodiments. In some embodiments, the methods further comprise: comparing the amount of antibody or antibody derivative produced by the first cell line with the amount of antibody or antibody derivative produced by the second cell line; and based on the comparison, selecting the cell line that produced the higher amount of the antibody or antibody derivative. In some embodiments, the methods further comprise: detecting an amount of one or more weight variant species produced by the first cell line according to the method of any one of the above

embodiments; and detecting an amount of one or more weight variant species produced by the second cell line according to the method of any one of the above embodiments. In some embodiments, the methods further comprise: after detecting the 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 based on the comparison, selecting the cell line that produced the higher ratio of antibody or antibody derivative to one or more weight variant species, or selecting the cell line that produced a higher relative proportion of a single weight variant species relative to a total amount of antibody or antibody derivative and one or more weight variant species produced. In some embodiments, the amount of an antibody or antibody derivative and/or one or more weight variant species according to any of the embodiments herein refers to a relative amount ( e.g ., as compared to one or more other species, or as compared to a total amount of antibody/species produced).

[0021] 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 the one or more weight variant species comprise species with a free cysteine (e.g., not disulfide bonded with another cysteine of the antibody or antibody derivative) that has been cysteinylated, N-acetyl cysteinylated, or glutathionylated. 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 (e.g, prior to SE-UPLC-MS), removing the antibody N-glycosylation. In some embodiments, removing the 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 the MS. In some embodiments, the methods further comprise, prior to detection, providing or obtaining (e.g, from a cell line that produces the antibody or antibody derivative and one or more weight variant species) a cell culture medium comprising the antibody or antibody derivative and one or more weight variant species. In some embodiments, the methods further comprise, prior to detection, separating the cell culture medium from a cell line that produces the antibody or antibody derivative and one or more weight variant species. In some embodiments, prior to detection, the cell culture medium is separated from the cell line by centrifugation. In some embodiments, the cell culture medium is subjected to SE-UPLC without a prior chromatographic separation. In some embodiments, the cell culture medium is subjected to SE-UPLC without prior protein A affinity chromatography. In some embodiments, the MS is intact MS. In some embodiments, the MS is quadrupole time-of-flight (QToF) MS. In some embodiments, the SE-UPLC is denaturing 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 species of the antibody or antibody derivative comprising a chemically modified cysteine residue. In some embodiments, the antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 119 Da. In some embodiments, the MS is capable of resolving a mass difference among cysteinylated (119 Da mass shift), N-acetyl cysteinylated (161 Da mass shift), and glutathionylated (305 Da mass shift) species the antibody or antibody derivative and one or more weight variant species differ in molecular weight by at least 162 Da. In some embodiments, the antibody or antibody derivative is N-glycosylated (e.g, in the antibody Fc region), and the one or more weight variant species represent gly coforms of the antibody or antibody derivative. In some embodiments, the MS is capable of resolving a mass difference of about 162 Da between the antibody or antibody derivative and one of 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.

[0022] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that bind three target proteins: CD28, CD3, and HER2. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28 (CODY), and a second pair of polypeptides

possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HER2 (Fab). The trispecific binding protein shown in FIG. 1A uses a constant region with a “knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.

[0024] FIG. IB illustrates species resulting from heavy chain (left) or light chain (right) mispairing. On left, shown are species that result from mispairing of two Fab heavy chains (top) or two CODV heavy chains (bottom). On right, shown are species that result from mispairing of two Fab light chains (bottom left), two CODV light chains (bottom right), or mispairing of Fab and CODV light chains to the wrong heavy chains (top right), as well as the correctly paired trispecific binding protein (top left).

[0025] FIGS. 2A & 2B show deconvoluted mass spectrometry (MS) spectra for intact (FIG. 2A) and F(ab’)2 fragments in IdeS-digested (FIG. 2B) anti-CD38 trispecific binding proteins, with different species labeled. Species depicted include correctly paired trispecific binding protein (H1L1/H2L2), mispaired species with two Fab light chains (H1L1/H2L1), and mispaired species with two CODV light chains (H1L2/H2L2).

Annotations show experimental mass vs. theoretical mass (in parenthesis).

[0026] FIGS. 3A & 3B show production of trispecific binding protein from a series of clones. FIG. 3A shows a ranking of anti-CD38 trispecific binding protein-producing clones by percentage of correct tsAb mass (purity). FIG. 3B shows the productivity of each clone (normalized titer) in the same order as depicted in FIG. 3A ( i.e ranked by purity).

[0027] FIG. 3C shows pie charts depicting contributions of mispaired and half antibody impurities, as well as correctly paired species, in randomly selected subset of anti-CD38 trispecific binding protein-producing clones generated by batch culture.

[0028] FIGS. 4A-4C show analysis of trispecific binding proteins by SEC-intact MS.

FIG. 4A shows a representative base peak chromatogram for denaturing SEC-intact MS analysis of a tsAb clarified harvest fluid. FIG. 4B shows combined spectra under each chromatographic peak. FIG. 4C shows a zoom-in of deconvoluted mass spectrum for a mixture of anti-CD38 trispecific binding protein and anti-HER2 trispecific binding protein resolving light chain mispaired species from two constructs with Amass = 302 Da.

[0029] FIG. 5A shows relative quantitation of correctly paired and mispaired species obtained for different column load (pg protein) from 4.4-21.8pg for anti-HER2 trispecific binding protein and 11.0-55. Opg for anti-CD38 trispecific binding protein, demonstrating the robustness and reliability of this method for analysis of low and high titer harvest samples. For anti-HER2 trispecific binding protein, column loads are shown as 4.4pg, 8.7pg, 13.1pg, 17.4pg, and 21.8pg (left to right). For anti-CD38 trispecific binding protein, column loads are shown as 1 l.Opg, 22.0pg, 33.0pg, 44.0pg, and 55.0pg (left to right).

[0030] FIG. 5B & 5C show raw and deconvoluted mass spectra obtained by SEC-LC-MS analysis of clarified harvest (FIG. 5B) and ProA purified (FIG. 5C) anti-CD38 trispecific binding protein showing comparability of the two methods. Annotations on the deconvoluted spectra show the experimental masses and % intensities of each species.

[0031] FIG. 5D & 5E show ranking of anti-HER2 trispecific binding protein-producing clones based on percentage of correct tsAb mass (purity) (FIG. 5D) and productivity measured by titer for the same clones (FIG. 5E).

[0032] FIGS. 6A & 6B show yield of correct tsAb mass in different clones of anti-CD38 trispecific binding protein (FIG. 6A) and anti-HER2 trispecific binding protein (FIG. 6B) grown under different cell culture conditions (ambr or batch, as indicated).

[0033] FIGS. 6C-6E show the effects of different growth conditions on chain mispairing and half antibody levels in anti-CD38 TCE clones grown under two different ambr conditions (FIGS. 6C & 6D) and batch culture conditions (FIG. 6E).

[0034] FIGS. 6F & 6G show the effects of different growth conditions on chain mispairing levels in anti-HER2 TCE clones grown under ambr (FIG. 6F) or batch (FIG. 6G) culture conditions.

[0035] FIG. 7 illustrates an exemplary workflow for high-throughput, intact MS, in accordance with some embodiments. In addition to the high-throughput capability, additional advantages include: (1) no need for protein A purification, saving time and cost; (2) more direct insights into cell line performance by providing information on potential wasted biomass on free chains, half antibodies, and other subspecies that may not pass protein A purification; and (3) applicability to Fc-less modalities, such as Fabs, scFvs, and other antibody fragments.

[0036] FIGS. 8A & 8B show the deconvoluted intact mass spectra obtained for anti-HIV\CD28\CD3 trispecific antibody-producing cell clones with the highest and lowest levels of chain mispairing, respectively.

[0037] FIGS. 9A & 9B show productivity (assessed by titer, pg/mL; FIG. 9A) and production of correctly paired product (% correct mass; FIG. 9B) of 50 cell clones producing anti-HIV\CD28\CD3 trispecific grown under batch culture conditions.

[0038] FIGS. 10A & 10B show the detailed glycan profiles obtained for the reference mass (HILI/H2L2) in a selected clone producing anti-HIV\CD28\CD3 trispecific antibody.

The clone was cultured under two different conditions: in a spin tube (batch condition;

FIG. 10A) and in an ambrl5 bioreactor (fed-batch condition; FIG. 10B).

[0039] FIGS. 11A & 11B show MS analysis of harvests of antibodies with an engineered cysteine (Cys293) for cysteine capping status. FIG. 11A shows identification of Cys293 capping status of species using direct intact MS analysis. This antibody contained an N300A mutation that ablated N-glycosylation of the Fc region. FIG. 11B shows identification of Cys293 capping status using direct intact MS analysis of an antibody treated with PNGase F to remove N-glycans prior to analysis.

DETAILED DESCRIPTION

[0040] The disclosure provides, inter alia , methods for monitoring production of a multispecific binding protein and one or more mispaired species, e.g ., a multispecific antibody, antibody fragment, Fc fusion protein, or other multispecific binding protein that comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain, and one or more mispaired species that comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein. Similar methods can be applied to production of a multispecific binding protein, screening cell lines for production of a multispecific binding protein, or in the production of antibodies or Fc fusion proteins, as well as production of an antibody or antibody derivative (e.g, produced with one or more weight variant species), or screening cell lines for production of an antibody or antibody derivative (e.g, produced with one or more weight variant species). Advantageously, these methods allow for 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. As such, these methods can be used to rapidly screen a large number of clones for potential production cell lines.

[0041] The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

Definitions

[0042] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0043] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0044] The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either 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, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0045] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0046] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free from

polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0047] Naturally occurring antibodies typically comprise a tetramer. 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). The terms "heavy chain" and "light chain" as used herein 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 includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and Cm), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0048] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See , e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable 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.

[0049] The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Rabat (Rabat et al. , SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Rabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al ., 1989, Nature 342: 877-83) found that certain sub-portions within Rabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI,

L2, and L3 or HI, H2, and H3 where the "L" and the "H" designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Rabat CDRs. Other boundaries defining CDRs overlapping with the Rabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp.

Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Rabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Rabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Rontermann R., Diibel S., eds. Springer-Verlag, Berlin, p.

33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.

[0050] The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl and IgG2 are preferred. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class ( e.g ., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0051] A F(ab) fragment typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CHI heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CHI domain.

[0052] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and Cm domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.

[0053] The term "binding protein" as used herein 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 infra. 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, an Fc fusion protein). In some embodiments, the binding protein is a dual variable domain (DVD) immunoglobulin, e.g., as described in WO2012061558. In some embodiments, the binding protein comprises dual variable domains having a cross over orientation, e.g. , as described in WO2012135345. In some embodiments, the binding protein comprises four polypeptide chains that form four antigen binding sites, wherein two polypeptide chains have a structure represented by the formula:

VLI-LI-VL2-L2-CL [I]

and two polypeptide chains have a structure represented by the formula:

VH2 -L3- VHI~L4~CH i -F c [11]

wherein:

VLJ is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

Vm is a first immunoglobulin heavy chain variable domain;

VR2 is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

Cm is the immunoglobulin Cm heavy chain constant domain;

Fc is the immunoglobulin hinge region and Cm, Cm immunoglobulin heavy chain constant domains; and

Li, L2, L3, and L4 are amino acid linkers; and

wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.

[0054] A trispecific binding protein of the present disclosure, unless otherwise specified, typically comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:

VL2- LI-VLI- L2-CL [I]

and a second polypeptide chain has a structure represented by the formula:

VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II]

and a third polypeptide chain has a structure represented by the formula:

VH3-CHI [III]

and a fourth polypeptide chain has a structure represented by the formula:

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is the immunoglobulin CHI heavy chain constant domain; and

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains;

Li, L2, L3 and L4 are amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formula II form a cross over light chain-heavy chain pair.

[0055] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.

[0056] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).

[0057] The term "swapability" as used herein refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity. "Full swapability" refers to the ability to swap the order of both VHI and VH2 domains, and therefore the order of VLI and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while maintaining full functionality of the binding protein as evidenced by the retention of binding affinity. Furthermore, it should be noted that the designations VH and VL refer only to the domain's location on a particular protein chain in the final format. For example, VHI and VH2 could be derived from VLI and VL2 domains in parent antibodies and placed into the VHI and Vm positions in the binding protein. Likewise, VLI and VL2 could be derived from Vm and Vm domains in parent antibodies and placed in the VHI and V positions in the binding protein. Thus, the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable." [0058] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes.

With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0059] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0060] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site.

[0061] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

[0062] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0063] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

[0064] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular embodiments 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.

[0065] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (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 sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using

Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.

[0066] The terms "substantially pure" or "substantially purified" as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0067] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics 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 is said to specifically bind an antigen when the equilibrium dissociation constant is < 108 M, more preferably when the equilibrium dissociation constant is < 109 M, and most preferably when the dissociation constant is < 10 10 M.

[0068] 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 ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the 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). The term "KD," as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0069] The term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 106 M, 1 x 107 M, 1 x 108 M, 1 x 109 M, 1 x 10 10 M, 1 x 10 11 M, 1 x 10 12 M, or more, and/or to bind to an epitope with an affinity that is at least two fold greater than its affinity for a nonspecific antigen.

[0070] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. The linkers described herein are referred to as Li, which is located on the light chain between the C-terminus of the VL2 and the N-terminus of the VLI domain; and L2, which is located on the light chain between the C-terminus of the VLI and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C-terminus of the Vm and the N-terminus of the VH2 domain; and L4, which is located between the C-terminus of the Vm and the N-terminus of the CHI domain.

CLAIMS

What is claimed is:

1. A method for monitoring production of a multispecific binding protein and one or more mispaired species, the method comprising:

detecting, by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of a multispecific binding protein and one or more mispaired species in a cell culture medium comprising the multispecific binding protein and the one or more mispaired species, thereby monitoring production of the multispecific binding protein and one or more mispaired species;

wherein the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain; and

wherein the one or more mispaired species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

2. The method 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 different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain.

3. The method of claim 2, wherein the one or more mispaired species comprises one or more of:

(i) an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody heavy chains;

(ii) an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody heavy chains;

(iii) an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody light chains; and

(iv) an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody light chains.

4. The method of claim 1, wherein the multispecific binding protein comprises four polypeptide chains that form the three antigen binding sites,

wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

VL2-LI-VLI-L2-CL [I]

and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II]

and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

Vm-Cm-hinge-Cm-Cm [III]

and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin Cm heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross over light chain-heavy chain pair, wherein VHI and VLI form a first antigen binding site, wherein Vm and VL2 form a second antigen binding site, and wherein VH3 and VL3 form a third antigen binding site.

5. The method of claim 4, wherein the one or more mispaired species comprise one or more of:

(i) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula I;

(ii) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula II;

(iii) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula III; and

(iv) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula IV.

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

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

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

9. The method of claim 7, wherein a relative amount of the multispecific binding protein as compared to an overall amount of mispaired species is detected.

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

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

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 1-12, wherein the cell culture medium is subjected to SE-UPLC without a prior chromatographic separation.

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

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

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

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

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

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

20. The method of claim 19, wherein SE-UPLC is performed with an initial flow rate of about O.lmL/min.

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

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

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

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

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 1-25, wherein detection is accomplished in about 33 minutes or less.

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

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

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

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

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

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

33. A method for producing a multispecific binding protein, the method comprising:

(a) culturing a cell line comprising one or more polynucleotides encoding the multispecific binding protein in a cell culture medium under conditions suitable for production of the multispecific binding protein and one or more mispaired species by the cell line;

(b) separating, from the cell line, the cell culture medium comprising the multispecific binding protein and one or more mispaired species;

(c) detecting an amount of the 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

(d) removing at least a portion of one or more of the mispaired species from the multispecific binding protein produced by the cell line, or determining one or both of quality and purity of the multispecific binding protein produced by the cell line;

wherein the multispecific binding protein comprises an association of two or more polypeptide chains comprising at least a first polypeptide chain and a second polypeptide chain different from the first polypeptide chain; and

wherein the one or more mispaired species comprise two or more polypeptide chains comprising at least one of the first and second polypeptide chains in an association other than that of the multispecific binding protein.

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 different from the first antibody heavy chain, and a second antibody light chain different from the first antibody light chain.

35. The method of claim 34, wherein the one or more mispaired species comprises one or more of:

(i) an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody heavy chains;

(ii) an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody heavy chains;

(iii) an association of four polypeptide chains of the multispecific antibody that comprises two of the first antibody light chains; and

(ii) an association of four polypeptide chains of the multispecific antibody that comprises two of the second antibody light chains.

36. The method of claim 33, wherein the multispecific binding protein comprises four polypeptide chains that form the three antigen binding sites,

wherein a first polypeptide chain of the binding protein comprises a structure represented by the formula:

VL2-LI-VLI-L2-CL [I]

and a second polypeptide chain of the binding protein comprises a structure represented by the formula:

VHi-L3-VH2-L4-Cm-hinge-CH2-CH3 [II]

and a third polypeptide chain of the binding protein comprises a structure represented by the formula:

Vm-Cm-hinge-Cm-Cm [III]

and a fourth polypeptide chain of the binding protein comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VLI is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VHI is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CHI is an immunoglobulin CHI heavy chain constant domain;

CH2 is an immunoglobulin Cm heavy chain constant domain;

CH3 is an immunoglobulin Cm heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CHI and Cm domains; and

Li, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair, wherein VHI and VLI form a first antigen binding site, wherein Vm and VL2 form a second antigen binding site, and wherein V and VL3 form a third antigen binding site.

37. The method of claim 36, wherein the one or more mispaired species comprise one or more of:

(i) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula I;

(ii) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula II;

(iii) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula III; and

(iv) an association of four polypeptide chains of the binding protein that comprises two polypeptide chains according to formula IV.

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

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

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

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

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

43. The method of claim 41 , wherein a relative amount of the multi specific binding protein as compared to an overall amount of mispaired species is detected.

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

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

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

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

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

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

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

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

52. The method of claim 51, wherein SE-UPLC is performed with an initial flow rate of about O.lmL/min.

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

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

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

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

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

58. The method of any one of claims 33-57, wherein (c) is accomplished in about 33 minutes or less.

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

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

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

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

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

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

65. A method for screening a plurality of cell lines for production of a multi specific binding protein, the method comprising:

(a) detecting an amount of a multispecific binding protein produced by a first cell line of the plurality according to the method of any one of claims 33-64; and

(b) detecting an amount of the multispecific binding protein produced by a second cell line of the plurality according to the method of any one of claims 33-64.

66. The method of claim 65, further comprising, after (a) and (b):

(c) comparing the amount of multispecific binding protein produced by the first cell line with the amount of multispecific binding protein produced by the second cell line; and

(d) based on the comparison, selecting the cell line that produced the higher amount of the multispecific binding protein.

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

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

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

70. The method of claim 68, wherein the cell line is selected based on a higher ratio of multispecific binding protein to the overall amount of mispaired species.

71. A method for monitoring production of an antibody or antibody derivative and one or more weight variant species, the method comprising:

detecting, by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS), an amount of an antibody or antibody derivative and one or more weight variant species in a cell culture medium comprising the antibody or antibody derivative and the one or more weight variant species, thereby monitoring production of the antibody or antibody derivative and one or more weight variant species;

wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight.

72. A method for producing a multispecific binding protein, the method 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) separating, from the cell line, the cell culture medium comprising the antibody or antibody derivative and one or more weight variant species;

(c) detecting an amount of the antibody or antibody derivative and one or more weight variant species in the cell culture medium by size-exclusion ultra-performance liquid chromatography and mass spectrometry (SE-UPLC-MS); and

(d) removing at least a portion of one or more of the weight variant species from the antibody or antibody derivative produced by the cell line, or determining one or both of quality and purity of the antibody or antibody derivative produced by the cell line;

wherein the antibody or antibody derivative and one or more weight variant species differ in molecular weight.

73. The method of claim 71 or claim 72, wherein the antibody or antibody derivative and the one or more weight variant species comprise species with a free cysteine that has been cysteinylated, N-acetyl cysteinylated, or glutathionylated.

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

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

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

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

78. The method of claim 77, wherein the antibody or antibody derivative comprises an N300A mutation, numbering according to EU index.

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

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

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

82. The method of any one of claims 71-81, wherein the method is capable of resolving a mass difference among cysteinylated, N-acetyl cysteinylated, and glutathionylated species.

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

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

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

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

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

88. The method of any one of claims 71-87, 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 the MS.

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

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

91. The method of claim 89, wherein a relative amount of the antibody or antibody derivative as compared to an overall amount of weight variant species is detected.

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

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

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

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

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

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

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

99. The method of any one of claims 71-98, wherein the MS is quadrupole time-of-flight

(QToF) MS.

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

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

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

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 72-103, wherein the cell line is cultured in a continuous, stirred tank bioreactor culture.

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

106. A method for screening a plurality of cell lines for production of an antibody or antibody derivative, the method comprising:

(a) detecting an amount of an antibody or antibody derivative produced by a first cell line of the plurality according to the method of any one of claims 72-105; and

(b) detecting an amount of the antibody or antibody derivative produced by a second cell line of the plurality according to the method of any one of claims 72-105.

107. The method of claim 106, further comprising, after (a) and (b):

(c) comparing the amount of antibody or antibody derivative produced by the first cell line with the amount of antibody or antibody derivative produced by the second cell line; and

(d) based on the comparison, selecting the cell line that produced the higher amount of the antibody or antibody derivative.

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

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 based on the comparison, selecting the cell line that produced the higher ratio of antibody or antibody derivative to one or more weight variant species.

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

111. The method of claim 109, wherein the cell line is selected based on a higher ratio of antibody or antibody derivative to the overall amount of weight variant species.