Affinity chromatography-coupled native mass spectrometry for antibody analysis

Abstract

To provide a high throughput analysis method and system for improving a manufacturing process of biopharmaceutical products.SOLUTION: There is provided rapid, sensitive high-throughput methods and systems for characterizing peptides or proteins using affinity-based chromatography-coupled native mass spectrometry to improve manufacturing process of biopharmaceutical products, such as identifying impurities during antibody purification, monitoring post-translational modification variants during production, or characterizing drug-to-antibody ratio of antibody-drug conjugates. The separation profiles of the peptides or proteins are generated and compared to identify or qualify the peptides or proteins, where the separation profile is based on differential affinity binding.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Field The present invention relates generally to methods and systems for characterizing peptides or proteins using affinity-based chromatography coupled native mass spectrometry. The present invention provides rapid, sensitive, and high-throughput methods and systems for characterizing peptides or proteins. [Background technology]

[0002] background Therapeutic peptides or proteins are typically expressed in cell culture suspension for production. The peptides or proteins are then purified to remove process-related impurities. The product quality attributes of the purified therapeutic peptides or proteins are extensively characterized to ensure preservation of their associated safety, efficacy, and shelf-life profiles related to pharmacokinetics.

[0003] Modifications of therapeutic peptides or proteins can occur at any time during and after the peptide or protein is produced and / or purified. Therapeutic peptides or proteins can be heterogeneous due to various post-translational modifications, proteolysis, enzymatic modifications, and chemical modifications. These modifications to the biophysical characteristics of biopharmaceutical products can affect their associated safety, efficacy, and shelf life.

[0004] It will be appreciated that there is a need to develop high-throughput analytical methods and systems that provide insights for improving biopharmaceutical product manufacturing processes. It is highly desirable that analytical methods be able to be performed in a short time to achieve rapid, sensitive, high-throughput analytical tools, as these methods provide significant improvements for controlling the manufacturing and purification of high-quality biopharmaceutical products. Summary of the Invention

[0005] overview Developing high-throughput analytical methods and systems can be important for improving biopharmaceutical manufacturing processes by monitoring the production and purification of biopharmaceutical products. The present disclosure provides methods and systems that meet the aforementioned needs by providing rapid, sensitive, high-throughput analytical methods and systems based on affinity-based chromatography coupled to native mass analysis to improve biopharmaceutical manufacturing processes.

[0006] The present disclosure provides a method for identifying at least one peptide or protein in a sample, the method comprising the steps of: contacting the sample with a solid surface, wherein the solid surface comprises an affinity binding molecule for the at least one peptide or protein; washing the solid surface with a mobile phase to generate at least one eluate, wherein the eluate comprises the at least one peptide or protein; and characterizing the at least one peptide or protein in the at least one eluate under native conditions using a mass spectrometer.

[0007] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises generating at least one separation profile.

[0008] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises identifying or quantifying the at least one peptide or protein based on the at least one separation profile.

[0009] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises identifying or quantifying a level of post-translational modification or post-translational modification diversity of the at least one peptide or protein based on the at least one separation profile or comparison with another separation profile.

[0010] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises identifying or quantifying a level of glycosylation or glycosylation diversity of the at least one peptide or protein based on the at least one separation profile or comparison with another separation profile, wherein the glycosylation is terminal galactose, Fc glycan occupancy, core fucose, bisecting GlcNAc, or Man5.

[0011] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises separating or identifying an impurity in the sample based on at least one separation profile or comparison with another separation profile.

[0012] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further includes the at least one peptide or protein being a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0013] In some embodiments, the method for identifying at least one peptide or protein in a sample further comprises quantifying the drug-to-antibody ratio of the antibody-drug complex based on at least one separation profile or comparison with another separation profile.

[0014] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises a chromatography column comprising a solid surface and affinity binding molecules for at least one peptide or protein.

[0015] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises a mass spectrometer directly coupled to the chromatography column.

[0016] In some exemplary embodiments, the at least one peptide or protein affinity binding molecule is protein A, protein G, an Fcγ receptor, FcγRIIIa, an anti-human Fc antibody, a fetal Fc receptor, Fc epsilon RI, an anti-idiotypic antibody, or complement component C1q.

[0017] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises a splitter used to connect the mass spectrometer and the chromatography column.

[0018] In some embodiments, the method for identifying at least one peptide or protein in a sample further comprises a splitter used to split the lower flow rate to a mass spectrometer and the higher flow rate to a detector.

[0019] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises a mobile phase that is an acidic solution, and the resulting eluate is characterized using a mass spectrometer under native conditions without pretreatment.

[0020] In some embodiments, the method for identifying at least one peptide or protein in a sample further comprises a mobile phase comprising ammonium acetate, acetic acid, or a combination thereof.

[0021] In other aspects, the method for identifying at least one peptide or protein in a sample further comprises a mobile phase used to wash the chromatography column and having a flow rate of about 0.2 to 0.6 mL / min.

[0022] In other embodiments, the method for identifying at least one peptide or protein in a sample further comprises a mass spectrometer that is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, a quadrupole mass spectrometer, or an ultra-high mass range hybrid quadrupole mass spectrometer.

[0023] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises an orbitrap mass spectrometer.

[0024] The present disclosure provides, at least in part, a system for identifying at least one peptide or protein, the system including: a sample containing at least one peptide or protein; a chromatography column containing an affinity binding molecule for the at least one peptide or protein, the chromatography column being washable with a mobile phase to produce an eluate; and a mass spectrometer capable of characterizing or quantifying the at least one peptide or protein, the mass spectrometer operating under native conditions and being directly coupled to the chromatography column.

[0025] In some embodiments, the system for identifying at least one peptide or protein further comprises a splitter used to connect the mass spectrometer and the chromatography column.

[0026] In some embodiments, the system for identifying at least one peptide or protein further comprises a splitter used to split the lower flow rate to a mass spectrometer and the higher flow rate to a detector.

[0027] In some exemplary embodiments, the system for identifying at least one peptide or protein further comprises a mobile phase that is an acidic solution, and the resulting eluate is characterized using a mass spectrometer without pretreatment.

[0028] In some embodiments, the system for identifying at least one peptide or protein further comprises a mobile phase comprising ammonium acetate, acetic acid, or a combination thereof.

[0029] In other embodiments, the system for identifying at least one peptide or protein further comprises a mobile phase having a flow rate of about 0.2 to 0.6 mL / min.

[0030] In some exemplary embodiments, the system for identifying at least one peptide or protein includes a diode array detector or a photodiode array detector.

[0031] In some exemplary embodiments, the system for identifying at least one peptide or protein further includes at least one peptide or protein that is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0032] In some embodiments, the system for identifying at least one peptide or protein further comprises a mass spectrometer that is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, or an ultra-high mass range hybrid quadrupole mass spectrometer.

[0033] In some exemplary embodiments, the system for identifying at least one peptide or protein further comprises an Orbitrap mass spectrometer.

[0034] In some exemplary embodiments, the system for identifying at least one peptide or protein comprises at least one peptide or protein affinity binding molecule, wherein the at least one peptide or protein affinity binding molecule is Protein A, Protein G, an Fcγ receptor, FcγRIIIa, an anti-human Fc antibody, a fetal Fc receptor, Fc epsilon RI, an anti-idiotypic antibody, or complement component C1q.

[0035] [The present invention 1001] 1. A method for identifying at least one peptide or protein in a sample, comprising: contacting the sample with a solid surface, the solid surface comprising an affinity binding molecule for the at least one peptide or protein; washing the solid surface with a mobile phase to produce at least one eluate, wherein the eluate comprises the at least one peptide or protein; characterizing said at least one peptide or protein in said at least one eluate using a mass spectrometer under native conditions; A method comprising: [The present invention 1002] 1001. The method of claim 1001, further comprising generating at least one separation profile. [The present invention 1003] 1002. The method of claim 10, further comprising identifying or quantifying said at least one peptide or protein based on said at least one separation profile. [The present invention 1004] The method of claim 1002, further comprising identifying or quantifying the level of post-translational modification or post-translational modification diversity of said at least one peptide or protein based on said at least one separation profile or comparison with another separation profile. [The present invention 1005] The method of claim 1002, further comprising identifying or quantifying the level of glycosylation or glycosylation diversity of said at least one peptide or protein based on said at least one separation profile or comparison with another separation profile. [The present invention 1006] 1005. The method of claim 10, wherein said glycosylation is terminal galactose, Fc glycan occupancy, core fucose, bisecting GlcNAc, or Man5. [The present invention 1007] 1002. The method of claim 10, further comprising the step of separating or identifying impurities in said sample based on said at least one separation profile or comparison with another separation profile. [The present invention 1008] 1002. The method of claim 1002, wherein said at least one peptide or protein is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product. [The present invention 1009] 1009. The method of claim 10, further comprising the step of quantifying a drug-to-antibody ratio of said antibody-drug conjugate based on said at least one separation profile or comparison with another separation profile. [The present invention 1010] 1001. The method of claim 10, wherein said solid surface comprising said affinity binding molecule of said at least one peptide or protein is comprised in a chromatography column. [The present invention 1011] 10. The method of claim 10, wherein said mass spectrometer is directly coupled to said chromatography column. [The present invention 1012] 1001. The method of claim 1001, wherein said affinity binding molecule of said at least one peptide or protein is protein A, protein G, an Fc gamma receptor, Fc gamma RIIIa, an anti-human Fc antibody, a fetal Fc receptor, Fc epsilon RI, an anti-idiotypic antibody, or complement component C1q. [The present invention 1013] The method of claim 1010, wherein a splitter is used to connect said mass spectrometer and said chromatography column. [The present invention 1014] 1013. A method according to claim 1013, wherein said splitter is used to split a lower flow rate to said mass spectrometer and a higher flow rate to a detector. [The present invention 1015] 1001. The method of claim 1001, wherein said mobile phase is an acidic solution and said eluate is characterized using said mass spectrometer under native conditions without pretreatment. [The present invention 1016] 1001. The method of claim 1001, wherein said mobile phase comprises ammonium acetate, acetic acid, or a combination thereof. [The present invention 1017] 10. The method of claim 10, wherein the mobile phase is used to wash the chromatography column and has a flow rate of about 0.2 to 0.6 mL / min. [The present invention 1018] 1001. The method of claim 1001, wherein said mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, a quadrupole mass spectrometer, or an ultra-wide mass range hybrid quadrupole mass spectrometer. [The present invention 1019] 1001. The method of claim 1001, wherein said mass spectrometer comprises an Orbitrap mass spectrometer. [The present invention 1020] 1. A system for identifying at least one peptide or protein, comprising: a sample comprising said at least one peptide or protein; a chromatography column comprising the at least one peptide or protein affinity binding molecule, the chromatography column being capable of being washed with a mobile phase to produce an eluate; a mass spectrometer capable of characterizing or quantifying said at least one peptide or protein, said mass spectrometer being capable of running under native conditions and being directly coupled to said chromatographic column; Including, the system. [The present invention 1021] The system of the present invention 1020, wherein a splitter is used to connect said mass spectrometer and said chromatography column. [The present invention 1022] 1021. The system of claim 1021, wherein the splitter is used to split a lower flow rate to the mass spectrometer and a higher flow rate to a detector. [The present invention 1023] The system of the present invention 1020, wherein the mobile phase is an acidic solution and the eluate is characterized using the mass spectrometer under native conditions without pretreatment. [The present invention 1024] The system of the present invention 1020, wherein the mobile phase comprises ammonium acetate, acetic acid, or a combination thereof. [The present invention 1025] The system of the present invention 1020, wherein the mobile phase has a flow rate of about 0.2 to 0.6 mL / min. [The present invention 1026] The system of the present invention 1020 includes a diode array detector or a photodiode array detector. [The present invention 1027] The system of the present invention 1020, wherein said at least one peptide or protein is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product. [The present invention 1028] The system of the present invention 1020, wherein said mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, or an ultra-wide mass range hybrid quadrupole mass spectrometer. [The present invention 1029] The system of the present invention 1020, wherein the mass spectrometer comprises an Orbitrap mass spectrometer. [The present invention 1030] The system of the present invention 1020, wherein said affinity binding molecule of said at least one peptide or protein is protein A, protein G, Fc gamma receptor, Fc gamma RIIIa, anti-human Fc antibody, fetal Fc receptor, Fc epsilon RI, anti-idiotypic antibody, or complement component C1q. These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments thereof and numerous specific details, is given by way of illustration and not limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief explanation of the drawings]

[0036] [Figure 1A] Figure 1 shows multiple binding sites in the molecular structure of an antibody for affinity intermolecular interactions and binding affinity values ​​between an antibody and a biological molecule. [Figure 1B] 1 shows a bispecific antibody and its parent monospecific antibody subjected to characterization or purification according to exemplary embodiments. The bispecific antibody format involves pairing two different heavy chains with two common light chains, which allows for two unique antigen-binding sites that target two different antigens according to exemplary embodiments. One arm of the heavy chain of the bispecific antibody has two amino acid substitutions, e.g., a substitution of RF for HY, referred to as a star substitution or Fc* according to exemplary embodiments. [Figure 2] Figure 2A shows a system in which a mass spectrometer is directly coupled to an affinity-based chromatography column, and in accordance with an exemplary embodiment, a splitter is used to connect the mass spectrometer and the affinity-based chromatography column. Figure 2B shows the pH range and profile during separation, such as in screening and resolution modes, in accordance with an exemplary embodiment. Mass spectrometry-compatible mobile phases containing ammonium acetate and / or acetic acid were used for separation in accordance with an exemplary embodiment. [Figure 3] Figure 3A shows raw mass spectrometry data from screening NISTmAb reference material using rapid pH gradient elution without sample pretreatment according to an exemplary embodiment. Figure 3B shows raw mass spectrometry data deconvoluted using INTACT MASS™ software according to an exemplary embodiment. The diversity of post-translational modifications of NISTmAb was characterized according to an exemplary embodiment. [Figure 4]

[0033] Figure 1 shows the results of evaluating variants of NISTmAb under oxidative stress in the presence of approximately 0.005% to 0.05% (v / v) hydrogen peroxide (HO). The treated NISTmAb was then analyzed by the Protein A chromatography-coupled native mass spectrometry method and system of the present application according to an exemplary embodiment. [Figure 5] 1 shows the results of separating and identifying components in a mixture containing a bispecific antibody and its parent monospecific antibody using the Protein A chromatography coupled native mass spectrometry method and system of the present application according to an exemplary embodiment. [Figure 6]

[0023] Figure 1 shows the results of characterizing the drug-to-antibody ratio (DAR) of a lysine-linked antibody-drug conjugate using the Protein A chromatography-coupled native mass spectrometry method and system of the present application according to an exemplary embodiment. The top figure shows the raw mass spectrum. The bottom figure shows the deconvoluted mass spectrum. [Figure 7] 1 shows a side-by-side comparison characterizing the drug-to-antibody ratio (DAR) of lysine-linked antibody-drug conjugates using Protein A chromatography-coupled native mass spectrometry methods and systems of the present application, native SEC-MS (size exclusion chromatography-mass spectrometry), and RPLC-MS (reverse phase liquid chromatography-mass spectrometry) according to exemplary embodiments. The comparison is demonstrated by corresponding extracted ion chromatograms (XICs) according to exemplary embodiments. [Figure 8] 1 shows results of characterizing the drug-to-antibody ratio (DAR) of cysteine-linked antibody-drug conjugates using Protein A chromatography-coupled native mass spectrometry methods and systems of the present application and SEC-MS (size exclusion chromatography-mass spectrometry) according to exemplary embodiments. [Figure 9] 1 shows the results of characterizing cysteine-linked antibody-drug conjugates using the Protein A chromatography-coupled native mass spectrometry method and system of the present application and SEC-MS (size exclusion chromatography-mass spectrometry) on released light chains according to an exemplary embodiment. [Figure 10-1]1 shows the detection of MAB4 in cell culture time course samples using Protein A chromatography coupled to native mass spectrometry (ProA-MS) of the present application according to an exemplary embodiment. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11] 10 shows the results of analyzing cell culture time course samples containing MAB4 for glycoform changes over the cell culture cycle using ProA-MS of the present application according to an exemplary embodiment. [Figure 12-1] 1 shows the detection of MAB5 in cell culture time course samples using ProA-MS of the present application according to an exemplary embodiment. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13] 1 shows the results of analyzing cell culture time course samples containing MAB5 for glycoform changes over the cell culture cycle using ProA-MS of the present application according to an exemplary embodiment. [Figure 14-1] 1 shows high speed screening of NISTmAb reference material with baseline resolution of glycoforms and accurate mass measurement using the FcγRIIIa chromatography coupled native mass spectrometry (FcγRIIIa-MS) method and system of the present application according to an exemplary embodiment. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15] 1 shows quantification of FcγRIIIa-MS binding affinity of various IgG formats by relative retention time according to an exemplary embodiment. [Figure 16] 1 shows an analysis of NISTmAb using the FcγRIIIa-MS of the present application to determine the effect of terminal galactose on affecting FcγRIIIa binding according to an exemplary embodiment. [Figure 17-1] 1 shows the analysis of MAB8 (IgG4) using FcγRIIIa-MS of the present application to study the effect of Fc glycan occupancy in IgG on FcγRIIIa binding, according to an exemplary embodiment. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1] 1 shows the analysis of MAB10 (IgG4S) using FcγRIIIa-MS of the present application to study the effect of Fc glycan occupancy in IgG on FcγRIIIa binding according to an exemplary embodiment. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19-1] 1 shows the analysis of MAB8 (IgG4) using FcγRIIIa-MS of the present application to study the influence of core fucose in IgG on affecting FcγRIIIa binding, according to an exemplary embodiment. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20-1] 1 shows the analysis of MAB9 (IgG4) using FcγRIIIa-MS of the present application to study the effect of bisecting GlcNAc on FcγRIIIa binding according to an exemplary embodiment. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 21] 1 shows the analysis of MAB8 (IgG4) using FcγRIIIa-MS of the present application to study the effect of Man5 on affecting FcγRIIIa binding according to an exemplary embodiment. [Figure 22] 1 shows the analysis of MAB9 C1P2 Lot A and MAB9 C2P1 Lot B using the FcγRIIIa-MS of the present application investigating glycan-based separation for intact mass analysis under native conditions according to an exemplary embodiment. [Figure 23A] 1 shows an analysis of MAB9 C1P2 DS Lot A in comparing FcγRIIIa-MS and RPLC-MS of the present application for glycan-based separation according to an exemplary embodiment. [Figure 23B-1] 1 shows the analysis of MAB9 C1P2 DS Lot A using the FcγRIIIa-MS of the present application in comparing FcγRIIIa-MS and RPLC-MS for glycan-based separation according to an exemplary embodiment. [Figure 23B-2] This is a continuation of Figure 23B-1. [Figure 24A](FIG. 24A) Analysis of MAB9 C2P1 FDS lot B when comparing FcγRIIIa-MS and RPLC-MS for glycan-based separation according to an exemplary embodiment. (FIG. 24B) Analysis of MAB9 C2P1 FDS lot B using the FcγRIIIa-MS of the present application when comparing FcγRIIIa-MS and RPLC-MS for glycan-based separation according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description The generation and manufacturing of biopharmaceutical products encompasses a variety of processes and technologies. Following expression and production of a therapeutic peptide or protein in cell culture suspension, the peptide or protein can be purified to remove process-related impurities. The purified therapeutic peptide or protein can be extensively characterized to ensure preservation of their associated safety, efficacy, and shelf-life profiles related to pharmacokinetics and product quality attributes.

[0038] Therapeutic peptides or proteins can be heterogeneous due to various post-translational modifications (PTMs), proteolysis, enzymatic modifications, and chemical modifications that can be introduced at any time during or after the production and purification of the peptide or protein. Identification and characterization of heterogeneous variants is important for controlling the quality attributes of biophysical characteristics of biopharmaceutical products. There is a need in the biopharmaceutical industry for rapid, sensitive, and high-throughput analytical methods to control and monitor the production and purification of therapeutic peptides or proteins, such as the production of monoclonal antibodies or antibody-drug conjugates.

[0039] Bispecific antibodies are highly valuable biopharmaceutical products because they can target two different antigens. Bispecific antibody design can target multiple tissue-specific antibodies in combination with the use of small molecule drugs, such as combining multiple tissue-specific antibodies with a cytotoxic drug to release the drug in close proximity to the tumor. Small drug molecules can be conjugated to purified bispecific antibodies to generate antibody-drug conjugates (ADCs). Expression and purification of bispecific antibodies can be challenging due to the need to remove impurities, such as the parent monospecific antibody. Monitoring and determining the drug-to-antibody ratio of ADCs is important for quality control of ADCs.

[0040] The present disclosure provides methods and systems that meet the aforementioned needs by providing high-throughput analytical methods and systems based on affinity-based chromatography coupled to native mass spectrometry to improve biopharmaceutical product manufacturing processes, such as identifying impurities during antibody purification, monitoring post-translational modification variants during production, or characterizing the drug-to-antibody ratio of antibody-drug conjugates. In particular, the analytical methods and systems of the present application can be sensitive and can be performed in a short time to achieve rapid, sensitive, high-throughput analytical tools to provide significant improvements in the control of biopharmaceutical product production and purification.

[0041] Native mass spectrometry is an approach to studying intact biomolecular structures in their native or near-native state. The term "native" refers to the biological state of the analyte in solution before undergoing ionization. Several parameters, such as the pH and ionic strength of the solution containing the biological analyte, can be controlled to maintain the native folding state of the biological analyte in solution. Generally, native mass spectrometry is based on electrospray ionization, in which the biological analyte is sprayed from a non-denaturing solvent. Other terms, such as non-covalent, native spray, electrospray ionization, non-denaturing, macromolecular, or supramolecular mass spectrometry, can also refer to native mass spectrometry. (Leney et al., J. Am. Soc. Mass Spectrom, 2017, 28, pages 5-13, Native Mass Spectrometry: what is in the name)

[0042] The present application provides affinity-based chromatographic separation coupled with native mass spectrometry, which provides a powerful analytical tool for rapid, sensitive, high-throughput screening or identification of peptides or proteins. In some embodiments, the high-throughput analytical methods and systems of the present application are based on a rapid direct coupling approach that couples an affinity-based chromatographic column to a mass spectrometer. In the methods and systems of the present application, peptide or protein separation profiles can be generated based on differential affinity binding, such as differential Protein A affinity binding or differential FcγRIIIa affinity binding, and mass spectrometry can then be used to characterize the intact biomolecular structure of the peptides or proteins in their native or near-native state.

[0043] In exemplary embodiments, affinity-based chromatography can involve various affinity intermolecular interactions between biological molecules with various binding affinity values, such as affinity interactions involving multiple binding sites in the molecular structure of an antibody, including binding sites for Protein A, Protein G, Fcγ receptor (FcγR), complement component C1q, or fetal Fc receptor (FcRn), as shown in FIG. 1A (Irani et al., Molecular Immunology, 67 (2015) 171-182; Guilliams et al., Nature Reviews Immunology 14 (2014) 94-108).

[0044] In certain embodiments, the affinity-based chromatography is Protein A chromatography, Protein G chromatography, Fcγ receptor (FcγR) chromatography, FcγRIIIa chromatography, anti-human Fc antibody chromatography, fetal Fc receptor (FcRn) chromatography, Fc epsilon RI (FcεRI) chromatography, anti-idiotypic antibody chromatography, or complement component C1q chromatography.

[0045] The fragment crystallizable (Fc) region of antibodies interacts with various molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) (Irani et al.). Among these affinity molecular interactions, FcγRs are involved in ADCC, an immune mechanism in which Fc receptor-bearing effector cells recognize and kill antibody-coated target cells expressing tumor- or pathogen-derived surface antigens. Because the natural killer cell FcγRIIIa receptor can recognize cell-bound antibodies, signaling through FcγRIIIa can trigger the release of cytokines and cytotoxic granules that mediate tumor cell apoptosis. Modulating antibody interactions through FcγRIIIa can contribute to cancer immunotherapy. As shown in Figure 1A, FcγRs exhibit different binding affinity values ​​for different IgG subclasses, so one immunotherapeutic approach is to enhance ADCC functionality by modifying the affinity binding of the Fc region to increase the binding affinity of activating FcγRIIIa. These approaches include site-directed mutagenesis, modifying the glycosylation of the Fc domain, or removing fucosylation from the Fc domain.

[0046] The present application provides a direct FcγRIIIa affinity chromatography coupled to a native mass spectrometry method for rapidly assessing FcγRIIIa affinity or ADCC activity, which may vary across various IgG formats. In some exemplary embodiments, IgG1 (fucose minus) has the greatest FcγRIIIa affinity or ADCC activity, followed by IgG1, IgG4, or IgG4S (e.g., IgG1 (fucose minus) > IgG1 > IgG4 > IgG4S). Different glycoforms and species with different glycan occupancies can be separated and characterized using the FcγRIIIa affinity chromatography coupled to a native mass spectrometry method and system of the present application. In some aspects, increased glycan occupancy, increased terminal galactose, or reduced core fucose resulted in increased FcγRIIIa affinity or ADCC activity. In some aspects, reduced bisecting GlcNAc or increased Man5 resulted in reduced FcγRIIIa affinity or ADCC activity. The FcγRIIIa affinity chromatography coupled native mass spectrometry methods and systems of the present application can reduce sample complexity to provide glycan-based separations.

[0047] Among these affinity molecular interactions, Protein A affinity binding can be used to facilitate antibody purification or separation. Substitution of two amino acids in the Fc region of an antibody heavy chain, for example, substitution of HY with RF, referred to as a star substitution or Fc*, as shown in Figure 1B, inhibits Protein A binding. This star substitution contributes to differential binding to Protein A, which can facilitate antibody purification or separation between a bispecific antibody and its parent monospecific antibody based on Protein A affinity chromatography.

[0048] Among the various detection modes that can be coupled with affinity-based chromatography, mass spectrometry enables precise and accurate identification of individual components in complex samples. Some parameters, such as the pH range of the solution containing the biological analyte, should be controlled to maintain the native folding state of the biological analyte for native mass analysis. It is unexpected that the biological state of the analyte in solution, e.g., a peptide or protein, is maintained in a native or native-like folding state after elution from an affinity-based chromatography column and before being subjected to the ionization step of mass analysis. In some exemplary embodiments, the mobile phase is an acidic solution, and the eluate from an affinity-based chromatography column, such as a Protein A column, can be directly characterized using a mass spectrometer without pretreatment to modify the mobile phase or adjustment of the mobile phase pH. Despite the acidic conditions required to elute peptides or proteins from an affinity-based chromatography column, such as a Protein A column, the native or native-like charge state of the peptide or protein can be maintained throughout the elution profile, indicating negligible denaturation using the methods and systems of the present application.

[0049] The methods and systems of the present application are advantageous for providing high-throughput methods and systems that provide mechanistic insights for improving the manufacturing process of therapeutic peptides or proteins. In particular, the present application can provide rapid, sensitive, and high-throughput methods and systems for characterizing antibodies, antibody variants, or antibody-drug conjugates by combining affinity-based chromatography with intact native mass spectrometry.

[0050] In one aspect, monoclonal antibodies or antibody variants containing specific post-translational modifications are evaluated using the high-throughput methods and systems of the present application by combining affinity-based chromatography with intact-native mass spectrometry. In some preferred aspects, the methods and systems of the present application can be used to identify or quantify the level of post-translational modifications or post-translational modification diversity of monoclonal antibodies or antibody variants.

[0051] In one aspect, monoclonal antibodies or antibody variants containing specific glycosylation are evaluated using the high-throughput methods and systems of the present application by combining affinity-based chromatography with intact native mass spectrometry. In some preferred aspects, the methods and systems of the present application can be used to identify or quantify the level of glycosylation or glycosylation diversity of monoclonal antibodies or antibody variants.

[0052] In one aspect, the present application provides sensitive, high-throughput analytical methods and systems for characterizing the effects of different amino acid modifications on therapeutic proteins, such as bispecific monoclonal antibodies, when Protein A is used to purify the therapeutic protein. In some preferred aspects, the methods and systems of the present application are used to separate or identify impurities in a sample based on comparison of at least one separation profile, where the separation profile is based on differential affinity binding, such as differential Protein A affinity binding or differential FcγRIIIa affinity binding.

[0053] In one aspect, the present application provides a sensitive, high-throughput analytical method and system for identifying or quantifying the drug-to-antibody ratio of antibody-drug conjugates using the high-throughput methods and systems of the present application by combining affinity-based chromatography with intact native mass spectrometry. In some preferred aspects, the antibody-drug conjugates analyzed are lysine-linked or cysteine-linked antibody-drug conjugates. The present application is particularly advantageous by providing high peak capacity coupled with uniform elution of species with different drug-to-antibody ratios in combination with sensitive mass spectrometry detection under native conditions.

[0054] In one aspect, the present application provides a sensitive, high-throughput analytical method and system for identifying or quantifying monoclonal antibodies or antibody variants containing specific glycosylation by combining FcγRIIIa affinity chromatography with intact native mass spectrometry. In some preferred aspects, the methods and systems of the present application can be used to identify or quantify the level of glycosylation or glycosylation diversity of monoclonal antibodies or antibody variants using glycan-based separation or glycoform variation, where the glycosylation is terminal galactose, Fc glycan occupancy, core fucose, bisecting GlcNAc, or Man5.

[0055] Given the limitations of existing methods, the exemplary embodiments disclosed herein fulfill a long-felt need to provide rapid, sensitive, and high-throughput analytical methods and systems based on affinity-based chromatography coupled to native mass spectrometry to improve biopharmaceutical product manufacturing processes, including identifying impurities during antibody purification, monitoring post-translational modification variants during production, or characterizing the drug-to-antibody ratio of antibody-drug conjugates.

[0056] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations as understood by one of ordinary skill in the art, and when ranges are provided, endpoints are included.

[0057] As used herein, the terms "include," "includes," and "including" are meant to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0058] In some exemplary embodiments, the present disclosure provides a method for identifying at least one peptide or protein in a sample, the method comprising: contacting the sample with a solid surface, wherein the solid surface comprises an affinity binding molecule for the at least one peptide or protein; washing the solid surface with a mobile phase to generate at least one eluate, wherein the eluate comprises the at least one peptide or protein; and characterizing the at least one peptide or protein in the at least one eluate using a mass spectrometer under native conditions.

[0059] In some exemplary embodiments, the present disclosure provides a system for identifying at least one peptide or protein, the system comprising: a sample comprising at least one peptide or protein; a chromatography column comprising an affinity binding molecule for the at least one peptide or protein, the chromatography column being capable of being washed with a mobile phase to produce an eluate; and a mass spectrometer capable of characterizing or quantifying the at least one peptide or protein, the mass spectrometer being operated under native conditions and being directly coupled to the chromatography column.

[0060] As used herein, the terms "affinity" or "affinity binding molecule" refer to affinity intermolecular interactions, such as the strength of interaction between a single biomolecule and its binding partner, or ligand. Intermolecular interactions can include non-covalent intermolecular interactions, such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. Shape complementarity is also important for affinity intermolecular interactions. Potential affinity for a target molecule can be achieved with a ligand that mirrors the shape of the target surface with a complementary charge distribution. Binding affinity, e.g., interaction strength, can be measured by the equilibrium dissociation constant (Kd) to rank the strength of bimolecular interactions. Affinity binding between two molecules can be considered the strength of the interaction for reversible binding. The dissociation constant defines the probability that the interaction between two molecules will break. (Eaton et al., Let's get specific: the relationship between specificity and affinity, Chemistry & Biology, October 1995, volume 2, No. 10, pages 633-638, Current Biology Ltd, ISSN 1074-5521; Panagiotis et al., 2013, On the binding affinity of macromolecular interactions: daring to ask why proteins interact, Journal of the Royal Society Interface, 10:20120835, http: / / dx.doi.org / 10.1098 / rsif.2012.0835). Affinity binding molecules can be immobilized on a solid surface or solid phase. By "solid surface" or "solid phase" is meant a non-aqueous matrix to which affinity binding molecules can adhere. Solid phases of interest herein can include glass or silica surfaces. The solid phase can be a purification column or a discontinuous phase of discrete particles.

[0061] As used herein, the term "native" in the description "using a mass spectrometer under native conditions" refers to the biological state of the analyte in solution before undergoing ionization. As used herein, the terms "native conditions" or "native mass spectrometry" can include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see the following 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).

[0062] As used herein, the term "mass spectrometer" includes devices that can identify specific molecular species and measure their exact mass. The term is meant to include any molecular detector from which polypeptides or peptides can be eluted for detection and / or characterization. A mass spectrometer can include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create gas-phase ions. Analyte atoms, molecules, or clusters can be transferred into the gas phase and simultaneously ionized (as in electrospray ionization). The choice of ion source is highly dependent on the application.

[0063] In some exemplary embodiments, in the method for identifying at least one peptide or protein in a sample, the at least one peptide or protein is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product. In some preferred embodiments, the at least one peptide or protein contains an Fc region of an antibody, and the Fc region provides affinity interaction with an affinity-based chromatography column.

[0064] As used herein, the term "peptide" or "protein" includes any amino acid polymer having covalently bonded amide bonds. A protein comprises one or more amino acid polymer chains, commonly known in the art as "peptides" or "polypeptides." A protein may contain one or more polypeptides to form a single functional biomolecule. In some exemplary embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a host-cell protein, or a combination thereof.

[0065] As used herein, a "protein pharmaceutical product" includes an active ingredient that may be completely or partially biological in nature. In some exemplary embodiments, a protein pharmaceutical product can include a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. In some other exemplary aspects, a protein pharmaceutical product can include a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof.

[0066] As used herein, "antibody fragment" includes a portion of an intact antibody, such as the Fc region, antigen-binding region, or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Fv fragments are a combination of the variable regions of immunoglobulin heavy and light chains, while ScFv proteins are recombinant single-chain polypeptide molecules in which the immunoglobulin light and heavy chain variable regions are connected by a peptide linker. Antibody fragments can be produced by various means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments may be wholly or partially synthetically produced. Antibody fragments may optionally comprise single-chain antibody fragments. Alternatively or additionally, antibody fragments may comprise multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally comprise multimolecular complexes.

[0067] 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) that can be covalently attached to the side chains of amino acid residues on 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 bind with sufficient affinity for selective accumulation and sustained retention at the target site. Most ADCs can have Kd values ​​in the nanomolar range. The payload can have potency in the nanomolar / picomolar range and reach achievable intracellular concentrations after distribution of the ADC to the target tissue. Finally, the linker forming the connection between the payload and the antibody should be sufficiently stable in circulation to take advantage of the pharmacokinetic properties (e.g., long half-life) of the antibody moiety and allow the payload to remain bound to the antibody as it distributes within tissues, while allowing efficient release of the biologically active drug once the ADC is internalized within the target cells. The linker can be non-cleavable during cellular processing or cleavable once the ADC reaches the target site. With a non-cleavable linker, the biologically active drug released within the cell contains all elements of the linker still attached to the payload and antibody amino acid residues, typically lysine or cysteine ​​residues, after complete proteolysis of the ADC within the lysosome. A cleavable linker is one whose structure includes a cleavage site between the payload and the amino acid binding site on the antibody. Cleavage mechanisms include hydrolysis of acid-labile bonds in acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by intracellular proteases or esterases, and reductive cleavage of disulfide bonds in the reducing environment within the cell.

[0068] As used herein, "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions 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 VL can be composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes, but is not limited to, those prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. IgG includes a subset of antibodies.

[0069] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises identifying or quantifying a level of post-translational modification or post-translational modification diversity of the at least one peptide or protein based on the at least one separation profile or comparison with another separation profile.

[0070] As used herein, the general term "post-translational modification" or "PTM" refers to a covalent modification that polypeptides undergo either during (co-translational modification) or after (post-translational modification) their ribosomal synthesis. PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at the site of specific characteristic protein sequences (signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some aspect of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853).Various post-translational modifications include cleavage, N-terminal elongation, proteolysis, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (addition of an acetyl group, usually at the N-terminus of a protein), alkylation (addition of an alkyl group (e.g., methyl, ethyl, or propyl) usually at a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylation and carboxy-terminal amidation), and vitamin K-dependent modifications in which vitamin K is a cofactor in the carboxylation of glutamic acid residues resulting in the formation of γ-carboxyglutamate. These modifications include, but are not limited to, hydroxyl modifications (glu residues), glutamylation (covalent attachment of glutamic acid residues), glycylation (covalent attachment of glycine residues), glycosylation (addition of glycosyl groups to either asparagine, hydroxylysine, serine, or threonine, resulting in glycoproteins), isoprenylation (addition of isoprenoid groups such as farnesol and geranylgeraniol), lipoylation (addition of a lipoate functional group), phosphopantetheinylation (addition of a 4'-phosphopantetheinyl moiety from coenzyme A as in fatty acid, polyketide, nonribosomal peptide, 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 that involve structural changes include, but are not limited to, disulfide bridge formation (covalent bonding of two cysteine ​​amino acids) and proteolytic cleavage (breakage of proteins at peptide bonds).Certain post-translational modifications involve 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-related modifier)), and ubiquitination (covalent attachment to the protein ubiquitin). For a more detailed controlled vocabulary of PTMs curated by UniProt, see the European Bioinformatics Institute Protein Information Resource, SIB Swiss Institute of Bioinformatics, European Bioinformatics Institute, Drs - Drosomycin precursor - Drosophila melanogaster (Fruit fly) - Drs gene & protein, http: / / www.uniprot.org / docs / ptmlist (last visited January 15, 2019).

[0071] In some exemplary embodiments, the method for identifying at least one peptide or protein in a sample further comprises separating or identifying an impurity in the sample based on at least one separation profile or comparison with another separation profile. In some preferred exemplary embodiments, the impurity does not contain an Fc region of an antibody. In some preferred exemplary embodiments, the impurity does not provide affinity binding to at least one peptide or protein, protein A, or an affinity binding molecule for FcγRIIIa.

[0072] As used herein, the term "impurity" may include any undesired protein present in a protein biopharmaceutical product. In particular, impurities do not contain the Fc region of an antibody or do not provide affinity binding to at least one peptide or protein, protein A, or FcγRIIIa affinity binding molecule. Impurities may include process- and product-related impurities. Impurities may further be of known structure, partially characterized, or unidentified. Process-related impurities may originate from the manufacturing process and include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vector, or total DNA) derived from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymatic, chemical, and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables. Product-related impurities (e.g., precursors, specific degradation products) may be molecular variants arising during production and / or storage that do not have equivalent properties to those of the desired product in terms of activity, efficacy, and safety. Such variants may require significant isolation and characterization efforts to identify the type of modification. Product-related impurities may include truncated forms, modified forms, and aggregates. Cleaved forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modified forms include, but are not limited to, deamidation, isomerization, mismatched S-S bonds, oxidation, or altered complexation forms (e.g., glycosylation, phosphorylation). Modified forms may also include any post-translational modifications. Aggregates include dimers and higher multiples of the desired product.(Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products, ICH August 1999, USDept. of Health and Humans Services).

[0073] In some exemplary embodiments, in the methods for identifying at least one peptide or protein in a sample, the solid surface comprising Protein A is comprised in a chromatography column.

[0074] As used herein, the term "Protein A" refers to Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and Protein C. H 2 / C H The term "protein A" encompasses variants thereof that retain the ability to bind to proteins having three domains. Non-limiting examples of commercial manufacturers of protein A include Repligen, Pharmacia, and Fermatech. Protein A can be immobilized on a solid surface or solid phase. By "solid surface" or "solid phase" is meant a non-aqueous matrix to which protein A can adhere. Solid phases of interest herein can include glass or silica surfaces. The solid phase can be a purification column or a discontinuous phase of discrete particles.

[0075] As used herein, the term "chromatography" refers to a process by which a liquid or gas-borne chemical mixture can be separated into components as a result of the differential distribution of chemical entities flowing around or over a stationary liquid or solid phase. Non-limiting examples of chromatography include conventional reversed-phase (RP), ion-exchange (IEX), mixed-mode chromatography, and normal-phase chromatography (NP).

[0076] In some exemplary embodiments, in the methods for identifying at least one peptide or protein in a sample, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, a quadrupole mass spectrometer, or an ultra-high mass range hybrid quadrupole mass spectrometer.

[0077] As used herein, the term "electrospray ionization" or "ESI" refers to the process of electrospray ionization in which either cations or anions in solution are transferred to the gas phase via the atmospheric pressure formation and desolvation of a stream of highly charged droplets resulting from the application of a potential difference between the tip of an electrospray needle containing the solution and a counter electrode. Generally, there are three major steps in the generation of gas-phase ions from electrolyte ions in solution. These are: (a) generation of charged droplets at the ES injection tip; (b) shrinkage of the charged droplets due to solvent evaporation and repeated droplet collapse, resulting in small, highly charged droplets that can generate gas-phase ions; and (c) the mechanism by which gas-phase ions are generated from very small, highly charged droplets. Steps (a) through (c) are typically performed in the atmospheric pressure region of the instrument.

[0078] As used herein, the term "nanoelectrospray" refers to electrospray ionization at very low solvent flow rates, typically no more than a few hundred nanoliters per minute of sample solution, often without the use of external solvent delivery. Electrospray injection setups that form nanoelectrosprays can use static or dynamic nanoelectrospray emitters. Static nanoelectrospray emitters perform continuous analysis of small sample (analyte) solution volumes over extended periods of time. Dynamic nanoelectrospray emitters use a capillary column and solvent delivery system to perform chromatographic separation on mixtures prior to analysis by mass spectrometer.

[0079] In some exemplary embodiments, in the methods for identifying at least one peptide or protein in a sample, the mass spectrometer comprises an Orbitrap mass spectrometer.

[0080] As used herein, the term "mass spectrometer" includes a device 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 sequencing are time-of-flight (TOF), magnetoelectric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and also accelerator mass spectrometry (AMS) techniques.

[0081] Illustrative Embodiments Embodiments disclosed herein provide compositions, methods, and systems for identifying at least one peptide or protein in a sample based on affinity chromatography coupled native mass spectrometry.

[0082] In some exemplary embodiments, the present disclosure provides a method for identifying at least one peptide or protein in a sample, the method comprising: contacting the sample with a solid surface, the solid surface comprising an affinity binding molecule for the at least one peptide or protein; washing the solid surface with a mobile phase to generate at least one eluate, the eluate comprising the at least one peptide or protein; and characterizing the at least one peptide or protein in the at least one eluate using a mass spectrometer under native conditions. In some exemplary aspects, the present disclosure provides a system for identifying at least one peptide or protein, the system comprising: a sample comprising at least one peptide or protein; a chromatography column comprising an affinity binding molecule for the at least one peptide or protein, the chromatography column can be washed with a mobile phase to generate an eluate; and a mass spectrometer capable of characterizing or quantifying the at least one peptide or protein, the mass spectrometer operating under native conditions and capable of being directly coupled to the chromatography column.

[0083] In some exemplary embodiments, in a method or system for identifying at least one peptide or protein in a sample, the mobile phase comprises an alkaline solution, an acidic solution, or a combination thereof. In some embodiments, the alkaline solution has a pH value of about pH 5.0-9.0, about pH 6.0-8.0, about pH 6.5-7.5, preferably about pH 6.5, or preferably about pH 7.0. In some embodiments, the acidic solution has a pH value of about pH 1.0-4.6, about pH 2.0-4.6, about pH 2.5-3.5, preferably about pH 4.5, or preferably about pH 3.0.

[0084] In some exemplary embodiments, a method or system for identifying at least one peptide or protein in a sample is based on FcγRIIIa chromatography coupled to native mass spectrometry, in which an FcγRIIIa chromatography column is directly coupled to a native mass spectrometer, and a splitter is used to connect the mass spectrometer and the chromatography column. To perform FcγRIIIa chromatography, a high-performance liquid chromatography (HPLC) system equipped with an FcγRIIIa column is used for front-end separation. A mass spectrometry-compatible mobile phase containing ammonium acetate (about pH 5.0-9.0, about pH 6.0-8.0, about pH 6.0-7.5, or preferably about pH 6.5) and / or ammonium acetate (about pH 1.0-5.0, about pH 2.0-5.0, about pH 2.5-5.0, or preferably about pH 4.5) is used for FcγRIIIa analysis. In some exemplary embodiments, the concentration of ammonium acetate is about 50-200 mM, about 100-200 mM, about 120-170 mM, or preferably about 150 mM.

[0085] In some exemplary embodiments, a method or system for identifying at least one peptide or protein in a sample is based on Protein A chromatography-coupled native mass spectrometry. The Protein A chromatography column is directly coupled to a native mass spectrometer, and a splitter is used to connect the mass spectrometer and the chromatography column. To perform Protein A chromatography, an HPLC equipped with a Protein A column is used for front-end separation. A mass spectrometry-compatible mobile phase containing ammonium acetate (about pH 5.0-9.0, about pH 6.0-8.0, about pH 6.5-7.5, or preferably about pH 7.0) and / or acetic acid (about pH 1.0-4.0, about pH 2.0-4.0, about pH 2.5-3.5, or preferably about pH 3.0) is used for Protein A applications. In some exemplary embodiments, the concentration of ammonium acetate or acetic acid is about 50-200 mM, about 100-200 mM, about 120-170 mM, or preferably about 150 mM.

[0086] In some exemplary embodiments, the mass analysis is performed using an Orbitrap mass spectrometer with electrospray ionization (ESI). In some exemplary embodiments, the mobile phase for washing the affinity column has a flow rate of about 0.1-0.8 mL / min, about 0.2-0.6 mL / min, about 0.3-0.5 mL / min, or preferably about 0.4 mL / min. A post-column splitter is used to divert low flow rates (ID 25 μm), such as 0.5-3 μL / min, or preferably about 1-2 μL / min, to a mass spectrometer equipped with a nanospray ion source. Higher flow rates, such as about 0.3-0.5 mL / min, or preferably about 0.4 mL / min, are diverted to a diode array detector (DAD) or photodiode array detector (PDA) for monitoring the separation at 280 nm and an in-line pH monitor for tracking the pH range of the elution.

[0087] It is understood that the methods or systems of the present application are not limited to any of the aforementioned pharmaceutical products, peptides, proteins, antibodies, antibody-drug conjugates, biopharmaceutical products, chromatography columns, or mass spectrometers.

[0088] The sequential numerical and / or letter labeling of method steps provided herein is not meant to limit the method or any embodiment thereof to the particular indicated order.

[0089] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and journal articles, are cited throughout this specification. Each of these cited references is incorporated herein by reference in its entirety and for all purposes.

[0090] The present disclosure will be more fully understood by reference to the following examples, which are provided to more fully illustrate the present disclosure and are intended to be illustrative and should not be construed as limiting the scope of the disclosure. [Example]

[0091] Material and Reagent Preparation. 1.1 Antibody reference material NISTmAb was used as the antibody reference material. NISTmAb is a recombinant humanized IgG1κ expressed in mouse suspension culture. It is a homodimer of two identical light chains and two identical heavy chains. NISTmAb has low abundance post-translational modifications, including methionine oxidation, deamidation, and glycosylation. The heavy chain of NISTmAb has N-terminal pyroglutamation, C-terminal lysine clipping, and glycosylation. NISTmAb has been extensively characterized and produced in mouse suspension cell culture and has undergone industry-standard upstream and downstream purification to remove process-related impurities.

[0092] 2.1 Bispecific antibodies and their parent monospecific antibodies The bispecific antibodies and their parent monospecific antibodies were subjected to characterization or purification. As shown in Figure 1B, MAB1 (Fc / Fc*, where Fc* indicates a star substitution) was derived by combining a single heavy chain from MAB2 (Fc* / Fc*) with a single heavy chain from MAB3 (Fc / Fc) (Tustian et al., mAbs, Vol. 8, No. 4, pages 828-838, 2016, Development of purification processes for fully human bispecific antibodies based on modification of protein A binding avidity). The bispecific antibody format involves pairing two different heavy chains with two common light chains, allowing for two unique antigen-binding sites that target two different antigens. For example, MAB1 (HH*L2, FcFc*) is a bispecific antibody that targets both ANTIGEN A and ANTIGEN B. The parent monospecific antibody MAB2 (H*2L2, Fc*Fc*) targeting ANTIGENA has two heavy chains, both modified by two amino acid substitutions. The parent monospecific antibody MAB3 (H2L2, FcFc) targeting ANTIGENB has no amino acid substitutions on its heavy chain. The ANTIGENA arm in the heavy chain of bispecific antibody MAB1 has two amino acid substitutions. A two-amino acid substitution in the Fc region of the heavy chain of MAB2, e.g., a substitution of RF for HY, called a star substitution or Fc*, abrogates Protein A binding. This star substitution contributes to differences in binding to Protein A, which may facilitate antibody purification or separation between bispecific antibodies and their parent monospecific antibodies based on Protein A affinity chromatography.

[0093] Instruments and workflows for peptide or protein identification 1.1. Affinity-based chromatography coupled to native mass spectrometry The present application provides affinity-based chromatography-coupled native mass spectrometry methods and systems, in which an affinity-based chromatography column is directly coupled to a native mass spectrometer, and a splitter is used to connect the mass spectrometer and the affinity-based chromatography column, as shown in Figure 2A according to an exemplary embodiment. Mass spectrometry in the methods or systems is performed under native conditions.

[0094] To perform affinity-based chromatography, such as with a Protein A chromatography column, a Dionex Ultimate 3000 HPLC (high-performance liquid chromatography, Thermo Fisher Scientific, Waltham, MA) equipped with a Bio-Monolith Protein A column (Agilent Technologies, Inc., Santa Clara, CA) was used for front-end separation in an exemplary embodiment. To perform FcγRIIIa affinity chromatography, a TSKgel FcγRIIIa column (Tosoh Biosciences LLC) with a Dionex Ultimate 3000 HPLC was used. Mobile phase A was 150 mM ammonium acetate at pH 6.5, and mobile phase B was 150 mM ammonium acetate at pH 4.5, with a flow rate of 0.4 mL / min in the combined system of the TSKgel FcγRIIIa column and native mass spectrometer.

[0095] A Thermo Scientific™ Q-Exactive™ UHMR (ultra-high mass range) mass spectrometer was used to perform native mass analysis. The mass spectrometer has an Orbitrap mass analyzer and uses electrospray ionization (ESI) as shown in Figure 2A. A mass spectrometry-compatible mobile phase containing ammonium acetate (e.g., about 150 mM at about pH 7.0) and / or acetic acid (e.g., about 150 mM at about pH 3.0) was used for Protein A applications as shown in Figure 2B. As an example, the pH range and pH profile of the mobile phase are shown in Figure 2B for the screening mode (rapid) and the degradation mode (long-term). The mobile phase used to wash the column had a flow rate of about 0.2-0.6 mL / min, e.g., about 0.4 mL / min. A post-column splitter was used to shunt the low flow rate (approximately 1-2 μL / min, flow rate at 25 μm ID) to a mass spectrometer equipped with a Nanospray Flex™ ion source, allowing the sensitivity and resolution of the nanospray ion source to be achieved. The high flow rate (approximately 0.4 mL / min flow rate) was shunted to a diode array detector (DAD), such as a photodiode array detector (PDA), to monitor the separation at 280 nm, and an in-line pH monitor to track the pH range of the elution. The raw mass spectrometry data was deconvoluted using INTACT MASS™ software from Protein Metrics.

[0096] Example 1. Screening of NISTmAb using ProA-MS The present application's Protein A chromatography-coupled native mass spectrometry (ProA-MS) method and system were used to identify and screen NISTmAb. Mass spectrometer analysis was performed under native conditions. The mass spectrometer was directly coupled to the Protein A chromatography column, and a splitter was used to connect the mass spectrometer and the chromatography column as shown in Figure 2A and described in the Instrumentation and Workflow section. To perform Protein A chromatography, an HPLC equipped with a Protein A column was used for front-end separation. A mass spectrometry-compatible mobile phase containing ammonium acetate (approximately pH 7.0) and acetic acid (approximately pH 3.0) was used for Protein A separation to generate an eluate containing NISTmAb, which was then subjected to native mass analysis. The mobile phase used to wash the Protein A column had a flow rate of approximately 0.4 mL / min. NISTmAb reference material was separated and screened using the present application's rapid, high-throughput analytical method. The eluate from the Protein A column was subjected to native mass analysis without sample pretreatment.

[0097] Rapid screening of NISTmAb reference material with baseline glycoform resolution and accurate mass measurement was achieved using rapid pH gradient elution without sample pretreatment. The raw mass spectrometry data is shown in Figure 3A. The raw mass spectrometry data was deconvoluted using INTACT MASS™ software, as shown in Figure 3B. NISTmAb is a recombinant humanized IgG1κ expressed in mouse suspension culture with low abundance post-translational modifications, including methionine oxidation, deamidation, and glycation. In addition, the heavy chain of NISTmAb has N-terminal pyroglutamation, C-terminal lysine clipping, and glycosylation. The diversity of NISTmAb's post-translational modifications and glycosylation was fully characterized with baseline resolution, as shown in Figure 3B. Despite the need for acidic conditions for elution from the Protein A column, the native-like charge state of NISTmAb was maintained throughout the elution profile, indicating negligible sample denaturation using the methods and systems described herein.

[0098] Example 2. Evaluation of antibody variants under oxidative stress using ProA-MS NISTmAb was subjected to increasing levels of oxidative stress in the presence of approximately 0.005% to 0.05% (v / v) hydrogen peroxide (HO) and then analyzed by ProA-MS in this application. A stepwise reduction in Protein A affinity was observed as a function of increasing oxidative stress, e.g., increasing hydrogen peroxide concentration, as shown in Figure 4. Partial separation of oxidized variants was achieved based on differential Protein A affinity binding. As shown in Figure 4, identification of oxidized antibody variants in the treated NISTmAb was achieved due to the high spectral quality and accurate mass determinations shown in the associated deconvoluted mass spectra.

[0099] Example 3. Detection of bispecific antibodies and their parent monospecific antibodies using ProA-MS The ProA-MS of the present application was used to separate and identify components in a mixture containing a bispecific antibody and its parent monospecific antibody, as shown in Figure 5. The bispecific antibody and its parent monospecific antibody were subjected to characterization. The bispecific antibody format involves pairing two different heavy chains with two common light chains, allowing for two unique antigen-binding sites targeting two different antigens. As shown in Figure 5, a bispecific antibody (bsAb, H*HL2) targeting two different antigens was derived by combining a single heavy chain with a star substitution (H*) from the parent monospecific antibody H*2L2 and a single heavy chain without the star substitution (H) from the parent monospecific antibody H2L2. Two amino acid substitutions, called star substitutions, in the Fc region of the heavy chain of the parent monospecific antibody H*2L2 abrogate Protein A binding. The parent monospecific antibody H2L2, which does not have any star substitutions, has the strongest binding to Protein A among the antibodies in the mixture.

[0100] The star substitution contributes to differences in binding to Protein A, facilitating antibody purification or separation between bispecific antibodies and their parent monospecific antibodies based on Protein A affinity chromatography. Due to the presence of the star substitution, H*2L2 showed no binding to a Protein A column. As shown in Figure 5 , the present application's Protein A chromatography-coupled native mass spectrometry method and system were used to monitor a mixture of bispecific molecules and corresponding monospecific antibodies (bsAb Mixture 1) based on their differential Protein A affinity binding. However, under certain conditions, H*2L2 showed some affinity for Protein A based on its subsequent elution time, which was likely due to nonspecific interactions between the Fab region and the Protein A stationary phase, as shown in Figure 5 for the analysis of bsAb Mixture 2. Therefore, the present application's method and system enabled rapid screening of undesired Fab binding that interfered with Protein A-based purification of bispecific antibodies. The present application's method and system provided a sensitive, high-throughput analytical tool to characterize the effects of different amino acid modifications on therapeutic monoclonal antibodies when Protein A is used to purify them. The methods and systems of the present application have been used to separate or identify impurities in samples with satisfactory results during Protein A-based purification to purify bispecific antibodies based on differential Protein A affinity binding.

[0101] Example 4. Characterization of lysine-linked antibody-drug conjugates using ProA-MS The present application's ProA-MS was used to characterize antibody-drug conjugates (ADCs). Lysine-linked ADCs were subjected to characterization and identification of the drug-to-antibody ratio (DAR). Native SEC-MS (size exclusion chromatography-mass spectrometry) and RPLC-MS (reverse phase liquid chromatography-mass spectrometry) were performed in parallel for comparison.

[0102] As shown in Figure 6, high-speed direct-coupled Protein A separation of lysine-linked ADCs using ProA-MS demonstrated uniform elution profiles across all DAR species. The top figure shows the raw mass spectrum, while the bottom figure shows the deconvoluted mass spectrum. In contrast, as shown in Figure 7, both native SEC-MS and native RPLC-MS analysis results showed shifted retention times and broader peak tailing for species with higher DARs compared to native ProA-MS analysis, as demonstrated by the corresponding extracted ion chromatograms (XICs). These results indicate that the ProA-MS method provided more reliable quantification of the average DAR. Native ProA-MS demonstrated improved sensitivity for species with higher DARs compared to both native SEC-MS and native RPLC-MS.

[0103] Example 5. Characterization of cysteine-linked antibody-drug conjugates using ProA-MS The present application's ProA-MS was used to characterize antibody-drug conjugates (ADCs). Cysteine-linked ADCs were subjected to characterization and identification of the drug-to-antibody ratio (DAR). SEC-MS (size exclusion chromatography-mass spectrometry) was performed in parallel as a comparison. As shown in Figure 8, improved elution profiles across all DAR species were observed for Protein A-based separations compared to SEC-based separations. The average DAR values ​​calculated by native ProA-MS and native SEC-MS were highly consistent. As shown in Figure 9, native ProA-MS showed slightly higher levels of released light chains compared to SEC-MS, likely due to the low pH elution from the Protein A column.

[0104] Example 6. Direct Enrichment and Direct Analysis Using ProA-MS Cell culture time-lapse samples containing MAB4 were analyzed using the ProA-MS of the present application. MAB4 is a bispecific monoclonal antibody (e.g., Fc / Fc*, HH*L2, where Fc* indicates a star substitution). The cell culture time-lapse samples were spun down at 14,000 × g for 5 minutes and loaded directly into injection vials. The ProA-MS was set to resolution mode. MAB4 was directly analyzed using direct enrichment in a complex matrix. As shown in Figure 10, the relative abundance of MAB4 to the medium background increased as a function of time during the separation. MAB4 could be enriched and detected directly from the cell culture medium, with MAB4 titers as low as approximately 0.005 mg / mL. The H2L2 parent monoclonal antibody has strong affinity for Protein A. The H*2L2 parent monoclonal antibody has disrupted affinity. The HH*L2 bispecific monoclonal antibody has intermediate affinity. In addition, the level of unglycosylated MAB4 increased over time. Furthermore, glycoform changes in MAB4 samples over the cell culture cycle were evaluated, as shown in Figure 11. The results demonstrate that the ProA-MS of the present application can facilitate the direct enrichment and analysis of low-abundance monoclonal antibodies in complex matrices.

[0105] Cell culture time-course samples containing MAB5 were analyzed using the ProA-MS of the present application, as shown in Figure 12. MAB5 is a monospecific monoclonal antibody. Cell culture time-course samples were spun down at 14,000 x g for 5 minutes and loaded directly into injection vials. The ProA-MS was set to resolution mode. MAB5 was directly analyzed using direct enrichment in a complex matrix. In addition, glycoform changes in MAB5 samples over the cell culture cycle were evaluated, as shown in Figure 13.

[0106] Example 7. Screening of NISTmAb using FcγRIIIa-MS The present FcγRIIIa chromatography-coupled native mass spectrometry (FcγRIIIa-MS) method and system were used to identify and screen NISTmAb. Mass spectrometer analysis was performed under native conditions. The mass spectrometer was directly coupled to the FcγRIIIa chromatography column, and a splitter was used to connect the mass spectrometer and the chromatography column as shown in Figure 2A and described in the Instrumentation and Workflow section. To perform FcγRIIIa chromatography, an HPLC equipped with a TSKgel FcγRIIIa column was used for front-end separation. Mass spectrometry-compatible mobile phase A containing 150 mM ammonium acetate (approximately pH 6.5) and mobile phase B containing 150 mM ammonium acetate (approximately pH 4.5) were used for FcγRIIIa separation to generate an eluate containing NISTmAb, which was then subjected to native mass spectrometry. The mobile phase used to wash the FcγRIIIa column had a flow rate of approximately 0.4 mL / min. The NISTmAb reference material was isolated and screened using the rapid, high-throughput analytical method of this application.

[0107] High-throughput screening of NISTmAb reference material with baseline resolution of glycoforms and accurate mass measurements was achieved. Good resolution was achieved by liquid chromatography. Exceptional quality of MS data was obtained, as shown in Figure 14. The post-translational modifications and glycosylation diversity of NISTmAb were well characterized with baseline resolution, as shown in Figure 14.

[0108] Example 8. Ranking the binding affinity of IgG using FcγRIIIa-MS The FcγRIIIa-MS of the present application was used to quantitate the binding affinity of various IgG formats by relative retention time. The results, shown in Figure 15, indicate that IgG4 exhibited weaker FcγRIIIa affinity compared to IgG1, the IgG4 stealth (IgG4s) format exhibited even reduced FcγRIIIa affinity, and IgG1, which lacks core fucose, exhibited the strongest FcγRIIIa affinity.

[0109] Example 9. Effect of terminal galactose The FcγRIIIa-MS method and system of the present application were used to investigate the effect of terminal galactose in IgG on FcγRIIIa binding by analyzing NISTmAb (IgG1). As shown in Figure 16, increasing the number of terminal galactoses resulted in increased binding to FcγRIIIa (or ADCC). The contribution of terminal galactose to binding affinity with FcγRIIIa can be determined by a single FcN-glycan.

[0110] Example 10. Effect of Fc glycan occupancy The FcγRIIIa-MS method and system of the present application were used to investigate the influence of Fc glycan occupancy in IgG on FcγRIIIa binding by analyzing MAB8 (IgG4). As shown in Figure 17, higher glycan occupancy resulted in increased binding to FcγRIIIa (or ADCC). The FcγRIIIa-MS method and system of the present application were used to further investigate the influence of Fc glycan occupancy in IgG on FcγRIIIa binding by analyzing the stealth format of MAB10 (IgG4S). As shown in Figure 18, IgG4S showed very weak binding to FcγRIIIa (or ADCC). However, Fc glycan occupancy-based separation was still achieved.

[0111] Example 11. Effect of core fucose The FcγRIIIa-MS method and system of the present application were used to investigate the effect of core fucose in IgG on FcγRIIIa binding by analyzing MAB8 (IgG4). As shown in Figure 19, increasing the number of fucoses resulted in decreased binding to FcγRIIIa (or ADCC).

[0112] Example 12. Effect of bisecting GlcNAc The FcγRIIIa-MS method and system of the present application were used to investigate the effect of bisecting GlcNAc on FcγRIIIa binding by analyzing MAB9 (IgG4). As shown in Figure 20, a reduced number of bisecting GlcNAc resulted in reduced binding FcγRIIIa (or ADCC).

[0113] Example 13. Effect of Man5 The effect of Man5 on FcγRIIIa binding was investigated by analyzing MAB8 (IgG4) using the FcγRIIIa-MS method and system of the present application. As shown in Figure 21, compared with G0F / G0F, Man5 / Man5 resulted in slightly reduced affinity binding.

[0114] Example 14. Comparative study The FcγRIIIa-MS method and system of the present application were compared with RPLC-MS for glycan-based separation. MAB9 C1P2 lot A and MAB9 C2P1 lot B were analyzed using the FcγRIIIa-MS method and system of the present application, as shown in Figure 22. The results show that the FcγRIIIa-MS of the present application is a good alternative to intact mass spectrometry under native conditions, providing glycan-based separation with more detailed identification.

[0115] The FcγRIIIa-MS method and system of the present application were compared to RPLC-MS by analyzing MAB9 C1P2 DS Lot A. Compared to RPLC-MS, the FcγRIIIa-MS of the present application allows for better glycan-based separation by better distinguishing between non-glycosylated, partially glycosylated, G0F / G0F-2GlcNAc, and G0F / G0F-GlcNAc peaks, as shown in Figures 23A and 23B.

[0116] The FcγRIIIa-MS method and system of the present application were compared to RPLC-MS by analyzing MAB9 C2P1 FDS lot B. Compared to RPLC-MS, the FcγRIIIa-MS of the present application allows for better glycan-based separation by better distinguishing between non-glycosylated, partially glycosylated, G0F / G0F-2GlcNAc, and Man5 / Man5 peaks, as shown in Figures 24A and 24B.

Claims

1. 1. A method for identifying at least one peptide or protein in a sample, comprising: contacting the sample with a solid surface, the solid surface comprising an affinity binding molecule of the at least one peptide or protein; washing the solid surface with a mobile phase to produce at least one eluate, wherein the eluate comprises the at least one peptide or protein; characterizing said at least one peptide or protein in said at least one eluate using a mass spectrometer under native conditions; A method comprising:

2. The method of claim 1 further comprising generating at least one separation profile.

3. The method of claim 2, further comprising identifying or quantifying said at least one peptide or protein based on said at least one separation profile.

4. The method of claim 2, further comprising identifying or quantifying a level of post-translational modification or post-translational modification diversity of the at least one peptide or protein based on comparison with the at least one separation profile or another separation profile.

5. The method of claim 2, further comprising identifying or quantifying the level of glycosylation or glycosylation diversity of said at least one peptide or protein based on said at least one separation profile or comparison with another separation profile.

6. 6. The method of claim 5, wherein the glycosylation is terminal galactose, Fc glycan occupancy, core fucose, bisecting GlcNAc, or Man5.

7. 3. The method of claim 2, further comprising separating or identifying impurities in the sample based on the at least one separation profile or comparison with another separation profile.

8. 3. The method of claim 2, wherein the at least one peptide or protein is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

9. 9. The method of claim 8, further comprising quantifying the drug-to-antibody ratio of the antibody-drug conjugate based on comparison with the at least one separation profile or another separation profile.

10. 10. The method of claim 1, wherein the solid surface comprising the affinity binding molecule of the at least one peptide or protein is comprised in a chromatography column.

11. The method of claim 10 , wherein the mass spectrometer is directly coupled to the chromatography column.

12. 2. The method of claim 1, wherein the affinity binding molecule of the at least one peptide or protein is protein A, protein G, an Fc gamma receptor, Fc gamma RIIIa, an anti-human Fc antibody, a fetal Fc receptor, Fc epsilon RI, an anti-idiotypic antibody, or complement component C1q.

13. The method of claim 10, wherein a splitter is used to connect the mass spectrometer and the chromatography column.

14. 14. The method of claim 13, wherein the splitter is used to split a lower flow rate to the mass spectrometer and a higher flow rate to a detector.

15. 10. The method of claim 1, wherein the mobile phase is an acidic solution and the eluate is characterized using the mass spectrometer under native conditions without pretreatment.

16. 10. The method of claim 1, wherein the mobile phase comprises ammonium acetate, acetic acid, or a combination thereof.

17. 11. The method of claim 10, wherein the mobile phase is used to wash the chromatography column and has a flow rate of about 0.2 to 0.6 mL / min.

18. 10. The method of claim 1, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, a quadrupole mass spectrometer, or an ultra-high mass range hybrid quadrupole mass spectrometer.

19. The method of claim 1 , wherein the mass spectrometer comprises an Orbitrap mass spectrometer.

20. 1. A system for identifying at least one peptide or protein, comprising: a sample comprising said at least one peptide or protein; a chromatography column comprising said at least one peptide or protein affinity binding molecule, said chromatography column being capable of being washed with a mobile phase to produce an eluate; a mass spectrometer capable of characterizing or quantifying said at least one peptide or protein, said mass spectrometer being capable of running under native conditions and being directly coupled to said chromatographic column; Including, the system.

21. 21. The system of claim 20, wherein a splitter is used to connect the mass spectrometer and the chromatography column.

22. 22. The system of claim 21, wherein the splitter is used to split a lower flow rate to the mass spectrometer and a higher flow rate to a detector.

23. 21. The system of claim 20, wherein the mobile phase is an acidic solution and the eluate is characterized using the mass spectrometer under native conditions without pretreatment.

24. 21. The system of claim 20, wherein the mobile phase comprises ammonium acetate, acetic acid, or a combination thereof.

25. 21. The system of claim 20, wherein the mobile phase has a flow rate of about 0.2 to 0.6 mL / min.

26. 21. The system of claim 20, comprising a diode array detector or a photodiode array detector.

27. 21. The system of claim 20, wherein the at least one peptide or protein is a drug, an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

28. 21. The system of claim 20, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, or an ultra-high mass range hybrid quadrupole mass spectrometer.

29. The system of claim 20 , wherein the mass spectrometer comprises an Orbitrap mass spectrometer.

30. 21. The system of claim 20, wherein the affinity binding molecule of the at least one peptide or protein is protein A, protein G, an Fc gamma receptor, Fc gamma RIIIa, an anti-human Fc antibody, a fetal Fc receptor, Fc epsilon RI, an anti-idiotypic antibody, or complement component C1q.

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