On-line chromatography and electrospray ionization mass spectrometry

Abstract

A method and system are provided for the characterization of proteins using online chromatography and electrospray ionization mass spectrometry.

Description

Technical Field

[0001] Field The present invention generally relates to a method for characterizing protein biopharmaceuticals using online chromatography and electrospray ionization mass spectrometry.

Background Art

[0002] Background Protein biopharmaceuticals have emerged as important drugs for the treatment of cancer, autoimmune diseases, infectious diseases, and cardio-metabolic disorders, and they are one of the most rapidly growing product segments in the pharmaceutical industry.

[0003] Protein biopharmaceuticals must meet very high purity standards. Therefore, it may be important to monitor and characterize protein biopharmaceuticals during various stages of drug development and manufacturing. Analytical methods for testing to characterize such protein biopharmaceuticals need to show sufficient accuracy and sensitivity for the detection and quantification of the product of interest. Protein biopharmaceuticals can be difficult to evaluate because their structural and physicochemical properties are similar compared to their variant, modified, or cleaved forms. In direct analysis, it may be necessary to isolate a sufficient amount of the product for the test, which is undesirable and possible only in selected cases.

[0004] Methods and / or systems for characterizing protein biopharmaceuticals have been needed in the art for many years.

Summary of the Invention

[0005] Summary The growth of the development, manufacture, and sale of protein biopharmaceuticals has led to an increasing need to characterize protein biopharmaceuticals along with possible impurities, binding stoichiometry, and their overall composition.

[0006] Exemplary embodiments disclosed herein meet the foregoing requirements by providing methods for characterizing, identifying, and / or quantifying protein biopharmaceuticals along with any impurities, linkages, and overall composition that may be present therein.

[0007] The present disclosure provides, at least in part, methods for characterizing proteins. In one exemplary embodiment, a 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 eluate containing the protein, and characterizing the protein in the eluate using an electrospray ionization mass spectrometer.

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

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

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

[0011] In one aspect, the method for characterizing a protein may include 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 nanoelectrospray ionization mass spectrometer.

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

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

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

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

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

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

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

[0020] In one aspect of this embodiment, a method for characterizing a protein can include washing the resin with a mobile phase to obtain an eluent containing the protein, and the eluent is introduced from at least one three-way splitter into the 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, a method for characterizing a protein can include a mobile phase containing a volatile salt.

[0022] In one aspect of this embodiment, a method for characterizing a protein can include a mobile phase containing ammonium acetate.

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

[0024] In one aspect of this embodiment, a method for characterizing a protein can include washing the resin with the 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, a method for characterizing a protein can include a mobile phase having a pH of about 6.8.

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

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

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

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

[0030] In one aspect of this embodiment, a method for characterizing a protein may 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, a method for characterizing a protein may include an electrospray ionization mass spectrometer in which the spray voltage of electrospray is about 0.8 kV to about 1.5 kV.

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

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

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

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

[0036] The present disclosure provides, at least in part, methods for characterizing antibody-drug conjugates. In one exemplary embodiment, a 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 eluate containing the antibody-drug conjugate, and characterizing the antibody-drug conjugate in the eluate using an electrospray ionization mass spectrometer.

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

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

[0039] In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include connecting 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 an antibody-drug conjugate may include an electrospray ionization mass spectrometer under non-denaturing conditions.

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

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

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

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

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

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

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

[0048] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate may include at least one three-way splitter for connecting 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, a method for characterizing an antibody-drug conjugate can include washing a resin using a mobile phase to obtain an eluent containing the antibody-drug conjugate, and the eluent is introduced into 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, a method for characterizing an antibody-drug conjugate can include a mobile phase containing a volatile salt.

[0051] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate can include a mobile phase containing ammonium acetate.

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

[0053] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate can include washing the resin with the 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, a method for characterizing an antibody-drug conjugate can include a mobile phase having a pH of about 6.8.

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

[0056] In one aspect of this embodiment, a method for characterizing an antibody-drug conjugate can include washing the resin using the 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, a method for characterizing an antibody-drug conjugate may include an electrospray ionization mass spectrometer having a flow rate of about 10 nL / min to about 50 nL / min.

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

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

[0060] In one aspect of this embodiment, a 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, a 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, a method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is a non-specific cysteine-based antibody-drug conjugate.

[0063] In one aspect of this embodiment, a method for characterizing an 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 may include identifying the antibody.

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

[0066] The present disclosure provides, at least in part, a system including 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, and the chromatography column can receive a mobile phase and a sample containing a protein.

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

[0068] In one aspect of this embodiment, the system may include an electrospray ionization mass spectrometer connectable to the chromatography column.

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

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

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

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

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

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

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

[0076] In one aspect of this embodiment, the system can enable the characterization of antigen - antibody complexes. [The present invention 1001] A step of contacting a sample containing a protein with a chromatography system having a size exclusion chromatography resin; A step of washing the size exclusion chromatography resin using a mobile phase to obtain an eluent containing the protein; and A step of characterizing the protein in the eluent using an electrospray ionization mass spectrometer under non-denaturing conditions A method for characterizing a protein, comprising: [The present invention 1002] The method of the present invention 1001, wherein the electrospray ionization mass spectrometer is connected to the chromatography system having the size exclusion chromatography resin. [The present invention 1003] The method of the present invention 1001, wherein the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer. [The present invention 1004] The method of the present invention 1001, wherein at least one three-way splitter is used to connect the electrospray ionization mass spectrometer to the chromatography system having the size exclusion chromatography resin. [The present invention 1005] The method of the present invention 1001, wherein at least one three-way splitter is used to connect an ultraviolet detector to the chromatography system having the size exclusion chromatography resin. [The present invention 1006] The method of the present invention 1005, wherein the eluent from the step of washing the size exclusion chromatography 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. [The present invention 1007] The method of the present invention 1001, wherein the mobile phase used to wash the size exclusion chromatography resin contains ammonium acetate. [The present invention 1008] The method of the present invention 1001, wherein the mobile phase used to wash the size exclusion chromatography resin contains a volatile salt. [The present invention 1009] The method of the present invention 1001, wherein the mobile phase used to wash the size exclusion chromatography resin has a total concentration of about 100 mM. [The present invention 1010] The method of the present invention 1001, 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. [The present invention 1011] The method of the present invention 1001, wherein the mobile phase used for washing the size exclusion chromatography resin has a pH of about 6.8. [The present invention 1012] The method of the present invention 1001, wherein the amount of the sample containing the protein contacted with the chromatography system is about 10 μg to about 100 μg. [The present invention 1013] The method of the present invention 1001, wherein the protein is an antibody. [The present invention 1014] The method of the present invention 1001, wherein the protein is a monoclonal antibody. [The present invention 1015] The method of the present invention 1001, wherein the protein is a therapeutic antibody. [The present invention 1016] The method of the present invention 1001, wherein the protein is an antigen-antibody complex. [The present invention 1017] The method of the present invention 1001, wherein the protein comprises an antibody contained in an antibody-drug conjugate. [The present invention 1018] The method of the present invention 1001, wherein the eluate obtained 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. [The present invention 1019] The method of the present invention 1001, wherein the eluate obtained from the step of washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer, and the flow rate of the electrospray from the electrospray ionization is about 10 nL / min to about 50 nL / min. [The present invention 1020] The method of the present invention 1001, wherein the eluate obtained from the step of washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer, and the spray voltage of the electrospray is about 0.8 kV to about 1.5 kV. [The present invention 1021] A step of contacting a sample containing an antibody-drug conjugate with a chromatography system having a size exclusion chromatography resin; A step of washing the size exclusion chromatography resin using a mobile phase to obtain an eluate containing the antibody-drug conjugate; and A step of characterizing the antibody-drug conjugate in the eluate using an electrospray ionization mass spectrometer under non-denaturing conditions A method for characterizing an antibody-drug conjugate. [The present invention 1022] The method of the present invention 1021, wherein the antibody-drug conjugate is an engineered cysteine-based antibody-drug conjugate. [The present invention 1023] The method of the present invention 1021, wherein the antibody-drug conjugate is a non-specific cysteine-based antibody-drug conjugate. [The present invention 1024] The method of the present invention 1021, wherein the step of characterizing the antibody-drug conjugate includes characterizing the drug-to-antibody ratio. [The present invention 1025] Contacting a sample containing a protein 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 the protein; and Characterizing the protein in the eluent using an electrospray ionization mass spectrometer under non-denaturing conditions A method for identifying a protein. [The present invention 1026] Contacting a sample containing a protein 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 the protein; and Characterizing the protein in the eluent using an electrospray ionization mass spectrometer under non-denaturing conditions A method for quantifying a protein. [The present invention 1027] A chromatography column having a size exclusion chromatography resin, the chromatography column capable of receiving a mobile phase and a sample containing a protein; and An electrospray ionization mass spectrometer connectable to the chromatography column and operable under non-denaturing conditions A system. [The present invention 1028] The system of the present invention 1027 capable of characterizing the drug-to-antibody ratio of an antibody-drug conjugate. [The present invention 1029] The system of the present invention 1027, wherein the chromatography column is connectable to the mass spectrometer using a T-splitter. [The present invention 1030] The system of the present invention 1027, wherein the chromatography column is further connectable to an ultraviolet detector using a T-splitter. [The present invention 1031] The system of the present invention 1027, wherein the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer. [The present invention 1032] A step of contacting a sample containing a protein with a chromatography system having a chromatography resin; A step of washing the size exclusion chromatography resin using a mobile phase to obtain an eluent containing the protein; and A step of characterizing the protein in the eluent using an electrospray ionization mass spectrometer under non-denaturing conditions A method for characterizing a protein, comprising: [Inventive concept 1033] The method of Inventive concept 1032, 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. [Inventive concept 1034] The method of Inventive concept 1032, wherein the chromatography system is selected from reverse phase (RP), ion exchange (IEX), or normal phase chromatography (NP).

Brief Description of the Drawings

[0077] [Figure 1] Shows spectra obtained from normal and native electrospray ionization mass spectrometry. [Figure 2] Illustrative embodiments of a system capable of characterizing protein biopharmaceuticals are shown. [Figure 3] Illustrative embodiments of a system capable of characterizing protein biopharmaceuticals are shown. [Figure 4] The configuration of a system capable of characterizing protein biopharmaceuticals according to an illustrative embodiment is shown. [Figure 5] Figures 5A and 5B show the analysis of antigen - antibody complexes using a system capable of characterizing protein biopharmaceuticals according to an illustrative embodiment. [Figure 6] Results of antigen - antibody titration between Bet v1 and Fab - 1 characterized according to an illustrative embodiment are shown. [Figure 7]Figure 7A shows the mass spectrometer signal as a result of double-detecting the antigen-antibody interaction between Bet v1 and Fab-1 under non-denaturing conditions by an ultraviolet detector and a native electrospray ionization mass spectrometer according to an exemplary embodiment. Figure 7B shows the result of the ultraviolet signal as a result of double-detecting the antigen-antibody interaction between Bet v1 and Fab-1 under non-denaturing conditions by an ultraviolet detector and a native electrospray ionization mass spectrometer according to an exemplary embodiment. [Figure 8A] Shows the raw spectrum of the parent mAb-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-1 by an online SEC-nano-ESI-MS device according to an exemplary embodiment. [Figure 8B] Shows the raw spectrum of the ADC-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-1 by an online SEC-nano-ESI-MS device according to an exemplary embodiment. [Figure 8C] Shows the convolution spectrum of the parent mAb-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-1 by an online SEC-nano-ESI-MS device according to an exemplary embodiment. [Figure 8D] Shows the convolution spectrum of the ADC-1 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-1 by an online SEC-nano-ESI-MS device according to an exemplary embodiment. [Figure 9] Shows the drug-to-antibody ratio analysis of site-specifically conjugated cysteine mAb-1 and ADC-1 by FabRICATOR digestion and an online SEC-nano-ESI-MS device according to an exemplary embodiment. [Figure 10A] Shows the raw spectrum of the parent mAb-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-2 by an online SEC-nano-ESI-MS device according to an exemplary embodiment, where N means the antibody has no glycan, S means glycosylation involves a single chain of the antibody, and D means glycosylation involves both chains of the antibody. [Figure 10B]Convolution spectra of the parental mAb-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-2 by an online SEC-nano-ESI-MS apparatus according to an exemplary embodiment, where N means the antibody has no glycan, S means glycosylation involves a single chain of the antibody, and D means glycosylation involves both chains of the antibody. [Figure 10C] Raw spectra of the ADC-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-2 by an online SEC-nano-ESI-MS apparatus according to an exemplary embodiment, where N means the antibody has no glycan, S means glycosylation involves a single chain of the antibody, and D means glycosylation involves both chains of the antibody. [Figure 10D] Convolution spectra of the parental ADC-2 drug-to-antibody ratio analysis of site-specifically conjugated cysteine ADC-2 by an online SEC-nano-ESI-MS apparatus according to an exemplary embodiment, where N means the antibody has no glycan, S means glycosylation involves a single chain of the antibody, and D means glycosylation involves both chains of the antibody.

Mode for Carrying Out the Invention

[0078] Detailed Description The identification and quantification of proteins in protein biopharmaceuticals can be very important during the manufacture and development of products. The presence of impurities and the binding methods of protein biopharmaceuticals may be essential for developing safe and effective products. Therefore, robust methods and / or workflows for the characterization of protein biopharmaceuticals, their binding methods, and the characterization of associated impurities may 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)). The separation of molecules by SEC depends on the various interactions of the molecules with a controlled porous structure on the stationary phase. Since SEC uses buffer conditions that maintain the native structure of the proteins in solution, it can characterize biomolecules without disturbing their native conformation. Among the various detection modes that can be combined with SEC, mass spectrometry (MS) can precisely and accurately identify individual components in complex samples.The combination of SEC and MS has been previously reported, including 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 10692-10699 (2014); C.F. Mcdonagh et al., Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment, 19 PROTEIN ENGINEERING DESIGN AND SELECTION 299-307(2006)). However, the practicality of linking these techniques for the analysis of non-covalent interactions is limited because harsh ionization conditions, which are often incompatible with native MS analysis, are required to directly ionize the high flow rates generated from SEC separation.Furthermore, the sensitivity of the mass spectrometer may be affected by the high salt concentrations used in the SEC buffer.

[0080] Since non-covalent protein interactions mediate such a wide range of biological functions, there is increasing interest in developing methods to facilitate the study of their structures, stoichiometries, and dynamics. Such methods may help to investigate non-covalent protein interactions that occur widely in nature and are also required to modulate the interactions of protein biopharmaceuticals with diverse molecules (such as 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 since proteins are maintained in their native conformations by buffer conditions, SEC is an ideal method for preserving non-covalent biomolecular complexes during separation. Among the various detection methods that can be combined with SEC analysis, mass spectrometry (MS) enables reliable identification and characterization of individual components from complex mixtures. However, the high flow rates and non-volatile salts used in SEC are often not compatible with downstream MS analysis.

[0081] Building on the limitations of existing methods, an effective and efficient method has been developed for analyzing protein biopharmaceuticals using online chromatography with an electrospray ionization MS platform.

[0082] Unless otherwise indicated, 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 thereof, the methods and materials are described hereinafter. All publications mentioned are hereby incorporated by reference into this specification.

[0083] The term "one (a)" should be understood to mean "at least one". The terms "about" and "approximately" should be understood to allow for standard variations that would be understood by one of ordinary skill in the art. When ranges are indicated, 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, "protein biopharmaceutical" includes an active ingredient that is essentially completely or partially a biologic. In some exemplary embodiments, protein biopharmaceuticals can include proteins, vaccines, allergens, nucleic acids, viruses, antibody-drug conjugates, cells, genes, tissues, or combinations thereof. In some other exemplary embodiments, protein biopharmaceuticals can include recombinant, engineered, modified, mutated, or truncated forms of proteins, vaccines, allergens, nucleic acids, viruses, antibody-drug conjugates, cells, genes, tissues, or combinations 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 that are commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and non-naturally occurring analogs thereof by synthesis. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase peptide synthesis methods are known. A protein can include one or more polypeptides to form a single functioning biomolecule. Proteins can include any of 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 aspect, 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., species of the genus Pichia (Pichia sp.)), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a 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, proteins include modifications, adducts, and other covalently linked moieties.These modifications, adducts, 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 tag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels, and other dyes. Since proteins can be classified based on composition and solubility, proteins can include simple proteins such as globular proteins and fibrous proteins; complex proteins such as nucleoproteins, glycoproteins, mucoproteins, pigment proteins, phosphoproteins, metalloproteins, and lipoproteins; and derivative proteins such as primary derivative proteins and secondary derivative proteins.

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

[0088] As used herein, the term "antibody" includes immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, which are four polypeptide chains, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H and a heavy chain constant region. The heavy chain constant region comprises three domains, C H1 , C H2 , and C H3 . Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L and a light chain constant region. The light chain constant region comprises one domain (C L1 ). The V H region and the V L region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and V L consists of three CDRs and four FRs, and is arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus towards the carboxy terminus. In various exemplary embodiments, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be modified naturally or artificially. Amino acid consensus sequences can be defined based on side-by-side analysis of two or more CDRs. As used herein, the term "antibody" includes antigen-binding fragments of complete antibody molecules. Terms such as "antigen-binding portion" of an antibody or "antigen-binding fragment" of an antibody include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from a complete antibody molecule using any suitable standard techniques, such as proteolytic or recombinant genetic engineering techniques (including 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 by chemical or molecular biological techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, form cysteine residues, modify, add, or delete amino acids, etc.

[0089] As used herein, the term "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, Fc fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetra-bodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of the heavy and light chains of an immunoglobulin, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains of an immunoglobulin are linked by a peptide linker. Antibody fragments can be produced by a variety of 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 genes encoding partial antibody sequences. Alternatively, or in addition thereto, antibody fragments can be produced wholly or partially by synthesis. Antibody fragments may optionally include single-chain antibody fragments. Alternatively, or in addition thereto, antibody fragments may include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes.

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

[0091] As used herein, the term "antibody-drug conjugate" or "ADC" can refer to an antibody linked to a biologically active drug by a linker having a labile bond. An ADC can contain several molecules of a biologically active drug (or payload) capable of covalently binding 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 an ADC can be made to bind with sufficient affinity for selective accumulation and sustained retention at the target site. Most ADCs can have Kd values in the nanomolar concentration range. The payload can have potency in the nanomolar / picomolar concentration range and be made to reach intracellular concentrations achievable after distribution of the ADC to the target tissue. Finally, the linker that forms the linkage between the payload and the antibody can be made sufficiently stable in circulation to utilize the pharmacokinetic properties of the antibody site (i.e., long half-life) and to allow the payload to remain bound to the antibody as it distributes within the tissue, but must be capable of efficient release of the biologically active drug after the ADC has been taken up by the target cell.

[0092] Linkers can be made non-cleavable during cell processing and cleavable when the ADC reaches the target site. In the case of non-cleavable linkers, the biologically active drug released intracellularly contains, after complete proteolysis of the ADC within the lysosome, the payload and all elements of the linker remaining bound to amino acid residues (typically lysine or cysteine residues) of the antibody. Cleavable linkers are those whose structure contains 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 intracellular reducing environment.

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

[0094] An ADC can be produced by conjugating an antibody to an endogenous amino acid residue while carefully controlling the average degree of modification so as to obtain an optimal drug-to-antibody ratio (DAR). This ratio can be selected based on (a) minimizing the amount of un-conjugated antibody and (b) avoiding species in mixtures with very high DARs, which can be problematic in manufacturing and formulation due to high hydrophobicity and low solubility and can also result in suboptimal pharmacokinetic properties. Too few bonds to the biologically active drug molecule results in reduced efficacy, while too many leads to an unstable ADC, altered pharmacokinetic properties, increased plasma clearance, shortened half-life, and increased systemic toxicity. The optimal DAR is often uncertain and highly dependent on other ADC variables. However, more generally, an ADC aims to achieve a DAR close to 4. Non-limiting examples of the conjugation of a biologically active drug to an antibody can include the conjugation of the biologically active drug to lysine or cysteine residues on the antibody. Conjugation to lysine can result in 0 to 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 the conjugation of a biologically active drug to an antibody can include the conjugation of cysteine that occurs after reduction of the four inter-chain disulfide bonds, such that the conjugation is limited to the eight exposed sulfhydryl groups, and thus the number of conjugated biologically active drug molecules per antibody can range from 0 to 8. Since these ADC species differ in drug load and binding site, the diversity of ADC mixture heterogeneity is doubled. Thus, each species can have different properties and, as a result, different in vivo PK properties. In addition, batch-to-batch consistency in ADC manufacturing can be difficult and may require faithful manufacturing capabilities.

[0095] Site-specific antibody-drug conjugates, in which a known number of biologically active drug molecules are consistently bound to defined sites, are one way to overcome these challenges. The heterogeneity is minimized, the properties of the ADCs become more predictable, and consistent conjugate production is carried out between batches. The drug-to-antibody ratio (DAR) is tightly controlled and can be adjusted for various conjugated bioactive drugs to produce 2- or 4-DAR site-specific ADCs. Non-limiting examples of site-specific conjugates include 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 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) for linking biologically active drug molecules to antibodies.

[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] As used herein, the term "impurity" can include any unwanted protein present in a protein biopharmaceutical. Impurities can include those related to the process and the product. Impurities can further be of known structure, partially characterized, or uncharacterized. Process-related impurities may originate from the manufacturing process and can include three main categories: those derived from the cell substrate, those derived from cell culture, and those derived downstream. Examples of impurities derived from the cell substrate include, but are not limited to, proteins derived from the host organism and nucleic acids (the genome of the host cell, vector, or total DNA). Examples of impurities derived from cell culture include, but are not limited to, inducers, antibiotics, serum, and other media components. Examples of impurities derived downstream include, but are not limited to, enzymes, chemical and biochemical processing reagents (such as cyanogen bromide, guanidine, oxidizing agents, and reducing agents), inorganic salts (such as heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (such as monoclonal antibodies), and other leachates. Product-related impurities (such as precursors, certain degradation products) can be molecular variants that occur during manufacturing and / or storage and do not have properties comparable to those of the desired product characteristics with respect to activity, efficacy, and safety. Such variants can require significant effort for separation and characterization in order to identify the type of modification. Product-related impurities include cleaved 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 modified complex forms (such as glycosylation, phosphorylation). Modified forms can also include any form of post-translational modification. Aggregates include dimers of the target product, as well as higher-order multimers.(Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products, ICH August 1999, U.S. Dept. of Health and Humans Services).

[0098] As used herein, the general terms "post-translational modification" or "PTM" refer to covalent modifications that polypeptides undergo either during their ribosomal synthesis (co-translational modification) or afterwards (post-translational modification). PTMs are typically introduced by specific enzymes or enzyme pathways. Many occur at sites of specific characteristic protein sequences (signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some aspect of the protein's 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, cleavage, 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 (usually the addition of an acetyl group at the N-terminus of a protein), alkylation (usually the addition of an alkyl group (e.g., methyl, ethyl, propyl) to a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, cross-linking by covalent bonds 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 cofactor in the carboxylation of glutamic acid residues to form γ-carboxyglutamic acid (Gla residues)), glutamylation (covalent attachment of glutamic acid residues), glycylation (covalent glycine residues), glycosylation (addition of a glycosyl group to any of asparagine, hydroxylysine, serine, or threonine to obtain a glycoprotein), isoprenylation (addition of an isoprenoid group such as farnesol and geranylgeraniol), lipoylation (addition of a lipoate functional group), phosphopantetheinylation (addition of a 4'-phosphopantetheinyl moiety from coenzyme A as in the case of fatty acids, polyketides, non-ribosomal peptides, and leucine biosynthesis), 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 involving structural changes include, but are not limited to, formation of disulfide bridges (covalent bond of two cysteine amino acids) and proteolytic cleavage (cleavage of a protein at a peptide bond).Certain post-translational modifications include the addition of other proteins or peptides such as ISGylation (covalent attachment to the ISG15 protein (interferon-stimulated gene)), SUMOylation (covalent attachment to the SUMO protein (small ubiquitin-like modifier)), and ubiquitination (covalent attachment to the protein ubiquitin). For more detailed controlled terms 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 accessed January 15, 2019).

[0099] As used herein, the term "product of interest" refers to a protein biopharmaceutical having a desired structure, function, or efficacy profile.

[0100] In some exemplary embodiments, the present disclosure also provides methods for characterizing the binding of protein biopharmaceuticals. For example, antibodies can bind to antigens through highly specific, high-affinity, and non-covalent interactions, which 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 therapies, it may be important to understand how antibody binding affects the function of the target protein.

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

[0102] In some specific exemplary embodiments, the present disclosure also provides a method for identifying an antigen to which an antibody binds. In some other exemplary embodiments, the method may include determining whether a cleaved, 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 a method 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, conjugates, or compositions may include contacting a sample containing the protein biopharmaceutical with a chromatography system having a chromatography resin.

[0105] As used herein, the term "chromatography" refers to a process capable of separating a chemical mixture carried by a liquid or gas into multiple components as a result of different distributions of the chemicals as they flow around or over a liquid stationary phase or a solid stationary phase. Non-limiting examples of chromatography include conventional reverse phase (RP), ion exchange (IEX), mixed-mode chromatography, and normal phase chromatography (NP).

[0106] As used herein, the terms "mixed-mode chromatography (MMC)" or "multimodal chromatography" include chromatographic methods in which a solute interacts with a stationary phase via multiple interaction modes or mechanisms. MMC can be used as an alternative or complementary tool to conventional reverse-phase (RP), ion-exchange (IEX), and normal-phase chromatography (NP). Unlike RP, NP, and IEX chromatography, in which hydrophobic interaction, hydrophilic interaction, and ionic interaction are the main interaction modes, respectively, in mixed-mode chromatography, two or more of these interaction modes can be used in combination. The media for mixed-mode chromatography can provide unique selectivity that cannot be reproduced in single-mode chromatography. Mixed-mode chromatography can also offer potential cost reduction and operational flexibility compared to affinity-based methods.

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

[0108] As used herein, the terms "SEC chromatography resin" or "SEC chromatography medium" are used interchangeably and can include any type of solid phase used in SEC to separate impurities from the target product (e.g., contaminating substances that are homodimers for a bispecific antibody product). The volume of the resin, the length and diameter of the column used, as well as the dynamic capacity and flow rate can depend on multiple parameters such as the volume of the fluid being processed and the concentration of protein in the fluid being treated.

[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 the protein, and characterizing the protein in the eluent using an electrospray ionization mass spectrometer.

[0110] As used herein, the term "mass spectrometer" includes an apparatus capable of identifying specific molecular species and measuring their exact masses. This term is intended to include any molecular detector capable of eluting polypeptides or peptides therein 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 to be analyzed can be transferred to the gas phase and simultaneously ionized (similar to electrospray ionization). The choice of ion source depends largely on the application.

[0111] As used herein, the term "electrospray ionization" or "ESI" refers to the process of spray ionization in which either cations or anions in solution are transferred to the gas phase by the formation and desolvation of a highly charged stream of droplets at atmospheric pressure obtained by applying a potential difference between an electrospray needle tip containing the solution and a counter electrode. There are usually three main stages in the generation of gas-phase ions from electrolyte ions in solution. These are: (a) the generation of charged droplets in the ES injection tip; (b) the reduction of the charged droplets by solvent evaporation and repeated droplet disintegration, resulting in the generation of small, highly charged droplets capable of generating gas-phase ions; and (c) the mechanism by which gas-phase ions are generated from very small and highly charged droplets. Stages (a)-(c) typically occur in the atmospheric pressure region of the apparatus.

[0112] As used herein, the term "electrospray infusion setup" refers to an electrospray ionization system that is compatible with a mass spectrometer used for protein mass spectrometry. In electrospray ionization, the electrospray needle has an orifice that is positioned near the inlet orifice of the spectrometer. A sample containing the protein of interest can be pumped from a syringe needle. A potential between the orifice of the syringe needle and the orifice leading to the mass spectrometer forms a spray of the solution ("electrospray"). The electrospray can be performed at atmospheric pressure and supplies highly charged droplets of the solution. The electrospray infusion setup can include an electrospray emitter, a nebulizing gas, and / or an ESI power supply. Optionally, the setup may be automated for performing aspiration of the sample, dispensing of the sample, feeding of the sample, and / or spraying of the sample.

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

[0114] As used herein, the term "nanoelectrospray" or "nanospray" refers to electrospray ionization in a sample solution with a very low solvent flow rate, typically less than a few hundred nanoliters per minute, and often without using an external solvent supply. The electrospray infusion setup for forming a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of a small volume of sample (analyte) solution over a long period of time. A dynamic nanoelectrospray emitter performs chromatographic separation of a mixture using a capillary column and a solvent supply system prior to analysis by a mass spectrometer.

[0115] The term "mass spectrometer" as used in the specification includes devices that can separate species, i.e., atoms, molecules, or clusters, according to their mass. Non-limiting examples of mass spectrometers that can be used for high-speed 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 native conditions.

[0117] The term "native 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 normal 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 that obtain structural information about sample molecules by using multiple stages of mass selection and mass separation. The prerequisites are that the sample molecules can be transferred to the gas phase and ionized intact, and that they can be induced to break in some predictable and controllable manner after the first mass selection stage. Multi-stage MS / MS, or MS n can be performed by first selecting and separating precursor ions (MS 2 ) and fragmenting them, separating the primary fragment ions (MS 3 ) and fragmenting them, separating the secondary fragment ions (MS 4 ), etc., as long as meaningful information can be obtained and fragment ion signals can be detected. Tandem MS has been successfully performed using a variety of combinations of analyzers. What analyzers are combined for a particular application is 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 there are also hybrids in which a temporal tandem analyzer is connected spatially or to 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 spectrometers. The specific m / z separation function can be designed such that ions are selected in one region of the device, 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 at the ion source can be trapped, separated, fragmented, and m / z separated within the same physical device.

[0121] Peptides identified by a mass spectrometer can be used as surrogates for intact proteins and their post-translational modifications. These can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter being generated from possible peptides within a protein sequence database. 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 homology analysis, or combinations thereof.

[0122] As used herein, the term "database" refers to a bioinformatics tool that provides the possibility to search uninterpreted MS-MS spectra against all possible sequences within 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 The disclosed embodiments 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 eluate containing the protein, and characterizing the protein in the eluate using an electrospray ionization mass spectrometer.

[0125] The present disclosure provides a method for characterizing an antibody-drug conjugate, comprising 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 eluate containing the antibody-drug conjugate, and characterizing the antibody-drug conjugate in the eluate using an electrospray ionization mass spectrometer.

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

[0127] In some exemplary embodiments, the chromatography system can include conventional reverse 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 nanoelectrospray ionization mass spectrometer.

[0130] In some exemplary embodiments, the electrospray ionization mass spectrometer can be coupled to a chromatography system having a chromatography 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 the 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 both the electrospray ionization mass spectrometer and the ultraviolet detector using a three-way splitter.

[0135] In some exemplary embodiments, the chromatography system can be coupled to both the electrospray ionization mass spectrometer and the ultraviolet detector using a three-way splitter, and the electrospray ionization mass spectrometer is a nanoelectrospray 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, and the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer under non-denaturing conditions.

[0138] In some exemplary embodiments, the eluent containing the protein or antigen-antibody complex or antibody-drug complex from the resin wash 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 mobile phase for washing 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 contain volatile salts. In some specific embodiments, the mobile phase may contain ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

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

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

[0143] In some exemplary embodiments, the sample can 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 an 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 from about 0.8 kV to about 1.5 kV.

[0146] In some exemplary embodiments, characterization can include identification and / or quantification of proteins. In one aspect, characterization can include protein sequencing, de novo protein sequencing, identification of post-translational modifications, or homology analysis, or combinations thereof. In another aspect, characterization can include quantification of the relative abundance of proteins.

[0147] In some exemplary embodiments, characterization can include identification and / or quantification of antibodies in an antibody-drug conjugate. In one aspect, characterization can include protein sequencing, de novo protein sequencing, identification of post-translational modifications, or homology analysis, or combinations thereof. In another aspect, characterization can include quantification of the relative abundance of antibodies in the antibody-drug conjugate.

[0148] In some exemplary embodiments, characterization can include identification and / or quantification of antibodies and / or antigens in an antigen-antibody complex. In one aspect, characterization can include protein sequencing of the antibody or antigen, de novo protein sequencing, identification of post-translational modifications, or homology analysis, or combinations thereof. In another aspect, characterization can include quantification of the relative abundance of antibodies and / or antigens in the antigen-antibody complex.

[0149] In some exemplary embodiments, the sample can contain 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, non-natural amino acid, glycan, 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, antibody, monoclonal antibody, polyclonal antibody, bispecific antibody, antibody fragment, fusion protein, or a combination thereof. In one aspect, the antibody fragments may include Fab fragments, Fab’ fragments, F(ab’)2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd’ fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, triabodies, tetra-bodies, 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 can be formed by a hydrolyzing agent. The digestion product may be an impurity associated with the product.

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

[0155] In some exemplary embodiments, the protein can have a pI in the range of 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 can include at least two proteins.

[0157] It is understood that the method is not limited to any of the proteins, impurities, and columns described above, and the method of identification or quantification can 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, and the chromatography column can receive a mobile phase and a sample containing a protein (see FIG. 2).

[0159] In some exemplary embodiments, the chromatography column 100 can 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, the 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 made to operate under native conditions.

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

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

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

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

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

[0167] In some exemplary embodiments, the three-way splitter 120 can be made to divide the flow from the chromatography column 100 to the ultraviolet detector 130 and the electrospray ionization mass spectrometer 110 unevenly.

[0168] In some exemplary embodiments, the system can be made to characterize the drug-to-antibody ratio of an antibody-drug conjugate.

[0169] In some exemplary embodiments, the system can be made to characterize a protein.

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

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

[0172] Another figure of the setup according to an exemplary embodiment is shown in Figure 4.

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

[0174] The sequential labeling by numbers and / or letters of the steps of the methods shown herein is not intended to limit the method or any of its embodiments to the particular order shown.

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

[0176] This disclosure will be more fully understood by reference to the following examples, which are shown to explain the disclosure 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 capillaries (inner diameter (ID) 150 μm, outer diameter (OD) 360 μm), three-way connectors, and sleeves were purchased from IDEX (Oak Harbour, 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 additional 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). The 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 mAb and ADC. The mobile phase was 100 mM ammonium acetate at pH 6.8. Each separation was performed for 30 minutes at a flow rate of 0.3 mL / min, and the injection volume was set at 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 large fraction was transferred to the UV detector via the fused silica tube, and the small fraction was directed to the MS via the SilicaTip. The following MS parameters were used for online SEC-nano-ESI-MS data acquisition. Each acquisition was 25 minutes starting immediately after sample injection. The sample was ionized in positive mode at a spray voltage of 3 kV, a capillary temperature of 200 °C, and an RF level of the 70S lens. The in-source CID was set at 75 eV. The full MS scan was acquired at a resolution of 15K in the mass range of m / z 2000 - 8000. For the full MS scan, a maximum injection time of 100 milliseconds, an automatic acquisition control target value of 3e6, and 10 microscans were used.

[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 ADC.

[0180] Example 1. Investigation of antigen-antibody interaction using online SEC-nano-ESI-MS To develop an effective antibody therapy, it may be important to understand how antibody binding affects the function of the target protein.

[0181] 1.1 Online SEC-Nano-ESI-MS apparatus SEC and MS technologies are routinely used to characterize protein samples. SEC enables the separation and characterization of proteins under conditions that minimize structural changes in the proteins, while MS enables the identification of individual components in complex samples. Although it is highly desirable to combine the individual functions of SEC and MS on a single platform, it has been found to be difficult because the high flow rates and non-volatile salts used in SEC analysis are incompatible with native MS. To overcome this limitation, the flow of the eluent from SEC was split using a post-column T-splitter to reduce the uptake of solvent and salts into the MS (see Figure 3). The setup is shown in Figure 4. Subsequently, the T-splitter was connected to the MS via a SilicaTip and simultaneously to a UV detector via a fused silica tube. This arrangement enabled simultaneous dual UV / MS detection of the SEC eluent. By varying the length and diameter of the fused silica tube, the flow rate to the MS via the SilicaTip can be adjusted (e.g., a longer / narrower tube can create more resistance and increase the flow rate to the SilicaTip and MS). The protein sample was separated on a 4.6 mm SEC column using a flow rate of 0.3 mL / min. A fused silica tube with a length of 140 cm and an inner diameter of 150 μm connecting the T-splitter and the UV detector resulted in a desirable flow rate of approximately 1 μL / min to the SilicaTip. The length and diameter of the fused silica tube also enabled nearly synchronous detection of molecules by UV and MS.

[0182] 1.2 Antigen-antibody complex Recombinant Bet v1 and previously reported antibodies against Bet v1 antigen (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)) formed a complex under native MS conditions.

[0183] In addition to the naked form, as seen in Figure 5, two major glycosylated forms, including G2S1F and G2S2F, were observed for Bet v1. After incubation with an equimolar amount 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 Figure 5A, from the deconvoluted native MS spectrum of Bet v1 antigen alone, three different Bet v1 species were revealed: naked Bet v1, and the two major glycosylated forms, Bet v1 G2S1F and G2S2F. Incubation of Bet v1 antigen with an equimolar amount of Bet v1 Fab-1 antibody showed that all three Bet v1 forms (naked, G2S1F, and G2S2F) formed a complex with Fab-1 at a 1 antigen to 1 Fab ratio (Figure 5B). Since the relative abundances of the MS signals for all Bet v1 forms were similar between the unbound and Fab-1-bound conditions, glycosylation does not seem to affect the formation of the antibody / antigen complex.

[0184] To further characterize the antigen-antibody interaction, titration experiments were performed on a dual detection platform of MS and UV. As shown in Figure 6, multiple different ratios of Bet v1 to Fab-1 were examined by UV. Bet v1 alone (black) eluted at 8.5 minutes, Fab-1 alone (teal), and the Bet v1:Fab-1 complex eluted at 9.8 minutes and 7.9 minutes, respectively. From the stoichiometry of the Bet v1:Fab-1 complex, a binding ratio of 1:1 was revealed. No additional stoichiometric ratios were observed where either the antigen or the antibody was in excess. Furthermore, when the Bet v1 antigen was mixed with Fab-1 in an exact 1:1 molar ratio, the amounts of free antigen and antibody were minimized (blue).

[0185] While the UV peak can represent the relative abundance of individual proteins in solution, MS enables the identification of individual components in complex samples. Dual detection by UV and MS allows determination of all species co-eluting within the same or different UV peaks. As shown in Figure 7, MS analysis of the UV peak eluting at 7.9 minutes revealed three different complexes formed by Fab-1 bound to each of three different Bet v1 species (naked, G2S1F, and G2S2F). The inventors found that the Fab-1:naked complex eluted after the forms of Fab-1 in complex with the glycosylated Bet v1 species. This may be due to the larger hydrodynamic radius imparted by the glycan. By minimizing the detection delay between UV and MS, fractions from the UV detector could be collected for sample recovery. This method is particularly useful for determining whether cleavage, modification, or variants of the antigen and / or antibody retain binding without the need to purify specific forms of the protein under study.

[0186] Example 2. Characterization of Cysteine ADC Using On-line SEC-Nano-ESI-MS Antibody-drug conjugates (ADCs) are a very powerful therapeutic modality capable of specifically delivering small molecule drugs to target tissues through binding to an antibody (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 an ADC can strongly depend on the number of small molecule drugs conjugated to each antibody. Therefore, it may be important to determine the drug-to-antibody ratio (DAR) of each ADC. Conjugation via interchain disulfides is one of the most common methods for attaching small molecule drugs to an antibody. In interchain disulfide-based ADCs, unpaired interchain cysteine residues can be introduced by engineering primary sequence mutations (site-specific interchain disulfide-based conjugates) or by partially reducing the antibody (random interchain disulfide-based conjugates). Site-specific conjugation of engineered mAbs better controls the DAR of the ADC, while conjugation with partially reduced mAbs yields a larger variable range of DARs (0-8). However, to understand and interpret the biological effects of these drug conjugates, it may still be necessary to determine the DAR of the ADCs generated by either method. The most common methods for determining 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 by RPLC-MS under reducing / denaturing conditions.However, recently, Debaene et al. reported an offline desalting SEC method combined 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 intact molecules.

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

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

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

[0190] An online size-exclusion chromatography-nanoelectrospray ionization (nano-ESI)-MS platform with dual detection of ultraviolet (UV) and MS was developed. The utility of this platform was verified by using it to investigate non-covalent protein interactions through the characterization of antigen-antibody complexes obtained from titration experiments and the determination of the drug-to-antibody ratio (DAR) of cysteine-based antibody-drug conjugates (ADCs). This platform can be easily modified and thus adapted for the analysis of other native MS projects, such as the characterization of charge variants of monoclonal antibodies (mAbs) or large aggregated protein complexes.

[0191] A three-way splitter was used to divide the SEC eluate unevenly between the MS and UV detectors, directing a small fraction to the MS and a large fraction to the UV detector. This platform enables 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 disulfide-linked ADCs. Further modification of this online SEC-nano-ESI-MS platform, such as changing the column chemistry or using a QExactive UHMR instrument, can adapt it for other applications such as the analysis of charge variants and very large protein complexes. The methods described herein have opened up 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 will be widely applicable to the analysis of protein biopharmaceuticals for various applications.

Claims

1. 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 an electrospray ionization mass spectrometer (ESI-MS) under non-denaturing conditions and using an ultraviolet (UV) detector, wherein the ESI-MS and the UV detector detect the antibody-drug conjugate in the eluate approximately synchronously. A method for characterizing an antibody-drug conjugate, comprising: The method, wherein the chromatography system having the size exclusion chromatography resin is coupled on-line to both the mass spectrometer and the ultraviolet detector by a splitter for near-synchronous ESI-MS and UV detection.

2. The method of claim 1, further comprising the step of recovering a fraction of the eluent from the UV detector to recover a sample.

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

4. 2. The method of claim 1, wherein at least one T-splitter is used to online couple the chromatography system having the size exclusion chromatography resin to both the mass spectrometer and the ultraviolet detector.

5. 5. The method of claim 4, wherein the eluent from the step of washing the size exclusion chromatography resin is introduced to the ultraviolet detector through at least one T-splitter at a flow rate of about 0.2 mL / min to about 0.4 mL / min.

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

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

8. 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.

9. 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.

10. 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.

11. 2. The method of claim 1, wherein the amount of the sample containing the antibody-drug conjugate contacted with the chromatography system is from about 10 μg to about 100 μg.

12. 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.

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

14. The method of claim 1, wherein the antibody-drug conjugate is an engineered cysteine-based antibody-drug conjugate.

15. The method of claim 1, wherein the antibody-drug conjugate is a non-specific cysteine-based antibody-drug conjugate.

16. 2. The method of claim 1, wherein the step of characterizing the antibody-drug conjugate comprises characterizing the drug-to-antibody ratio.

17. a chromatography column having a size exclusion chromatography resin, the chromatography column being capable of accepting a mobile phase and a sample comprising a protein; an electrospray ionization mass spectrometer configured to operate under non-denaturing conditions; and UV detector A system comprising: the chromatography column is directly coupled on-line to both the electrospray ionization mass spectrometer and the ultraviolet detector by a splitter; and The system, wherein the splitter, an electrospray ionization mass spectrometer (ESI-MS), and an ultraviolet (UV) detector are configured to detect eluent from the chromatography column with substantially synchronized ESI-MS and UV detection.

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