Online chromatography and electrospray ionization mass spectrometer

Abstract

To provide a method and system for protein characterization using online chromatography and electrospray ionization mass spectrometry.SOLUTION: A method for protein characterization comprises the steps of: contacting a sample including protein to a chromatography system with a size exclusion chromatography resin; cleaning the size exclusion chromatography resin using a mobile phase to obtain an eluent containing the protein; and characterizing the protein in the eluent using an electrospray ionization mass spectrometer under non-denaturing conditions.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] Field The present invention relates generally to methods for characterizing protein biopharmaceuticals using on-line chromatography and electrospray ionization mass spectrometry. [Background technology]

[0002] background Protein biopharmaceuticals have emerged as important drugs for the treatment of cancer, autoimmune diseases, infectious diseases, and cardiometabolic disorders and are one of the fastest growing product segments in the pharmaceutical industry.

[0003] Protein biopharmaceuticals must meet very high purity standards. It may therefore be important to monitor and characterize protein biopharmaceuticals during the various stages of drug development and manufacturing. Analytical methods for testing to characterize such protein biopharmaceuticals must exhibit sufficient accuracy and sensitivity for the detection and quantification of the product of interest. Protein biopharmaceuticals may be difficult to evaluate due to their similar structural and physicochemical properties compared to their mutant, modified or truncated forms. Direct analysis may require the isolation of a large enough amount of product for testing, which is undesirable and only possible in selected cases.

[0004] There is a long-felt need in the art for methods and / or systems for characterizing protein biopharmaceuticals. Summary of the Invention

[0005] overview The growth in the development, production, and sale of protein biopharmaceuticals has resulted in an increasing demand to characterize the protein biopharmaceutical along with possible impurities, binding stoichiometry, and its overall composition.

[0006] The exemplary embodiments disclosed herein fulfill the aforementioned needs by providing methods for characterizing, identifying, and / or quantifying protein biopharmaceuticals along with their possible impurities, conjugates, and overall composition.

[0007] The present disclosure provides, at least in part, a method for characterizing a protein. In one exemplary embodiment, the method for characterizing a protein includes contacting a sample containing the protein with a chromatography system having a chromatography resin, washing the resin with a mobile phase to obtain an eluent containing the protein, and characterizing the protein in the eluent using an electrospray ionization mass spectrometer.

[0008] In one aspect of this embodiment, the method for characterizing a protein can include a chromatography system having a size exclusion chromatography resin.

[0009] In one aspect of this embodiment, a method for characterizing a protein can include coupling an electrospray ionization mass spectrometer to a chromatography system having a chromatography resin.

[0010] In one aspect of this embodiment, a method for characterizing a protein may include coupling an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0011] In one aspect, the method for characterizing proteins may involve an electrospray ionization mass spectrometer operated under non-denaturing conditions.

[0012] In one aspect of this embodiment, the method for characterizing a protein may include a nano-electrospray ionization mass spectrometer.

[0013] In one aspect of this embodiment, the method for characterizing a protein may include a nano-electrospray ionization mass spectrometer operated under non-denaturing conditions.

[0014] In one aspect of this embodiment, the method for characterizing a protein can include at least one three-way splitter for coupling an electrospray ionization mass spectrometer to a chromatography system having a resin.

[0015] In one aspect of this embodiment, the method for characterizing a protein can include at least one three-way splitter for coupling an ultraviolet detector to a chromatography system having a resin.

[0016] In one aspect of this embodiment, the method for characterizing a protein may include at least one three-way splitter for coupling a chromatographic system having a resin to an ultraviolet detector and an electrospray ionization mass spectrometer.

[0017] In one aspect of this embodiment, the method for characterizing a protein may include at least one three-way splitter for coupling an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0018] In one aspect of this embodiment, the method for characterizing a protein can include at least one three-way splitter for coupling an ultraviolet detector to a chromatography system having a size exclusion chromatography resin.

[0019] In one aspect of this embodiment, the method for characterizing proteins may include at least one three-way splitter for coupling an ultraviolet detector and an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0020] In one aspect of this embodiment, the method for characterizing a protein can include washing a resin with a mobile phase to obtain an eluent containing the protein, the eluent being introduced from at least one three-way splitter to an ultraviolet detector at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0021] In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase that includes a volatile salt.

[0022] In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase that includes ammonium acetate.

[0023] In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase having a total concentration of ammonium acetate less than about 100 mM.

[0024] In one aspect of this embodiment, the method for characterizing a protein can include washing the resin with a mobile phase at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0025] In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase having a pH of about 6.8.

[0026] In one aspect of this embodiment, the method for characterizing a protein may include a sample containing protein in an amount of about 10 μg to about 100 μg of protein.

[0027] In one aspect of this embodiment, the method for characterizing a protein may involve a protein that is an antibody.

[0028] In one aspect of this embodiment, the method for characterizing a protein may involve a protein that is an antigen-antibody complex.

[0029] In one aspect of this embodiment, the method for characterizing a protein may include a protein that is an antibody-drug conjugate.

[0030] In one aspect of this embodiment, the method for characterizing proteins can include an electrospray ionization mass spectrometer having a flow rate of about 10 nL / min to about 50 nL / min.

[0031] In one aspect of this embodiment, the method for characterizing a protein can include an electrospray ionization mass spectrometer, where the electrospray spray voltage is about 0.8 kV to about 1.5 kV.

[0032] In one aspect of this embodiment, the method for characterizing may include identifying the protein.

[0033] In one aspect of this embodiment, the method for characterizing a protein may include quantitating the protein.

[0034] In one aspect of this embodiment, the method for characterizing a protein may include quantifying the relative abundance of the protein.

[0035] In one aspect of this embodiment, the method for characterizing a protein may include a sample comprising at least two proteins.

[0036] The present disclosure provides, at least in part, a method for characterizing an antibody-drug conjugate. In one exemplary embodiment, the method for characterizing an antibody-drug conjugate includes contacting a sample containing the antibody-drug conjugate with a chromatography system having a chromatography resin, washing the resin with a mobile phase to obtain an eluent containing the antibody-drug conjugate, and characterizing the antibody-drug conjugate in the eluent using an electrospray ionization mass spectrometer.

[0037] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a chromatography system having a size-exclusion chromatography resin.

[0038] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate may include coupling an electrospray ionization mass spectrometer to a chromatography system having a chromatography resin.

[0039] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate may include coupling an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0040] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include electrospray ionization mass spectrometry under non-denaturing conditions.

[0041] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a nano-electrospray ionization mass spectrometer.

[0042] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a nano-electrospray ionization mass spectrometer operated under non-denaturing conditions.

[0043] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include at least one three-way splitter for coupling an electrospray ionization mass spectrometer to a chromatography system having a resin.

[0044] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include at least one three-way splitter for coupling an ultraviolet detector to a chromatography system having the resin.

[0045] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include at least one three-way splitter for coupling a chromatographic system having a resin to an ultraviolet detector and an electrospray ionization mass spectrometer.

[0046] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way splitter for coupling an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0047] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way splitter for coupling an ultraviolet detector to a chromatography system having a size exclusion chromatography resin.

[0048] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way splitter for coupling an ultraviolet detector and an electrospray ionization mass spectrometer to a chromatography system having a size exclusion chromatography resin.

[0049] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate can include washing the resin with a mobile phase to obtain an eluent comprising the antibody-drug conjugate, the eluent being introduced to an ultraviolet detector through at least one three-way splitter at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0050] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a mobile phase that includes a volatile salt.

[0051] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a mobile phase that includes ammonium acetate.

[0052] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a mobile phase having a total concentration of ammonium acetate less than about 100 mM.

[0053] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate can include washing the resin with a mobile phase at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0054] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include a mobile phase having a pH of about 6.8.

[0055] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a sample containing protein in an amount of about 10 μg to about 100 μg of antibody-drug conjugate.

[0056] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include washing the resin with a mobile phase to obtain an eluent that is introduced into an electrospray ionization mass spectrometer at a flow rate of less than about 50 μL / min.

[0057] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include an electrospray ionization mass spectrometer with a flow rate of about 10 nL / min to about 50 nL / min.

[0058] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate can include an electrospray ionization mass spectrometer with an electrospray spray voltage of about 0.8 kV to about 1.5 kV.

[0059] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate can include an antibody-drug conjugate that is a site-specific antibody-drug conjugate.

[0060] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is not a site-specific antibody-drug conjugate.

[0061] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is an engineered cysteine-based antibody-drug conjugate.

[0062] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate can include an antibody-drug conjugate that is a non-specific cysteine-based antibody-drug conjugate.

[0063] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include characterizing the drug to antibody ratio.

[0064] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate can include identifying the antibody.

[0065] In one aspect of this embodiment, the method for characterizing the antibody-drug conjugate may include quantitating the antibody.

[0066] The present disclosure provides, at least in part, a system that includes a chromatography column having a chromatography resin. In another exemplary embodiment, the system includes a chromatography column having a chromatography resin and an electrospray ionization mass spectrometer, where the chromatography column is capable of receiving a mobile phase and a sample that includes a protein.

[0067] In one aspect of this embodiment, the system can include a chromatography column having a size exclusion chromatography resin.

[0068] In one aspect of this embodiment, the system can include an electrospray ionization mass spectrometer that can be coupled to the chromatography column.

[0069] In one aspect of this embodiment, the system can include an electrospray ionization mass spectrometer capable of operating under non-denaturing conditions.

[0070] In one aspect of this embodiment, the system may include a nano-electrospray ionization mass spectrometer.

[0071] In one aspect of this embodiment, the system can include a chromatography column that can be coupled to a mass spectrometer using a three-way splitter.

[0072] In one aspect of this embodiment, the system can include a chromatography column that can be coupled to an ultraviolet detector using a three-way splitter.

[0073] In one aspect of this embodiment, the system can include a chromatography column that can be coupled to an ultraviolet detector and a mass spectrometer using a three-way splitter.

[0074] In one aspect of this embodiment, the system can enable characterization of the drug-to-antibody ratio of an antibody-drug conjugate.

[0075] In one aspect of this embodiment, the system can enable characterization of proteins.

[0076] In one aspect of this embodiment, the system can enable characterization of antigen-antibody complexes. [Brief description of the drawings]

[0077] [Figure 1] Spectra obtained from conventional and native electrospray ionization mass spectrometry are shown. [Diagram 2] 1 illustrates an exemplary embodiment of a system capable of characterizing a protein biopharmaceutical. [Diagram 3] 1 illustrates an exemplary embodiment of a system capable of characterizing a protein biopharmaceutical. [Figure 4] 1 illustrates the configuration of a system capable of characterizing protein biopharmaceuticals, according to an exemplary embodiment. [Diagram 5] 5A and 5B show the analysis of antigen-antibody complexes using a system capable of characterizing protein biopharmaceuticals, according to an exemplary embodiment. [Figure 6] 1 shows the results of an antigen-antibody titration between Bet v1 and Fab-1 characterized according to an exemplary embodiment. [Figure 7] Figure 7A shows mass spectrometer signals resulting from dual detection of antigen-antibody interaction between Bet v1 and Fab-1 by a UV detector and a native electrospray ionization mass spectrometer under non-denaturing conditions according to an exemplary embodiment. Figure 7B shows UV signals resulting from dual detection of antigen-antibody interaction between Bet v1 and Fab-1 by a UV detector and a native electrospray ionization mass spectrometer under non-denaturing conditions according to an exemplary embodiment. [Figure 8A]1 shows raw spectra of parent mAb-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-1 by online SEC-nano-ESI-MS instrumentation according to an exemplary embodiment. [Figure 8B] 1 shows raw spectra of ADC-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-1 by an online SEC-nano-ESI-MS instrument according to an exemplary embodiment. [Figure 8C] 1 shows the convoluted spectrum of parent mAb-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-1 by online SEC-nano-ESI-MS instrumentation according to an exemplary embodiment. [Figure 8D] 1 shows a convoluted spectrum of ADC-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-1 by an online SEC-nano-ESI-MS instrument according to an exemplary embodiment. [Figure 9] 1 shows drug to antibody ratio analysis of site-specific conjugated cysteine ​​mAb-1 and ADC-1 by FabRICATOR digestion and online SEC-nano-ESI-MS instrumentation according to an exemplary embodiment. [Figure 10A] 1 shows raw spectra of parent mAb-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-2 by online SEC-nano-ESI-MS instrumentation according to an exemplary embodiment, where N means that the antibody has no glycans, S means that glycosylation involves a single chain of the antibody, and D means that glycosylation involves both chains of the antibody. [Figure 10B] 1 shows the convolution spectrum of parent mAb-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-2 by online SEC-nano-ESI-MS instrumentation according to an exemplary embodiment, where N means that the antibody has no glycans, S means that glycosylation involves a single chain of the antibody, and D means that glycosylation involves both chains of the antibody. [Figure 10C]1 shows raw spectra of ADC-2 drug to antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-2 by an online SEC-nano-ESI-MS instrument according to an exemplary embodiment, where N means that the antibody has no glycans, S means that glycosylation involves a single chain of the antibody, and D means that glycosylation involves both chains of the antibody. [Figure 10D] 1 shows a convolution spectrum of parent ADC-2 drug to antibody ratio analysis of site-specifically conjugated cysteine ​​ADC-2 by an online SEC-nano-ESI-MS instrument according to an exemplary embodiment, where N means that the antibody has no glycans, S means that glycosylation involves a single chain of the antibody, and D means that glycosylation involves both chains of the antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] Detailed Description Identification and quantification of proteins in protein biopharmaceuticals can be of great importance during product manufacturing and development. The presence of impurities and binding methods of protein biopharmaceuticals can be essential to develop a safe and effective product. Therefore, robust methods and / or workflows for characterization of protein biopharmaceuticals, their binding methods, and associated impurities can be beneficial.

[0079] One method involves the use of size-exclusion chromatography (SEC) to characterize the aggregation and fragmentation of biomolecules in the biotechnology industry (Hong Paule et al., Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates, 35 JOURNAL OF LIQUID CHROMATOGRAPHY AND RELATED TECHNOLOGY 2923-2950 (2012)). Separation of molecules by SEC relies on the various interactions of the molecules with the controlled porous structure on the stationary phase. SEC uses buffer conditions that preserve the native structure of proteins in solution, and therefore allows characterization of biomolecules without perturbing the native conformation. Among the various detection modes that can be combined with SEC, mass spectrometry (MS) allows for precise and accurate identification of individual components in complex samples.Combining SEC with MS involves collection of SEC peaks followed by direct infusion MS (Basak Kukrer et al., Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography, 27 PHARMACEUTICAL RESEARCH 2197-2204 (2010); Francois Debaene et al., Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment, 86 ANALYTICAL CHEMISTRY 10674-10683 (2014)) or online SEC-MS (Khaja Muneeruddin et al., Characterization of Small Protein Aggregates and Oligomers Using Size Exclusion Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry, 86 ANALYTICAL CHEMISTRY These have been reported previously, including by CFMcdonagh et al., Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment, 19 PROTEIN ENGINEERING DESIGN AND SELECTION 299-307 (2006). However, direct ionization of the high flow rates generated from SEC separations requires harsh ionization conditions that are often incompatible with native MS analysis, limiting the practicality of coupling these techniques to the analysis of non-covalent interactions.Additionally, mass spectrometer sensitivity can be affected by the high salt concentrations used in SEC buffers.

[0080] Because noncovalent protein interactions mediate such a wide range of biological functions, there is growing interest in developing methods to facilitate the study of their structure, stoichiometry, and dynamics. Such methods may help investigate noncovalent protein interactions that occur widely in nature, and are also needed to tune the interactions of protein biopharmaceuticals with a variety of molecules, including other proteins and peptides, nucleic acids, lipids, and inorganic and organic small molecules. Size exclusion chromatography (SEC) can separate biomolecules from heterogeneous mixtures of molecular components, and because the buffer conditions maintain proteins in their native conformation, SEC is an ideal method for preserving noncovalent biomolecular complexes during separation. Among the various detection methods that can be combined with SEC analysis, mass spectrometry (MS) allows reliable identification and characterization of individual components from complex mixtures. However, the high flow rates and nonvolatile salts used in SEC are often not compatible with downstream MS analysis.

[0081] Given the limitations of existing methods, an effective and efficient method was developed for the analysis of protein biopharmaceuticals using online chromatography with electrospray ionization MS platform.

[0082] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are described below. All publications mentioned are incorporated herein by reference.

[0083] The term "a" should be understood to mean "at least one." The terms "about" and "approximately" should be understood to allow for standard variations understood by one of ordinary skill in the art. When ranges are stated, the endpoints are included.

[0084] In some exemplary embodiments, the present disclosure provides methods for the characterization, identification, and / or quantification of protein biopharmaceuticals.

[0085] As used herein, a "protein biopharmaceutical" includes active ingredients that are entirely or partially biologic in nature. In some exemplary embodiments, a protein biopharmaceutical may include a protein, a vaccine, an allergen, a nucleic acid, a virus, an antibody-drug conjugate, a cell, a gene, a tissue, or a combination thereof. In some other exemplary embodiments, a protein biopharmaceutical may include a recombinant, engineered, modified, mutated, or truncated form of a protein, a vaccine, an allergen, a nucleic acid, a virus, an antibody-drug conjugate, a cell, a gene, a tissue, or a combination thereof.

[0086] As used herein, the term "protein" includes any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known. A protein can include one or more polypeptides to form a single functional biomolecule. Proteins can include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary embodiment, proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For an illustrative review of biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," (BIOTECHNOL. GENET. ENG. REV. 147-175 (2012)). In some exemplary embodiments, the proteins include modifications, additions, and other covalently attached moieties.These modifications, additions, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels and other dyes, etc. Proteins can be classified based on composition and solubility, and thus may include simple proteins, such as globular proteins and fibrous proteins; complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0087] In some exemplary embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a host cell derived protein, or a combination thereof.

[0088] As used herein, the term "antibody" includes immunoglobulin molecules, as well as multimers thereof (e.g., IgM), which are comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region comprises a C H1 , C H2 , and C H3 Each light chain comprises three domains: a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L1 ) is included. V H Area and V L The region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various exemplary embodiments, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of an intact antibody molecule. As used herein, terms such as "antigen-binding portion" of an antibody and "antigen-binding fragment" of an antibody include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolysis or recombinant genetic engineering techniques (including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains). Such DNA is known and / or readily available, for example from commercial DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, form cysteine ​​residues, modify, add or delete amino acids, etc.

[0089] An "antibody fragment" as used herein includes a portion of an intact antibody, such as, for example, an antigen-binding region or a variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are linked by a peptide linker. Antibody fragments can be produced by various means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody and / or they can be produced recombinantly from a gene encoding a partial antibody sequence. Alternatively, or in addition, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or in addition, the antibody fragment may comprise multiple chains linked together, for example, by sulfide bonds. The antibody fragment may optionally comprise a multimolecular complex.

[0090] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be produced using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0091] The term "antibody-drug conjugate" or "ADC" as used herein can refer to an antibody linked to a biologically active drug by a linker with a labile bond. An ADC can contain several molecules of a biologically active drug (or payload) that can be covalently attached to the side chains of amino acid residues of the antibody (Siler Panowski et al., Site-specific antibody drug conjugates for cancer therapy,6 MABS 34-45(2013)). The antibody used in the ADC can be capable of binding with sufficient affinity for selective accumulation and sustained retention at the target site. Most ADCs can have Kd values ​​in the nanomolar range. The payload can have a potency in the nanomolar / picomolar range and can enable the ADC to reach achievable intracellular concentrations after distribution to the target tissue. Finally, the linker forming the link between the payload and the antibody must be capable of being sufficiently stable in the circulation to take advantage of the pharmacokinetic properties of the antibody moiety (i.e., long half-life) and to allow the payload to remain associated with the antibody as it distributes into tissues, yet allow for efficient release of the biologically active drug after the ADC is taken up by target cells.

[0092] The linker can be non-cleavable during cellular processing and cleavable when the ADC reaches the target site. With a non-cleavable linker, the biologically active drug released within the cell includes the payload and all elements of the linker that remain attached to the amino acid residues (typically lysine or cysteine ​​residues) of the antibody after complete proteolysis of the ADC in the lysosome. A cleavable linker is one whose structure includes a cleavage site between the payload and the amino acid binding site on the antibody. Cleavage mechanisms can include hydrolysis of acid-labile bonds within acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by intracellular proteases or esterases, and reductive cleavage of disulfide bonds by the reducing environment within the cell.

[0093] In some particular embodiments, the present disclosure also provides methods for determining the drug-to-antibody ratio (DAR) of an antibody-drug conjugate.

[0094] ADCs can be made by conjugating antibodies to endogenous amino acid residues while carefully controlling the average degree of modification to obtain an optimal drug-to-antibody ratio (DAR). This ratio can be selected based on (a) minimizing the amount of unconjugated antibody, and (b) avoiding species in the mixture with very high DAR, which can be problematic in manufacturing and formulation due to their high hydrophobicity and low solubility, and can lead to poor pharmacokinetic properties. Conjugation of too little to the biologically active drug molecule reduces efficacy, while conjugation of too much can destabilize the ADC, altering its pharmacokinetic properties, increasing plasma clearance, shortening its half-life, and increasing systemic toxicity. The optimal DAR is often uncertain and highly dependent on other ADC variables. More commonly, however, ADCs aim to achieve a DAR close to 4. Non-limiting examples of conjugation of biologically active drugs to antibodies include conjugation of biologically active drugs to lysine or cysteine ​​residues on the antibody. Lysine conjugation can result in 0-8 conjugated biologically active drug molecules per antibody, which can occur on both the heavy and light chains at different lysine residues. Another non-limiting example of conjugation of biologically active drugs to antibodies can include cysteine ​​conjugation that occurs after reduction of the four interchain disulfide bonds, resulting in conjugation limited to eight exposed sulfhydryl groups, and thus can range from 0-8 linked biologically active drug molecules per antibody. Because these ADC species have different drug loadings and conjugation sites, the heterogeneity of the ADC mixture is two-fold diverse. Thus, each species can have different properties, which can result in various in vivo PK properties. In addition, batch-to-batch consistency in ADC manufacturing can be challenging, and faithful manufacturing capabilities may be required.

[0095] Site-specific antibody-drug conjugates, in which a known number of biologically active drug molecules are consistently attached to a defined site, are one way to overcome these challenges. Heterogeneity is minimized, making ADC properties more predictable and conjugate production consistent from batch to batch. Drug-to-antibody ratios (DARs) can be tightly controlled and tailored for a variety of linked bioactive drugs, generating 2- or 4-DAR site-specific ADCs. Non-limiting examples of site-specific conjugates include linking biologically active drug molecules to antibodies via engineered cysteine ​​residues, glutamine residues, unnatural amino acids (e.g., p-acetylphenylalanine, N6-((2-azidoethoxy)carbonyl)-L-lysine, p-azidomethyl-L-phenylalanine, selenocysteine), glycans, or short peptide tags as taught by Qun Zhou in the review "Site-Specific Antibody Conjugation for ADC and Beyond" (Qun Zhou, Site-Specific Antibody Conjugation for ADC and Beyond, Biomedicines 64 (2017), which is incorporated by reference).

[0096] In some exemplary embodiments, the present disclosure provides methods for characterizing, identifying, and / or quantifying at least one impurity in a protein biopharmaceutical.

[0097] The term "impurities" as used herein may include any undesired proteins present in a protein biopharmaceutical. Impurities may include process and product-related impurities. Impurities may further be of known structure, partially characterized, or unidentified. Process-related impurities may originate from the manufacturing process and may include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism and nucleic acids (host cell genome, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing agents, and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables. Product-associated impurities (e.g., precursors, certain degradation products) may be molecular variants arising during manufacture and / or storage that do not have properties comparable to those of the desired product in terms of activity, efficacy, and safety. Such variants may require significant efforts for separation and characterization to identify the type of modification. Product-associated impurities include truncated forms, modified forms, and aggregates. Cleaved forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modified forms include, but are not limited to, deamidation, isomerization, mismatched S-S bonds, oxidation, or altered complex forms (e.g., glycosylation, phosphorylation). Modified forms may also include any post-translationally modified forms. Aggregates include dimers and higher order multimers of the desired product.(Q6B Specifications:Test Procedures and Acceptance Criteria for Biotechnological / Biological Products,ICH August 1999,U.S.Dept.of Health and Humans Services)。

[0098] The general term "post-translational modification" or "PTM" as used herein refers to covalent modifications that polypeptides undergo either during (co-translational modification) or after (post-translational modification) their ribosomal synthesis. PTMs are usually introduced by specific enzymes or enzymatic pathways. Many occur at the site of specific characteristic protein sequences (signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some features of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853).Various post-translational modifications include, but are not limited to, truncation, N-terminal extension, proteolysis, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (addition of an acetyl group, usually at the N-terminus of a protein), alkylation (addition of an alkyl group, e.g., methyl, ethyl, propyl, usually at a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylation and carboxy-terminal amidation), vitamin K-dependent modifications (where vitamin K is a hydroxyl group that converts glutamic acid to form γ-carboxyglutamic acid (Glu) residues), and the like. These include covalent attachment of a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, nonribosomal peptide, and leucine biosynthesis, and the addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in the biosynthesis of fatty acids, polyketides, nonribosomal peptides, and leucine, phosphorylation (addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine), and sulfation (addition of a sulfate group, usually to a tyrosine residue). Post-translational modifications that change the chemical properties of amino acids include, but are not limited to, citrullination (conversion of arginine to citrulline by deimination) and deamidation (conversion of glutamine to glutamic acid or asparagine to aspartic acid).Post-translational modifications that involve structural changes include, but are not limited to, the formation of disulfide bridges (covalent bonding of two cysteine ​​amino acids) and proteolytic cleavage (breaking of a protein at a peptide bond).Specific post-translational modifications include the addition of other proteins or peptides, such as ISGylation (covalent attachment to the ISG15 protein (interferon-activated gene)), SUMOylation (covalent attachment to the SUMO protein (small ubiquitin-like modifier)), and ubiquitination (covalent attachment to the protein ubiquitin). For a more detailed controlled nomenclature of PTMs collected by UniProt, see European Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics,EUROPEAN BIOINFORMATICS INSTITUTE DRS-DROSOMYCIN PRECURSOR-DROSOPHILA MELANOGASTER (FRUIT FLY)-DRS GENE&PROTEIN,http: / / www.uniprot.org / docs / ptmlist (last visited January 15, 2019).

[0099] The term "desired product" as used herein refers to a protein biopharmaceutical having a desired structure, function, or efficacy profile.

[0100] In some exemplary embodiments, the present disclosure also provides a method for characterizing the binding of protein biopharmaceuticals. For example, antibodies can bind to antigens with high specificity, high affinity, and through non-covalent interactions, and this property has enabled the development of therapeutic antibodies that target disease-specific antigens in the treatment of various diseases (Andrew C. Chan & Paul J. Carter, Therapeutic antibodies for autoimmunity and inflammation, 10 NATURE REVIEWS IMMUNOLOGY 301-316 (2010)). To develop effective antibody therapeutics, it may be important to understand how antibody binding affects the function of target proteins.

[0101] In some exemplary embodiments, the present disclosure also provides methods for determining the binding ratio of an antibody to an antigen from an antigen-antibody complex.

[0102] In certain exemplary embodiments, the present disclosure also provides methods for identifying the antigen to which an antibody binds. In some further exemplary embodiments, the methods may include determining whether a truncated, modified, or mutated form of the antigen and / or antibody is involved in the antigen-antibody complex.

[0103] In some exemplary embodiments, the present disclosure also provides methods for quantifying the relative abundance of individual proteins in a solution.

[0104] In some exemplary embodiments, a method for characterizing, identifying, and / or quantifying a protein biopharmaceutical, its possible impurities, binding, or composition may include contacting a sample containing the protein biopharmaceutical with a chromatography system having a chromatography resin.

[0105] The term "chromatography" as used herein refers to a process by which a liquid or gas-borne chemical mixture can be separated into multiple components as a result of differential distribution of the chemicals as they flow around or over a liquid or solid stationary phase. Non-limiting examples of chromatography include conventional reversed-phase (RP), ion exchange (IEX), mixed-mode chromatography, and normal-phase chromatography (NP).

[0106] As used herein, the term "mixed-mode chromatography (MMC)" or "multimodal chromatography" includes chromatographic methods in which solutes interact with a stationary phase through multiple interaction modes or mechanisms. MMC can be used as an alternative or complementary tool to traditional reversed-phase (RP), ion-exchange (IEX), and normal-phase (NP) chromatography. Unlike RP, NP, and IEX chromatography, in which hydrophobic, hydrophilic, and ionic interactions, respectively, are the primary interaction modes, mixed-mode chromatography allows the use of two or more of these interaction modes in combination. Mixed-mode chromatographic media can provide unique selectivities that cannot be reproduced by single-mode chromatography. Mixed-mode chromatography can also offer potential cost savings and operational flexibility compared to affinity-based methods.

[0107] In some exemplary embodiments, the chromatography may be size exclusion chromatography.

[0108] As used herein, the terms "SEC chromatography resin" or "SEC chromatography media" are used interchangeably and can include any type of solid phase used in SEC to separate impurities from the desired product (e.g., homodimer contaminants for a bispecific antibody product). The volume of resin, length and diameter of the column used, as well as the dynamic capacity and flow rate, can depend on several parameters, such as the volume of fluid to be processed and the concentration of protein in the fluid undergoing processing.

[0109] In some exemplary embodiments, a method for characterizing, identifying, and / or quantifying a protein biopharmaceutical, its possible impurities, conjugates, or compositions may include contacting a sample containing the protein biopharmaceutical with a chromatography system having a size exclusion chromatography resin, washing the size exclusion chromatography resin with a mobile phase to obtain an eluent containing proteins, and characterizing the proteins in the eluent using an electrospray ionization mass spectrometer.

[0110] The term "mass spectrometer" as used herein includes devices that can identify specific molecular species and measure their exact mass. The term is intended to include any molecular detector in which a polypeptide or peptide can be eluted for detection and / or characterization. A mass spectrometer may include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to form gas phase ions. The atoms, molecules, or clusters of interest can be transferred to the gas phase and ionized simultaneously (similar to electrospray ionization). The choice of ion source is highly dependent on the application.

[0111] The term "electrospray ionization" or "ESI" as used herein refers to the process of spray ionization in which either positive or negative ions in a solution are transferred to the gas phase by formation and desolvation at atmospheric pressure of a stream of highly charged droplets obtained by applying a potential difference between an electrospray needle tip containing the solution and a counter electrode. There are typically three main steps in the generation of gas phase ions from electrolyte ions in solution. These are: (a) generation of charged droplets at the ES injection tip; (b) shrinkage of the charged droplets by solvent evaporation and repeated droplet collapse, resulting in the generation of small highly charged droplets that can generate gas phase ions; and (c) a mechanism by which gas phase ions are generated from very small and highly charged droplets. Steps (a)-(c) typically occur in the atmospheric pressure region of the device.

[0112] The term "electrospray injection setup" as used herein refers to an electrospray ionization system that is compatible with a mass spectrometer used for mass analysis of proteins. In electrospray ionization, an electrospray needle has its orifice located near the entrance orifice of the spectrometer. A sample containing the protein of interest can be pumped through a syringe needle. An electric potential between the orifice of the syringe needle and an orifice leading to the mass spectrometer forms a spray of solution ("electrospray"). Electrospray can be performed at atmospheric pressure and delivers highly charged droplets of the solution. The electrospray injection setup can include an electrospray emitter, a nebulizer gas, and / or an ESI power supply. The setup can optionally be automated to perform sample aspiration, sample dispensing, sample delivery, and / or sample nebulization.

[0113] In some exemplary embodiments, the electrospray ionization mass spectrometer may be a nano-electrospray ionization mass spectrometer.

[0114] The term "nanoelectrospray" or "nanospray" as used herein refers to electrospray ionization at very low solvent flow rates, typically a few hundred nanoliters per minute or less of sample solution, often without the use of an external solvent supply. Electrospray injection setups that form nanoelectrosprays can use static nanoelectrospray emitters or dynamic nanoelectrospray emitters. Static nanoelectrospray emitters perform continuous analysis of small sample (analyte) solution volumes over an extended period of time. Dynamic nanoelectrospray emitters use a capillary column and solvent supply system to perform chromatographic separation of mixtures prior to analysis by a mass spectrometer.

[0115] The term "mass analyzer" as used herein includes devices capable of separating species, i.e. atoms, molecules, or clusters, according to their mass. Non-limiting examples of mass analyzers that can be used for fast protein sequence analysis are time-of-flight (TOF), magnetic / electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and accelerator mass spectrometry (AMS).

[0116] In some exemplary embodiments, mass spectrometry can be performed under non-denaturing conditions.

[0117] The term "non-denaturing conditions" or "native MS" or "native ESI-MS" as used herein may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see Review: Elisabetta Boeri Erba&Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015). Some of the differences between native ESI and regular ESI are shown in Table 1 and Figure 1 (Hao Zhang et al., Native mass spectrometry of photosynthetic pigment-protein complexes, 587 FEBS Letters 1012-1020 (2013)).

[0118] [Table 1]

[0119] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer.

[0120] As used herein, the term "tandem mass spectrometry" includes techniques in which structural information about sample molecules is obtained by using multiple stages of mass selection and mass separation. The prerequisite is that the sample molecules can be transferred into the gas phase and ionized intact, and that they can be induced to break down in some predictable and controllable manner after the initial mass selection step. Multistage MS / MS, or MS n The first step is to extract the precursor ion (MS 2016) as long as meaningful information can be obtained or the fragment ion signal is detectable. 2 ) was selected and isolated to fragment the primary fragment ions (MS 3 ) is isolated and fragmented into secondary fragment ions (MS 4 ) and other methods. Tandem MS has been successfully performed using a wide variety of analyzer combinations. The analyzers that are combined for a particular application are determined by many different factors such as sensitivity, selectivity, and speed, as well as size, cost, and effectiveness. The two main categories of tandem MS methods are spatial tandem and temporal tandem, although hybrids exist in which a temporal tandem analyzer is coupled spatially or with a spatial tandem analyzer. A spatial tandem mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function can be designed such that ions are selected in one area of ​​the instrument, dissociated in an intermediate region, and then the product ions are sent to another analyzer for m / z separation and data acquisition. In a temporal tandem mass spectrometer, ions generated in the ion source can be trapped, separated, fragmented, and m / z separated in the same physical device.

[0121] The peptides identified by mass spectrometry can be used as representatives of intact proteins and surrogates of their post-translational modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter generated from possible peptides in protein sequence databases. Characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequencing, protein de novo sequencing, localization of post-translational modifications, or identification of post-translational modifications, or fit analysis, or a combination of these.

[0122] The term "database" as used herein refers to a bioinformatics tool that provides the possibility to search uninterpreted MS-MS spectra against all possible sequences in the database. Non-limiting examples of such tools are: Mascot (http: / / www.matrixscience.com), Spectrum Mill (http: / / www.chem.agilent.com), PLGS (http: / / www.waters.com), PEAKS (http: / / www.bioinformaticssolutions.com), Proteinpilot (http: / / download.appliedbiosystems.com / / proteinpilot), Phenyx (http: / / www.phenyx-ms.com), Sorcerer (http: / / www.sagenresearch.com), OMSSA (http: / / www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (http: / / www.thegpm.org / TANDEM / ), Protein Prospector (http: / / www.http: / / prospector.ucsf.edu / prospector / mshome.htm), Byonic (https: / / www.proteinmetrics.com / products / byonic), or Sequest (http: / / fields.scripps.edu / sequest).

[0123] Exemplary embodiments Embodiments disclosed herein provide compositions, methods, and systems for the rapid characterization of proteins in a sample.

[0124] The present disclosure provides a method for characterizing a protein, comprising contacting a sample containing the protein with a chromatography system having a chromatography resin, washing the resin with a mobile phase to obtain an eluent containing the protein, and characterizing the protein in the eluent using an electrospray ionization mass spectrometer.

[0125] The present disclosure provides a method for characterizing an antibody-drug conjugate comprising contacting a sample comprising the antibody-drug conjugate with a chromatography system having a chromatography resin, washing the resin with a mobile phase to obtain an eluent comprising the antibody-drug conjugate, and characterizing the antibody-drug conjugate in the eluent using an electrospray ionization mass spectrometer.

[0126] The present disclosure provides a method for characterizing an antigen-antibody complex, comprising contacting a sample containing an antigen-antibody complex with a chromatography system having a chromatography resin, washing the resin with a mobile phase to obtain an eluent containing the antigen-antibody complex, and characterizing the antigen-antibody complex in the eluent using an electrospray ionization mass spectrometer.

[0127] In some exemplary embodiments, the chromatography system may include conventional reversed phase (RP), ion exchange (IEX), or normal phase chromatography (NP).

[0128] In some exemplary embodiments, the chromatography resin can be selected from an affinity chromatography resin, an anion exchange resin, a cation exchange resin, an affinity resin, a mixed-mode chromatography resin, a hydrophobic interaction chromatography resin, or a size-exclusion chromatography resin.

[0129] In some exemplary embodiments, the electrospray ionization mass spectrometer may be a nano-electrospray ionization mass spectrometer.

[0130] In some exemplary embodiments, the electrospray ionization mass spectrometer can be coupled to a chromatography system having a chromatographic resin.

[0131] In some exemplary embodiments, the electrospray ionization mass spectrometer can be operated under non-denaturing conditions.

[0132] In some exemplary embodiments, the chromatography system can be coupled to an electrospray ionization mass spectrometer using a three-way splitter.

[0133] In some exemplary embodiments, the chromatography system can be coupled to an ultraviolet detector using a three-way splitter.

[0134] In some exemplary embodiments, the chromatography system can be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-way splitter.

[0135] In some exemplary embodiments, the chromatography system can be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-way splitter, and the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer.

[0136] In some exemplary embodiments, the chromatography system can be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-way splitter, and the mass spectrometer is an electrospray ionization mass spectrometer operated under non-denaturing conditions.

[0137] In some exemplary embodiments, the chromatography system can be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-way splitter, where the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer under non-denaturing conditions.

[0138] In some exemplary embodiments, the eluent containing the protein or antigen-antibody complexes or antibody-drug complexes from washing the resin is introduced into the ultraviolet detector through at least one three-way splitter at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0139] In some exemplary embodiments, the washing mobile phase has a flow rate of about 0.2 mL / min to about 0.4 mL / min.

[0140] In some exemplary embodiments, the mobile phase may include a volatile salt, hi some specific embodiments, the mobile phase may include ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0141] In some exemplary embodiments, the mobile phase used may be compatible with a mass spectrometer.

[0142] In some exemplary embodiments, the mobile phase can have a pH of about 6.0 to 8.0.

[0143] In some exemplary embodiments, a sample may be used in an amount of about 10 μg to about 100 μg of protein or antigen-antibody complex or antibody-drug complex.

[0144] In some exemplary embodiments, the flow rate in the electrospray ionization mass spectrometer can be from about 10 nL / min to about 50 nL / min.

[0145] In some exemplary embodiments, the electrospray ionization mass spectrometer can have a spray voltage of about 0.8 kV to about 1.5 kV.

[0146] In some exemplary embodiments, characterization may include protein identification and / or quantification. In one aspect, characterization may include protein sequencing, protein de novo sequencing, post-translational modification identification, or compatibility analysis, or a combination thereof. In another aspect, characterization may include quantification of relative abundance of proteins.

[0147] In some exemplary embodiments, characterization may include identification and / or quantification of the antibody in the antibody-drug complex. In one aspect, characterization may include protein sequencing, protein de novo sequencing, identification of post-translational modifications, or compatibility analysis, or a combination thereof. In another aspect, characterization may include quantification of the relative abundance of the antibody in the antibody-drug complex.

[0148] In some exemplary embodiments, characterization may include identification and / or quantification of the antibody and / or antigen in the antigen-antibody complex. In one aspect, characterization may include protein sequencing of the antibody or antigen, protein de novo sequencing, identification of post-translational modifications, or compatibility analysis, or a combination thereof. In another aspect, characterization may include quantification of the relative abundance of the antibody and / or antigen in the antigen-antibody complex.

[0149] In some exemplary embodiments, the sample may include at least two proteins.

[0150] In some exemplary embodiments, the antibody-drug conjugate may include a site-specific ADC or a non-site-specific ADC. In one aspect, the antibody-drug conjugate may include a non-site-specific ADC linked via a cysteine ​​or lysine residue on the antibody. In another aspect, the antibody-drug conjugate may include a site-specific ADC linked via a natural amino acid, a non-natural amino acid, a glycan, a short peptide tag, or a combination thereof.

[0151] In some exemplary embodiments, the electrospray ionization mass spectrometer may be a tandem mass spectrometer.

[0152] In some exemplary embodiments, the protein may be a therapeutic antibody, an antibody, a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment, a fusion protein, or a combination thereof. In one aspect, the antibody fragment may include a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a dAb fragment, an Fd' fragment, an Fd fragment, and isolated complementarity determining region (CDR) regions, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0153] In some exemplary embodiments, the protein may be a digestion product of an antibody. The digestion product may be formed by a hydrolysis agent. The digestion product may be a product-associated impurity.

[0154] In some exemplary embodiments, the protein may be a product-associated impurity present in a biopharmaceutical.

[0155] In some exemplary embodiments, the protein can have a pI ranging from about 4.5 to about 9.0. In one aspect, the protein can have a pI of about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0156] In an exemplary embodiment, the sample may include at least two proteins.

[0157] It will be appreciated that the methods are not limited to any of the proteins, impurities, and columns described above, and that the methods of identification or quantification may be performed by any suitable means.

[0158] In some exemplary embodiments, the present disclosure provides a system including a chromatography column 100 having a chromatography resin and an electrospray ionization mass spectrometer 110, where the chromatography column can accept a mobile phase and a sample containing proteins (see FIG. 2).

[0159] In some exemplary embodiments, the chromatography column 100 may have a resin selected from a hydrophobic interaction chromatography resin, an anion exchange resin, a cation exchange resin, an affinity chromatography resin, a size exclusion chromatography resin, a mixed mode resin, or a combination thereof.

[0160] In some exemplary embodiments, an electrospray ionization mass spectrometer 110 can be connectable to the chromatography column 100 .

[0161] In some exemplary embodiments, the electrospray ionization mass spectrometer 110 can be enabled to operate under native conditions.

[0162] In some exemplary embodiments, the electrospray ionization mass spectrometer 110 may be a nano-electrospray ionization mass spectrometer.

[0163] In some exemplary embodiments, the electrospray ionization mass spectrometer 110 may be a nano-electrospray ionization mass spectrometer operated under non-denaturing conditions.

[0164] In some exemplary embodiments, the chromatography column 100 can be made connectable to an electrospray ionization mass spectrometer 100 using a three-way splitter 120 .

[0165] In some exemplary embodiments, the chromatography column 100 can be connectable to an ultraviolet detector 130 using a three-way splitter 120 .

[0166] In some exemplary embodiments, the chromatography column 100 can be coupled to an ultraviolet detector 130 and an electrospray ionization mass spectrometer 110 using a three-way splitter 120 .

[0167] In some exemplary embodiments, the three-way splitter 120 can allow for an unequally split flow from the chromatography column 100 to the ultraviolet detector 130 and the electrospray ionization mass spectrometer 110 .

[0168] In some exemplary embodiments, the system may enable characterization of the drug-to-antibody ratio of an antibody-drug conjugate.

[0169] In some exemplary embodiments, the system may enable protein characterization.

[0170] In some exemplary embodiments, the system may enable characterization of antigen-antibody complexes.

[0171] An exemplary embodiment of the system is shown in Figure 3. A post-column three-way splitter is used to enable UV / MS dual detection. A major fraction is sent to the UV detector while a minor fraction is directed to the MS. The detectors share approximately the same retention time. The fraction from the UV detector can be collected for sample collection.

[0172] Another view of the setup according to an exemplary embodiment is shown in FIG.

[0173] It is understood that the system is not limited to any of the previously mentioned proteins, chromatography columns, mass spectrometers, antibody-drug conjugates, or antigen-antibody conjugates.

[0174] Sequential number and / or letter labeling of method steps presented herein is not intended to limit the method, or any embodiment thereof, to the particular order presented.

[0175] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and journal articles, are cited throughout the specification. Each of these cited references is incorporated herein by reference in its entirety for all purposes.

[0176] The present disclosure may be more fully understood by reference to the following examples, which are set forth to more fully illustrate the present disclosure, and are intended for illustrative purposes and should not be construed as limiting the scope of the disclosure. EXAMPLES

[0177] Materials and Reagents. Water was purchased from Honeywell (Muskegon, MI). Ammonium acetate was purchased from Sigma-Aldrich (St Louis, MO). 1 M Tris-HCl, pH 7.5 was purchased from Teknova (Hollister, CA). Fused silica tubing (inner diameter (ID) 150 μm, outer diameter (OD) 360 μm), three-way connectors, and sleeves were purchased from IDEX (Oak Harbor, WA). PicoTip EMITTER SilicaTip (FS360-20-10-D-20-7CT) was purchased from New Objective (Woburn, MA). ACQUITY UPLC Protein BEH SEC column, 200 Å, 1.7 μm, 4.6 × 300 mm, was purchased from Waters (Milford, MA). Hot pocket column heater was purchased from Thermo-Fisher (Waltham, MA). All reagents were used without further purification.

[0178] Online SEC-nano-ESI-MS analysis. For all online SEC-nano-ESI-MS analyses, an ACQUITY UPLC I-class system (Waters, Milford, MA) was coupled to a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). An ACQUITY UPLC Protein BEH SEC column (200 Å, 1.7 μm, 4.6 × 300 mm) was set at 30 °C and used for the separation of mAbs and ADCs. The mobile phase was 100 mM ammonium acetate at pH 6.8. Each separation was performed for 30 min at a flow rate of 0.3 mL / min, and the injection volume was set to 40 μg. A three-way splitter (T-splitter) was connected after the SEC column. A fused silica tube (L: 140 cm, ID: 150 μm) and a SilicaTip (L: 5 cm, ID: 10 μm) were connected to the T-splitter. The major fraction was transferred to the UV detector via a fused silica tube, while the minor fraction was directed to the MS via a SilicaTip. The following MS parameters were used for online SEC-nano-ESI-MS data acquisition: Each acquisition was 25 min immediately after sample injection. Samples were ionized in positive mode with a spray voltage of 3 kV, a capillary temperature of 200 °C, and an RF level of 70 S-lens. In-source CID was set at 75 eV. Full MS scans were acquired at a resolution of 15 K in the mass range of m / z 2000-8000. A maximum injection time of 100 ms, an automatic acquisition control target value of 3e6, and 10 microscans were used for the full MS scan.

[0179] Data analysis. Protein Metrics Intact Mass software was used for deconvolution of raw data. Thermo Xcalibur QualBrowser was used for extracted ion chromatogram analysis. Microsoft Excel was used for DAR calculation of ADCs.

[0180] Example 1. Investigating antigen-antibody interactions using online SEC-nano-ESI-MS To develop effective antibody therapeutics, it can be important to understand how antibody binding affects the function of target proteins.

[0181] 1.1 Online SEC-nano-ESI-MS instrument SEC and MS techniques are routinely used to characterize protein samples. SEC allows for the separation and characterization of proteins under conditions that minimize protein conformational changes, while MS allows for the identification of individual components in complex samples. Combining the individual features of SEC and MS in a single platform is highly desirable but has proven difficult as the high flow rates and non-volatile salts used in SEC analysis are not compatible with native MS. To overcome this constraint, a post-column T-splitter was used to split the eluent flow from SEC, thereby reducing the uptake of solvent and salts into the MS (see Figure 3). A diagram of the setup is shown in Figure 4. The T-splitter was then connected to the MS via a SilicaTip and simultaneously to a UV detector via fused silica tubing. This arrangement allowed for simultaneous dual UV / MS detection of the SEC eluent. The flow rate through the SilicaTip to the MS can be adjusted by varying the length and diameter of the fused silica tubing (e.g. longer / narrower tubing can create more resistance, increasing the flow rate to the SilicaTip and to the MS). Protein samples were separated on a 4.6 mm SEC column using a flow rate of 0.3 mL / min. A 140 cm length of 150 μm inner diameter fused silica tubing connecting the T-splitter and the UV detector allowed for a desired flow rate of approximately 1 μL / min to the SilicaTip. The length and diameter of the fused silica tubing also allowed for nearly synchronous detection of molecules by UV and MS.

[0182] 1.2 Antigen-antibody complex A complex was formed between recombinant Bet v1 and a previously reported antibody against Bet v1 antigen under native MS conditions (Qian Zhang et al., Epitope Mapping by HDX-MS Elucidates the Surface Coverage of Antigens Associated with High Blocking Efficiency of Antibodies to Birch Pollen Allergen, 90 ANALYTICAL CHEMISTRY 11315-11323(2018)).

[0183] Besides the naked form, two major glycosylated forms were observed for Bet v1, including G2S1F and G2S2F, as seen in FIG. 5. After incubation with equimolar amounts of Fab-1 antibody, all three Bet v1 forms (naked, G2S1F, and G2S2F) bound to one Fab-1 as one antigen to form one Fab complex. In FIG. 5A, the deconvoluted native MS spectrum of only Bet v1 antigen revealed three different Bet v1 species: naked Bet v1, and two major glycosylated forms, Bet v1 G2S1F and G2S2F. Incubation of Bet v1 antigen with equimolar amounts of Bet v1 Fab-1 antibody showed that all three Bet v1 forms (naked, G2S1F, and G2S2F) formed complexes with Fab-1 at one antigen to one Fab ratio (FIG. 5B). Glycosylation does not appear to affect antibody / antigen complex formation, as the relative abundance of MS signals for all Bet v1 forms was similar between unbound and Fab-1 bound conditions.

[0184] To further characterize the antigen-antibody interaction, titration experiments were performed on a dual detection platform with MS and UV. As shown in Figure 6, several different ratios of Bet v1 to Fab-1 were examined by UV. Bet v1 alone (black) eluted at 8.5 min, while Fab-1 alone (turquoise) and the Bet v1:Fab-1 complex eluted at 9.8 and 7.9 min, respectively. The stoichiometry of the Bet v1:Fab-1 complex revealed a 1:1 binding ratio. No additional stoichiometries were observed in which either the antigen or the antibody was in excess. Furthermore, mixing the Bet v1 antigen with Fab-1 in an exact 1:1 molar ratio minimized the amount of free antigen and antibody (blue).

[0185] While the UV peaks can represent the relative abundance of individual proteins in solution, MS allows for the identification of individual components in complex samples. Dual UV and MS detection allows the determination of all species co-eluting within the same UV peak or between different UV peaks. As shown in Figure 7, MS analysis of the UV peak eluting at 7.9 min revealed three different complexes formed by Fab-1 bound to each of the three different Bet v1 species (naked, G2S1F, and G2S2F). We found that the Fab-1:naked complex eluted after Fab-1 in complex with glycosylated Bet v1 species. This may be due to the larger hydrodynamic radius imparted by the glycans. Minimizing the detection delay between UV and MS allowed us to collect fractions from the UV detector for sample recovery. This method is particularly useful for determining whether truncated, modified, or mutated forms of antigens and / or antibodies retain binding without the need to purify the specific form of the protein under study.

[0186] Example 2. Characterization of Cysteine ​​ADCs using Online SEC-nano-ESI-MS Antibody-drug conjugates (ADCs) are highly powerful therapeutics that can specifically deliver small molecule drugs to target tissues by conjugation to antibodies (Francois Debaene et al., Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment, 86 ANALYTICAL CHEMISTRY 10674-10683 (2014)). The potency, efficacy, and toxicity of ADCs can be highly dependent on the number of small molecule drugs conjugated to each antibody. Therefore, it can be important to determine the drug-to-antibody ratio (DAR) of each ADC. Conjugation via interchain cysteines is one of the most common approaches for conjugating small molecule drugs to antibodies. In interchain cysteine-based ADCs, unpaired interchain cysteine ​​residues can be introduced by engineering primary sequence mutations (site-directed interchain cysteine-based conjugates) or by partially reducing the antibody (random interchain cysteine-based conjugates). Site-specific conjugation of engineered mAbs provides better control over the DAR of the ADC, while conjugation with partially reduced mAbs provides a larger and more variable range of DAR (0-8). However, determining the DAR of ADCs generated by either method may still be necessary to understand and interpret the biological effects of these drug conjugates. The most common methods to determine the DAR of cysteine-based ADCs are by HIC-UV (Laura R. Saunders et al., A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo, 7 SCIENCE TRANSLATIONAL MEDICINE (2015)) or RPLC-MS under reducing / denaturing conditions.However, Debaene et al. recently reported an offline desalting SEC method coupled with high-resolution native MS and IM-MS for average DAR measurement. Native MS analysis is the only method to analyze the DAR of cysteine-linked ADCs without destroying the intact molecule.

[0187] 1.1 Online SEC-nano-ESI-MS instrument The apparatus shown in 1.1 was used.

[0188] 1.2 Cysteine-based site-specific conjugation ADCs The DAR of cysteine-based site-specific conjugated ADCs was evaluated using an online SEC-nano-ESI-MS system. Mutation of the mAb heavy chain to interchain cysteines introduced two unpaired cysteines in the light chain of the mAb, which were conjugated to drugs to obtain the DAR2 antibody species. As shown in Figure 8, the raw and deconvoluted spectra of parent mAb-1 and ADC-1 indicate that only the DAR2 form is present in the ADC samples. The deconvoluted spectra in Figures 8A-C show that various glycans are present in both parent mAb-1 and conjugated ADC-1. Along with unglycosylated mAb-1, partially and fully glycosylated mAb-1 species with various combinations of G0F, G1F, and G2F were all conjugated with the two drugs. These results were confirmed by analyzing manufacturer-digested mAb-1 and ADC-1, as shown in Figure 9. Multiple ADCs with similar linkage chemistries were tested and only showed a DAR 2 form (data not shown).

[0189] 1.3 Non-site-specific cysteine-linked ADCs In addition to analyzing the site-specific ADCs, the DAR of drugs conjugated to the interchain cysteines of partially reduced mAbs was also investigated. Raw and deconvoluted spectra of non-site-specific cysteine-linked ADCs generated using similar chemistries are shown in Figure 10. ADC-2 was generated using unmodified parent antibody with normal glycosylation. Raw and deconvoluted spectra of parent mAb-2 (Figure 10A-B) and ADC-2 (Figure 10C-D) show DAR values ​​that vary from 2 to 8 for ADC-2. The deconvoluted spectra also show the various glycans present in mAb-2 (Figure 10B) and ADC-2 (Figure 10D).

[0190] We developed an online SEC-nanoelectrospray ionization (nano-ESI)-MS platform with dual ultraviolet (UV) and MS detection. The utility of this platform was verified by investigating non-covalent protein interactions by using it for the characterization of antigen-antibody complexes obtained from titration experiments and for the determination of drug-to-antibody ratios (DARs) of cysteine-based antibody-drug conjugates (ADCs). The platform can be easily modified and therefore adapted for the analysis of other native MS projects, such as the characterization of monoclonal antibody (mAb) charge variants, or large aggregated protein complexes.

[0191] A three-way splitter was used to split the SEC eluate unequal to the MS and UV detector, with the low-volume fraction directed to the MS and the high-volume fraction directed to the UV detector. This platform allows for complementary dual detection by UV and native MS with the possibility of fraction collection and can be applied to the characterization of antigen-antibody complexes and the DAR analysis of interchain cysteine-linked ADCs. Further modifications of this online SEC-nano-ESI-MS platform, such as changing the column chemistry or using a QExactive UHMR instrument, can make it adaptable for other applications, such as the analysis of charge variants and very large protein complexes. The method described herein opens the possibility of combining high salt separation techniques (i.e. HIC, WCX) with mass spectrometry-based detection. Finally, the online SEC-nano-ESI-MS platform could be widely applied to the analysis of protein biopharmaceuticals for various applications.

Claims

1. contacting a sample containing protein with a chromatography system having a size exclusion chromatography resin; washing the size exclusion chromatography resin with a mobile phase to obtain an eluate containing the protein; and characterizing the proteins in the eluate using electrospray ionization mass spectrometry under non-denaturing conditions. A method for characterizing a protein, comprising:

2. The method of claim 1 , wherein the electrospray ionization mass spectrometer is coupled to the chromatography system having the size exclusion chromatography resin.

3. The method of claim 1 , wherein the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer.

4. The method of claim 1 , wherein at least one three-way splitter is used to couple the electrospray ionization mass spectrometer to the chromatography system having the size exclusion chromatography resin.

5. 10. The method of claim 1, wherein at least one three-way splitter is used to couple an ultraviolet detector to the chromatography system having the size exclusion chromatography resin.

6. 6. The method of claim 5, wherein the eluent from the step of washing the size exclusion chromatography resin is introduced to the ultraviolet detector through at least one three-way splitter at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

7. 10. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin comprises ammonium acetate.

8. The method of claim 1 , wherein the mobile phase used to wash the size exclusion chromatography resin comprises a volatile salt.

9. 2. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin has a total concentration of about 100 mM.

10. 10. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin has a flow rate of about 0.2 mL / min to about 0.4 mL / min.

11. 10. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin has a pH of about 6.

8.

12. 2. The method of claim 1, wherein the amount of the sample containing the protein contacted with the chromatography system is from about 10 μg to about 100 μg.

13. The method of claim 1 , wherein the protein is an antibody.

14. The method of claim 1 , wherein the protein is a monoclonal antibody.

15. The method of claim 1 , wherein the protein is a therapeutic antibody.

16. The method of claim 1, wherein the protein is an antigen-antibody complex.

17. The method of claim 1 , wherein the protein comprises an antibody contained in an antibody-drug conjugate.

18. 2. The method of claim 1, wherein the eluent resulting from the step of washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer at a flow rate of less than about 50 μl / min.

19. 2. The method of claim 1, wherein the eluent obtained from the step of washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer, and the flow rate of electrospray from the electrospray ionization is from about 10 nL / min to about 50 nL / min.

20. 2. The method of claim 1, wherein the eluate obtained by washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer, and the electrospray voltage is from about 0.8 kV to about 1.5 kV.

21. contacting the sample containing the antibody-drug conjugate with a chromatography system having a size exclusion chromatography resin; washing the size-exclusion chromatography resin with a mobile phase to obtain an eluate containing the antibody-drug conjugate; and characterizing the antibody-drug conjugate in the eluate using electrospray ionization mass spectrometry under non-denaturing conditions. A method for characterizing an antibody-drug conjugate, comprising:

22. 22. The method of claim 21, wherein the antibody-drug conjugate is an engineered cysteine-based antibody-drug conjugate.

23. The method of claim 21 , wherein the antibody-drug conjugate is a non-specific cysteine-based antibody-drug conjugate.

24. 22. The method of claim 21, wherein the step of characterizing the antibody-drug conjugate comprises characterizing the drug-to-antibody ratio.

25. contacting a sample containing protein with a chromatography system having a size exclusion chromatography resin; washing the size exclusion chromatography resin with a mobile phase to obtain an eluate containing the protein; and characterizing the proteins in the eluate using electrospray ionization mass spectrometry under non-denaturing conditions.

23. A method for identifying a protein, comprising:

26. contacting a sample containing protein with a chromatography system having a size exclusion chromatography resin; washing the size exclusion chromatography resin with a mobile phase to obtain an eluate containing the protein; and characterizing the proteins in the eluate using electrospray ionization mass spectrometry under non-denaturing conditions. A method for quantifying a protein, comprising:

27. a chromatography column having a size exclusion chromatography resin, the chromatography column being capable of accepting a mobile phase and a sample containing a protein; and an electrospray ionization mass spectrometer that can be coupled to the chromatography column and operated under non-denaturing conditions; Including, the system.

28. The system of claim 27, capable of characterizing the drug-to-antibody ratio of an antibody-drug conjugate.

29. 28. The system of claim 27, wherein the chromatography column is connectable to the mass spectrometer using a T-splitter.

30. 28. The system of claim 27, wherein the chromatography column is further connectable to an ultraviolet detector using a T-splitter.

31. The system of claim 27 , wherein the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer.

32. contacting a sample containing the protein with a chromatography system having a chromatography resin; washing the size exclusion chromatography resin with a mobile phase to obtain an eluate containing the protein; and characterizing the proteins in the eluate using electrospray ionization mass spectrometry under non-denaturing conditions. A method for characterizing a protein, comprising:

33. 33. The method of claim 32, wherein the chromatography resin is selected from the group consisting of an affinity chromatography resin, an anion exchange resin, a cation exchange resin, an affinity resin, a mixed-mode chromatography resin, a hydrophobic interaction chromatography resin, or a size-exclusion chromatography resin.

34. 33. The method of claim 32, wherein the chromatography system is selected from reversed phase (RP), ion exchange (IEX), or normal phase chromatography (NP).

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