Sequence variant analysis using heavy peptides

Abstract

The present invention relates generally to methods for identifying sequence variants in a protein of interest, and in particular to the use of heavy peptide standards using liquid chromatography-mass spectrometry to specifically identify sequence variants of a protein of interest.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 310,626 (filed February 16, 2022).

[0002] The present application relates to methods for identifying sequence variants in a protein of interest. [Background technology]

[0003] Characterization of the critical quality attributes (CQAs) of therapeutic antibodies is important due to the large size and complex heterogeneity of this major therapeutic class. One such CQA is variation in the amino acid sequence of the protein, called sequence variants (SVs). The presence of high SVs contributes to product heterogeneity and may affect drug efficacy and safety, for example, when amino acid variants are present within the binding region or introduce non-human amino acid sequences.

[0004] Detection of sequence variants is challenging due to their typically low abundance. Sequence variants may be present, for example, at abundances of 0.001% to 0.1% of the corresponding non-variant peptide. Proper identification and isolation of variant amino acids requires high-quality MS sequences with near-complete backbone fragmentation. 2 Requires Spectra: In the absence of completely unambiguous and unequivocal spectral data, it is possible to identify false negatives or false positives for sequence variants.

[0005] Therefore, a need exists for methods and systems for identifying sequence variants in antibody products in a sensitive and specific manner. Summary of the Invention

[0006] A method has been developed for reliably identifying the amino acid sequence of a peptide of a protein of interest, for example to identify sequence variants in an antibody product. The method involves the novel use of heavy peptide standards that contain heavy isotopes at or near each peptide terminus. The heavy peptide standards can be selected to contain amino acid sequences that correspond to predicted amino acid sequences of digested peptides of the protein of interest. The predicted amino acid sequences can be, for example, wild-type amino acid sequences, mutant amino acid sequences, or sequence variants. Using liquid chromatography, the retention times of the heavy peptide standards match the retention times of the corresponding digested peptides, thereby controlling for any experimental variation in retention times. Using mass spectrometry, MS spectrometry from the heavy peptide standards can be used to determine the retention times of the corresponding digested peptides. 1 The peaks are shifted by the mass of the heavy isotope from the corresponding peptic peptides, allowing unambiguous confirmation of the mass of the peptic peptides. 2 The peaks are shifted from the corresponding peptic peptide by the mass of the heavy isotope at or near the peptide terminus involved, allowing unambiguous identification of each amino acid of the peptic peptide. Using the methods of the present invention, it is possible to reliably determine whether a peptic peptide is characterized by a predicted amino acid sequence (e.g., a wild-type amino acid sequence, a mutant amino acid sequence, or a sequence variant). Conversely, a false positive identification of an amino acid sequence can be refuted by comparison with a heavy peptide standard known to be characterized by that amino acid sequence.

[0007] The present disclosure provides a method for identifying the amino acid sequence of a digested peptide of a protein of interest. In some exemplary embodiments, the method includes: (a) combining a peptide digest having digested peptides of a protein of interest with at least one heavy peptide standard to form a mixture, the at least one heavy peptide standard including a heavy isotope at or near each peptide terminus, and the amino acid sequence of the at least one heavy peptide standard being a predicted amino acid sequence of a digested peptide of the protein of interest; (b) subjecting the mixture to analysis using liquid chromatography-mass spectrometry; (c) comparing the retention time and / or at least one mass spectrum of the at least one heavy peptide standard with the retention time and / or at least one mass spectrum of the digested peptide; and (d) identifying the amino acid sequence of the digested peptide of the protein of interest using the comparison of (c).

[0008] In one embodiment, the amino acid sequence is a sequence variant. In certain embodiments, the sequence variant is an important quality characteristic.

[0009] In one embodiment, the protein of interest can be an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, a host cell protein, or a protein pharmaceutical.

[0010] In one embodiment, the chromatography step comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0011] In one embodiment, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap®-based mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system.

[0012] In one embodiment, the at least one mass spectrum is MS 1 In another embodiment, the at least one mass spectrum is an MS 2 In a further embodiment, the at least one mass spectrum is a MS 3 It is a spectrum.

[0013] In one embodiment, the comparing step comprises determining whether the retention time of the at least one heavy peptide standard matches the retention time of the digested peptide. In another embodiment, the comparing step comprises determining whether the retention time of the at least one heavy peptide standard matches the retention time of the digested peptide. 1 In a further embodiment, the comparing step comprises determining whether the spectral peak is shifted by the mass of the heavy isotope compared to the digested peptides. 2 This involves determining whether the spectral peak is shifted by the additional mass of one of the heavy isotopes compared to the digested peptides.

[0014] In one embodiment, the comparing step further comprises comparing at least one chromatogram of the at least one heavy peptide standard with at least one chromatogram of the digested peptides. In a particular embodiment, the comparing step comprises determining whether a main peak of the at least one heavy peptide standard substantially overlaps with a main peak of the digested peptides. In a further specific embodiment, the comparing step comprises determining whether a main peak of the at least one heavy peptide completely overlaps with a main peak of the digested peptides.

[0015] In one embodiment, the molar ratio of peptide digest:heavy peptide standard is about 1:50 to 1:200, about 1:100, or 1:100.

[0016] These and other aspects of the invention will be more fully appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while setting forth various embodiments and many specific details thereof, is given by way of illustration and not by way of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the invention. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the mass difference between post-translational modifications and similar sequence variants, according to an exemplary embodiment. [Figure 2A-1] 1 shows mass spectra of previously identified sequence variants that were shown to be false positives using the methods of the present invention, according to an exemplary embodiment. [Figure 2A-2] Same as above. [Figure 2B-1] 1 shows mass spectra of previously identified sequence variants that were shown to be false positives using the methods of the present invention, according to an exemplary embodiment. [Figure 2B-2] Same as above. [Figure 2C-1] 1 shows mass spectra of previously identified sequence variants that were shown to be true positives using the methods of the present invention, according to an exemplary embodiment. [Figure 2C-2] Same as above. [Diagram 3] 2 illustrates a workflow of the method of the present invention according to an exemplary embodiment. [Figure 4] 1 shows a comparison of heavy peptide standards and digested peptides with liquid chromatography retention times, MS1 spectra, and MS2 spectra according to an exemplary embodiment. [Diagram 5] 1 shows amino acid sequences of heavy peptide standards, according to an exemplary embodiment. [Figure 6]1 shows regions of an antibody selected for analysis with heavy peptide standards, according to an exemplary embodiment. [Figure 7] 1 shows liquid chromatography and MS1 analysis in which sequence variants are not identified, according to an exemplary embodiment. [Figure 8A] 1 shows liquid chromatography and MS1 analysis in which sequence variants were identified, according to an exemplary embodiment. [Figure 8B] 1 shows MS2 analysis in which amino acid sequences of sequence variants were identified, according to an exemplary embodiment. [Figure 9A] 1 shows liquid chromatography and MS1 analysis in which unconfirmed sequence variants were identified, according to an exemplary embodiment. [Figure 9B] 1 shows an MS2 analysis in which an unidentified leucine or isoleucine sequence variant was identified, according to an exemplary embodiment. [Figure 9C] 1 shows the retention time differences of heavy peptide standards corresponding to leucine or isoleucine sequence variants according to an exemplary embodiment. [Figure 9D] 1 shows a liquid chromatography analysis in which a sequence variant was identified as isoleucine, according to an exemplary embodiment. [Figure 10A] 1 shows liquid chromatography and MS1 analysis with predicted sequence variants, according to an exemplary embodiment. [Figure 10B] 1 shows MS2 analysis with putative sequence variants featuring unconfirmed amino acid sequences, according to an exemplary embodiment. [Figure 10C] 1 shows liquid chromatography and MS1 analysis disproving false positive identification of putative sequence variants, according to an exemplary embodiment. [Figure 10D] 1 shows liquid chromatography and MS2 analysis disproving false positive identification of putative sequence variants, according to an exemplary embodiment. [Figure 10E]1 shows liquid chromatography and MS2 analysis identifying putative sequence variants as non-specific cleavage products according to an exemplary embodiment. [Figure 11] 1 shows previously identified sequence variants that were confirmed or disproved using the methods of the invention, according to exemplary embodiments. [Figure 12] 1 shows known NISTmAb sequence variants used to benchmark the liquid chromatography step of the method of the invention, according to an exemplary embodiment. [Figure 13] 1 shows the standard deviation of retention times of digested peptides across five gradient durations tested, according to an exemplary embodiment. [Figure 14] 1 shows the overall sequence variants identified across the five gradient durations tested, according to an exemplary embodiment. [Figure 15] 1 shows validation of previously identified sequence variants across four gradient durations tested, according to an exemplary embodiment. [Figure 16] 1 shows Byonic scores in MS2 spectra of sequence variants identified across five gradient durations tested, according to an exemplary embodiment. [Figure 17] 1 shows quantitative signals of sequence variants identified over five gradient durations tested, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Characterization of the critical quality attributes (CQAs) of therapeutic antibodies is important due to the large size and complex heterogeneity of this increasingly popular therapeutic class. One such CQA is sequence variants caused by amino acid substitutions. Sequence variants can be caused, for example, by DNA mutations in the production cell line or translation errors during protein production. High levels of sequence variants contribute to product heterogeneity and may affect efficacy or safety if amino acid variants are present in the binding region or introduce non-human amino acid sequences.

[0019] Detection of sequence variants is challenging due to their typically low abundance. Sequence variants may be present, for example, at abundances of 0.001% to 0.1% of the corresponding non-variant peptide. Proper identification and isolation of variant amino acids requires high-quality MS sequences with near-complete backbone fragmentation. 2 The difficulty of distinguishing sequence variants from other CQAs (e.g., non-specific cleavage or post-translational modifications (PTMs)) is illustrated in Figure 1. The mass difference between the PTM and the substituted amino acid can be in the ppm (parts per million) range or less, which necessitates the use of instruments with particularly high detection sensitivity. In Figure 1, the green rows show PTM masses that are very close to the mass of the sequence variant, while the white rows show PTM masses that are identical to the mass of the sequence variant and cannot be distinguished using mass spectrometry.

[0020] MS 2 Ambiguous or confusing spectra further reduce the reliability of identifying sequence variants. Examples of peptides identified as sequence variants based on mass spectra are shown in Figures 2A, 2B, and 2C. Two of these examples show false positive identifications, in one example (Figure 2A) caused by confounding non-specific cleavage, and in the other example (Figure 2B) caused by confounding PTM combinations. This shows that it can be difficult to distinguish false positives from true positives when performed without a standard of comparison.

[0021] The presence of multiple sequence variants provides a high degree of confidence in the identification of a sequence variant, whereas the identification of only one variant provides a low degree of confidence in the accuracy of the identification of a sequence variant.

[0022] The present disclosure provides a solution to confirm true positive identification of sequence variants and exclude false positive identification of sequence variants in a protein of interest. Heavy peptide standards containing heavy isotopes at or near both peptide termini provide a basis of comparison for putative sequence variant peptides that overlap in retention time in liquid chromatography systems but are clearly separable and comparable in mass spectra. The method of the present invention can be used to evaluate, for example, the CQAs of therapeutic antibodies (including, for example, sequence variants). Additionally, the method can be used to confirm peptide sequences and identities in the analysis of a protein of interest.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are described herein.

[0024] 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 that one of ordinary skill in the art would appreciate, and when ranges are stated, the endpoints are included. As used herein, the terms "include," "includes," and "including" are meant to be open-ended and should be understood to mean "comprise," "comprises," and "comprising," respectively.

[0025] As used herein, the term "protein" or "protein of interest" can include any amino acid polymer with covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs linked through peptide bonds. A "synthetic peptide or polypeptide" refers to a peptide or polypeptide that does not occur in nature. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those of skill in the art. A protein can include one or more polypeptides to form a functional biomolecule. In another exemplary embodiment, the protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest can include biological therapeutic proteins, recombinant proteins for research or therapeutic use, trap proteins, and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins can be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (eg, Pichia species), and mammalian systems (eg, CHO cells and CHO derivatives such as CHO-K1 cells).For a recent review of biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are incorporated herein by reference). In some exemplary embodiments, the proteins include modifications, additions, and other covalently attached moieties. These modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes. Proteins can be classified based on composition and solubility, and thus include simple proteins, such as globular proteins and fibrous proteins; complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0026] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried on a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein can be an antibody (e.g., a chimeric antibody, a humanized antibody, or a fully human antibody). In certain exemplary embodiments, the recombinant protein can be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., IgG1), or alternatively, the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

[0027] As used herein, the term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) that contain four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of the present invention, the FRs of the anti-big ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique (e.g., proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domains and, optionally, the constant domains). Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized.The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example to arrange one or more variable and / or constant domains in a suitable configuration, to introduce codons, to create cysteine ​​residues, to modify, add, or delete amino acids.

[0028] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. In some exemplary embodiments, an antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment to bind the same antigen as the parent antibody binds, and in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments can be produced by any means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody and / or recombinantly from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments can be produced in whole or in part synthetically. Antibody fragments may optionally include single chain antibody fragments. Alternatively or additionally, antibody fragments may include multiple chains linked together (e.g., by disulfide bonds). Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, more typically include at least about 200 amino acids.

[0029] The term "bispecific antibody" includes antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, each of which specifically binds to a different epitope (either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least 1-2 or 3-4 orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody may be present on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[0030] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen binding specificity but can bind to each heavy chain, or can associate with each heavy chain and bind to one or more of the epitopes bound by the heavy chain antigen binding region, or can associate with each heavy chain and allow binding of one or both of the heavy chains to one or both of the epitopes. bsAbs can be divided into two main classes, those that have an Fc region (IgG-like) and those that lack an Fc region, the latter being usually smaller than the Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs can have different formats such as, but not limited to, triomabs, kih IgG (knobs into holes IgG), crossMab, orth-Fab IgG, dual variable Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG single chain Fv (IgG-scFv), or κλ-bodies. Different non-IgG-like formats include tandem scFvs, diabody formats, single chain diabodies, tandem diabodies (TandAbs), dual specificity retargeting molecules (DART), DART-Fc, nanobodies, or antibodies produced by dock-and-lock (DNL) technology (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the teachings of which are incorporated herein by reference in their entirety).Methods for producing bsAbs include, but are not limited to, quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation involving chemical cross-linking agents, and genetic approaches using recombinant DNA technology.

[0031] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Typically, such molecules bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecific antibodies, can also be addressed by the systems and methods disclosed herein.

[0032] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including eukaryotic, prokaryotic, or phage clones, by any means available or known in the art. Monoclonal antibodies useful in the context of the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0033] As used herein, a "sample" can be obtained from any step of a bioprocess, e.g., cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in downstream processing, drug substance (DS), or drug product (DP), including the final formulated product. In some specific exemplary embodiments, the sample can be selected from any step of downstream processing of clarification, chromatography production, or filtration.

[0034] In some exemplary embodiments, a sample containing a protein of interest can be prepared prior to LC-MS analysis. Preparation steps can include denaturation, alkylation, dilution, and digestion.

[0035] As used herein, the term "protein alkylating agent" or "alkylating agent" refers to an agent used to alkylate certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents include iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0036] As used herein, "protein denaturation" or "denaturation" may refer to a process in which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation can be performed using a protein denaturant. Non-limiting examples of protein denaturants include exposure to heat, high or low pH, reducing agents such as DTT, or chaotropic agents. Several chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of a system by disrupting intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and salts thereof.

[0037] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to perform protein digestion in a sample using a suitable hydrolyzing agent, such as enzymatic digestion or non-enzymatic digestion. Digesting a protein into its component peptides results in a "peptide digest," which can be further analyzed using peptide mapping analysis.

[0038] As used herein, the term "digestive enzyme" refers to any of a number of different agents that can perform protein digestion.Non-limiting examples of hydrolytic agents that can perform enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), or outer membrane protein T (OmpT), immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or their biologically active fragments or homologues, or combinations thereof. For a recent review describing available techniques for protein digestion, see Switazar et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzer, Martin Giera & Wilfried MANiessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077(2013)).

[0039] As used herein, the term "protein reducing agent" or "reducing agent" refers to an agent used to reduce disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof. Reduced peptide mapping, a traditional method of protein analysis, involves protein reduction prior to LC-MS analysis. In contrast, non-reduced peptide mapping omits the sample preparation step of reduction to preserve endogenous disulfide bonds.

[0040] In some exemplary embodiments, heavy peptide standards can be added to the peptide digest. As used herein, the term "heavy peptide standard" refers to a peptide having a known amino acid sequence that includes at least one heavy isotope. The heavy peptide standard can be compared to a peptide of unknown amino acid sequence to identify the unknown amino acid sequence. For example, the heavy peptide standard can be compared to another peptide based on retention time using liquid chromatography or mass spectrum signal using a mass spectrometer. The heavy peptide standard is expected to have a chromatographic peak that substantially overlaps with a chromatographic peak from another peptide having the same amino acid sequence.

[0041] Heavy peptide standards that are particularly useful in the methods of the invention include heavy peptide standards that contain a heavy isotope at or near each peptide terminus. The heavy isotopes near the peptide termini can be, for example, one amino acid away from the terminus or two amino acids away from the terminus. By including a heavy isotope at or near each terminus, all or nearly all fragment ions in a tandem mass spectrum that includes either the N-terminus or C-terminus of a fragment peptide are shifted by the mass of the corresponding heavy isotope, thereby shifting the MS 2The analysis makes it possible to distinguish and compare different peptides with the same amino acid sequence.

[0042] As used herein, the term "liquid chromatography" refers to a process in which a liquid-borne biological / chemical mixture can be separated into components as a result of differences in the distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some embodiments, a sample containing at least one protein of interest or a peptide digest can be subjected to any one of the aforementioned chromatographic methods, or a combination thereof. Analytes separated using chromatography are characterized by a characteristic retention time that reflects the speed at which the analyte moves through a chromatographic column. Analytes can be compared using chromatograms that plot retention time on one axis and measured signal on the other axis, which can be obtained, for example, from UV or fluorescence detection.

[0043] As used herein, the term "mass spectrometer" includes devices capable of identifying and measuring the exact mass of a particular molecular species. The term is intended to include any molecular detector capable of characterizing a polypeptide or peptide. A mass spectrometer can include three main parts: an ion source, a mass spectrometer, and a detector. The role of the ion source is to generate gas phase ions. Analyte atoms, molecules, or clusters are transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or by a separate process. The choice of ion source depends on the application.

[0044] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques that obtain structural information of sample molecules by using multiple stages of mass selection and mass separation. The prerequisite is that the sample molecules are converted into the gas phase and ionized, so that after an initial mass selection step, fragments are formed in a predictable and controllable manner. MS / MS (or MS 2 ) is first detected as a precursor ion (MS 1 ) and fragmentation to obtain meaningful information. Tandem MS has been successfully implemented with a wide variety of analyzer combinations. The choice of which analyzer to pair for a particular application can be determined by many different factors, such as sensitivity, selectivity, and speed, as well as size, cost, and availability. Two major categories of tandem MS techniques are tandem-in-space and tandem-in-time, although hybrids also exist that couple tandem-in-time analyzers at intervals or with tandem-in-space analyzers. Tandem-in-space mass analyzers include an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A particular m / z separation function can be designed to select ions in one section of the instrument, dissociate in an intermediate region, and then send the product ions to another analyzer for m / z separation and data acquisition. In tandem-in-time, ions produced in an ion source in a mass spectrometer can be trapped, isolated, fragmented, and m / z separated within the same physical device.

[0045] The peptides identified by mass spectrometry can be used as surrogate representatives of the intact protein and its post-translational or other modifications (e.g., sequence variants). They can be used for protein characterization by correlating with experimental and theoretical MS / MS data, the latter generated from potential peptides in protein sequence databases. Characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequence determination, protein de novo sequencing determination, post-translational modification or sequence variant location, or post-translational modification or sequence variant identification, or comparability analysis, or a combination thereof.

[0046] In some exemplary embodiments, the mass spectrometer can operate with nanoelectrospray or nanospray. As used herein, the term "nanoelectrospray" or "nanospray" refers to electrospray ionization at very low solvent flow rates, typically a few hundred nanoliters per minute of sample solution or less, often without the use of external solvent delivery. The electrospray injection setup that forms the nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs sustained analysis of a small amount of sample (analyte) solution over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separation of the mixture prior to analysis by the mass spectrometer.

[0047] In some exemplary embodiments, mass spectrometry can be performed under native conditions. As used herein, the term "native conditions" can include performing mass spectrometry under conditions that preserve non-covalent interactions in analytes. 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).

[0048] As used herein, the term "database" refers to a compiled collection (e.g., in the form of a FASTA format file) of protein sequences that may be present in a sample. The relevant protein sequences may be derived from the cDNA sequence of the species being tested. Public databases that may be used to search for relevant protein sequences include, for example, databases maintained by Uniprot® or Swiss-prot®. The databases may be searched using what are referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra against all possible sequences in the database(s) and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools include Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0049] The present disclosure provides a method for identifying the amino acid sequence of a digested peptide of a protein of interest. In some exemplary embodiments, the method includes: (a) combining a peptide digest having digested peptides of a protein of interest with at least one heavy peptide standard to form a mixture, the at least one heavy peptide standard including a heavy isotope at or near each peptide terminus, and the amino acid sequence of the at least one heavy peptide standard being a predicted amino acid sequence of a digested peptide of the protein of interest; (b) subjecting the mixture to analysis using liquid chromatography-mass spectrometry; (c) comparing the retention time and / or at least one mass spectrum of the at least one heavy peptide standard with the retention time and / or at least one mass spectrum of the digested peptide; and (d) identifying the amino acid sequence of the digested peptide of the protein of interest using the comparison of (c).

[0050] In some exemplary embodiments, the comparing step can include determining whether the retention time of the at least one heavy peptide standard matches the retention time of the digested peptide. The retention times can be considered to match if they are identical or nearly identical (e.g., less than 1% difference, less than 0.5% difference, or less than 0.1% difference). The retention times can be compared by comparing at least one chromatogram of the at least one heavy peptide standard with at least one chromatogram of the digested peptide. The retention times can be considered to match if the main peaks of the chromatograms are completely or substantially overlapping. For example, peaks can be considered to substantially overlap if the area of ​​one peak is completely or nearly completely within the area of ​​the other peak, or if most of the area of ​​one peak (e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% of the area of ​​the peak) is within the area of ​​the other peak.

[0051] In some exemplary embodiments, the amount of peptide digest sample injected into the liquid chromatography column is about 100 fmol, about 200 fmol, about 300 fmol, about 400 fmol, about 500 fmol, about 600 fmol, about 700 fmol, about 800 fmol, about 900 fmol, about 1 pmol, about 2 pmol, about 3 pmol, about 4 pmol, about 5 pmol, about 6 pmol, about 7 pmol, about 8 pmol, about 9 pmol, about 10 pmol, or in the range of about 0.5 pmol to about 2 pmol. In some exemplary embodiments, the amount of heavy peptide standard applied to the liquid chromatography column is about 1 fmol, about 2 fmol, about 3 fmol, about 4 fmol, about 5 fmol, about 6 fmol, about 7 fmol, about 8 fmol, about 9 fmol, about 10 fmol, about 11 fmol, about 12 fmol, about 13 fmol, about 14 fmol, about 15 fmol, about 20 fmol, about 30 fmol, or about 5 fmol to about 20 fmol.

[0052] The present disclosure also provides a method for identifying sequence variants of an antibody. In some exemplary embodiments, the method includes: (a) combining a peptide digest having digested peptides of the antibody with at least one heavy peptide standard to form a mixture, the at least one heavy peptide standard comprising a heavy isotope at or near each peptide terminus, the amino acid sequence of the at least one heavy peptide standard being a predicted amino acid sequence of a digested peptide of the antibody characterized by a sequence variant; (b) subjecting the mixture to analysis using liquid chromatography-mass spectrometry; (c) comparing the retention time and / or at least one mass spectrum of the at least one heavy peptide standard with the retention time and / or at least one mass spectrum of the digested peptide; and (d) identifying the amino acid sequence of the sequence variant of the antibody using the comparison of (c).

[0053] In some exemplary embodiments, sequence variants may be important quality characteristics.

[0054] In some exemplary embodiments, the antibody may be a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, a biotherapeutic antibody, or an antibody pharmaceutical.

[0055] In some exemplary embodiments, the chromatography step can include reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0056] In some exemplary embodiments, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap®-based mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system.

[0057] In some exemplary embodiments, at least one mass spectrum is determined by MS 1 Spectra, MS 2 Spectrum (tandem mass spectrum), or MS 3 It can be a spectrum.

[0058] In some exemplary embodiments, the comparing step can include determining whether the retention time of the at least one heavy peptide standard matches the retention time of the digested peptide. The retention times can be considered to match if they are identical or nearly identical (e.g., less than 1% difference, less than 0.5% difference, or less than 0.1% difference). The retention times can be compared by comparing at least one chromatogram of the at least one heavy peptide standard with at least one chromatogram of the digested peptide. The retention times can be considered to match if the main peaks of the chromatograms are completely or substantially overlapping. For example, peaks can be considered to substantially overlap if the area of ​​one peak is completely or nearly completely within the area of ​​the other peak, or if most of the area of ​​one peak (e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% of the area of ​​the peak) is within the area of ​​the other peak.

[0059] In some exemplary embodiments, the comparing step comprises comparing the MS 1 In some exemplary embodiments, the step of comparing includes determining whether the spectral peak is shifted by the mass of the heavy isotope compared to the digested peptide. 1 This involves determining whether the spectral peak is shifted by the additional mass of one of the heavy isotopes compared to the digested peptides.

[0060] In some exemplary embodiments, the molar ratio of peptide digest to heavy peptide standard is about 1:50 to 1:200, about 1:100, or 1:100.

[0061] In some exemplary embodiments, the amount of peptide digest sample injected into the liquid chromatography column is about 100 fmol, about 200 fmol, about 300 fmol, about 400 fmol, about 500 fmol, about 600 fmol, about 700 fmol, about 800 fmol, about 900 fmol, about 1 pmol, about 2 pmol, about 3 pmol, about 4 pmol, about 5 pmol, about 6 pmol, about 7 pmol, about 8 pmol, about 9 pmol, about 10 pmol, or in the range of about 0.5 pmol to about 2 pmol. In some exemplary embodiments, the amount of heavy peptide standard applied to the liquid chromatography column is about 1 fmol, about 2 fmol, about 3 fmol, about 4 fmol, about 5 fmol, about 6 fmol, about 7 fmol, about 8 fmol, about 9 fmol, about 10 fmol, about 11 fmol, about 12 fmol, about 13 fmol, about 14 fmol, about 15 fmol, about 20 fmol, about 30 fmol, or about 5 fmol to about 20 fmol.

[0062] The methods of the invention can be applied to any protein of interest. In some exemplary embodiments, particular applications involve the analysis of a protein of interest that is an antibody. In some exemplary embodiments, the protein of interest is a monoclonal antibody. In some exemplary embodiments, the protein of interest is a bispecific antibody. In some exemplary embodiments, the protein of interest is a recombinant protein. In some exemplary embodiments, the protein of interest is a fusion protein (e.g., a receptor fusion protein). In some exemplary embodiments, the protein of interest is a host cell protein.

[0063] It is to be understood that the present invention is not limited to any of the aforementioned protein(s), protein(s), antibody(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s), and that any protein(s), protein(s), antibody(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s) may be selected by any suitable means.

[0064] The present invention may be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0065] Example 1. Heavy Peptide Standard Strategy A heavy peptide standard strategy was designed to reliably identify sequence variants of a protein of interest. An exemplary workflow is shown in Figure 3. It should be understood that a variety of proteins of interest, liquid chromatography systems, and mass spectrometry systems can be used in the methods of the present invention.

[0066] Peptide standards having the amino acid sequences of known digested peptide fragments with sequence variants are synthesized with heavy isotopes at or near both peptide termini. The resulting heavy peptide standards elute at substantially the same retention time as the corresponding sequence variant peptide fragments, allowing for one-to-one identification of the heavy peptide standards and the sequence variant peptides. Mass spectrometry can then be used to distinguish between the peptide standards and the sequence variant peptides, as well as to further confirm the exact amino acid sequences of the sequence variant peptides.

[0067] Heavy isotopes placed at or near both peptide termini improve retention time, MS 1 Signal and MS 2 Fragmentation validation is possible, resulting in more confident identification and compilation of sequence variant libraries. The heavy peptide standards and the corresponding endogenous sequence variant peptides always co-elute, eliminating run-to-run retention time variations. A novel feature of the method of the present invention is the use of heavy isotopes at or near both termini of the heavy peptide standards, which allows for more accurate identification and better identification of sequence variant libraries. 2 The analysis is used to produce heavy fragment ions, allowing for convenient separation and identification of heavy peptide standards and corresponding sequence variant peptides. 2 The spectrum allows for the simultaneous identification of light and heavy fragment ions in the spectrum. Using the methods of the invention, detailed determinations such as isoleucine vs. leucine substitutions and precise pinpoint detection of sequence variant residues with multiple potential sites can be made.

[0068] The methods of the invention use small amounts of sample (e.g., about 1 pmol on column) and standards (e.g., about 10 fmol on column) and can be used to validate sequence variants in historical samples. In some exemplary embodiments, variants can be separated and identified even with very short gradients of as little as 11 minutes, for example using Evosep One™.

[0069] The co-elution and subsequent separation of the heavy peptide standards and the corresponding experimental peptides (characterized by the same amino acid sequence) is shown in Figure 4. The heavy peptide standards and the corresponding sequence variant peptides have consistent retention times as indicated by the substantially overlapping chromatographic peaks. MS 1 In the analysis, the peaks of the heavy peptide standards and the corresponding sequence variant peptides are separated by the mass difference attributable to the heavy isotope. 2 In the analysis, the fragment ion (e.g., a, b, c, x, y, or z ion) peaks of the heavy peptide standard and the corresponding sequence variant peptides are separated by mass differences attributable to the heavy isotopes at the respective peptide termini.

[0070] Example 2. NISTmAb Case Study The method of the present invention was performed using the NISTmAb standard and experimental antibodies. Exemplary heavy peptide standards used for the NISTmAb standard and experimental antibodies are shown in Figure 5. Known sequence variants are highlighted in red. Additional masses that can be attributed to heavy isotopes are shown in blue numbers. Heavy peptide standard sequences were selected to correspond to known sequence variants across the heavy constant gamma and kappa light chains, as shown in Figure 6.

[0071] Figure 7 shows a negative control using a NISTmAb sample. No sequence variant peptides were detected from NISTmAb, only the wild-type peptide, which does not have the same retention time as the heavy peptide standard. In Figure 7 and subsequent figures, the scale of the extracted ion chromatogram (XIC) and mass spectrum signals are not 1:1.

[0072] Figure 8A shows the identification of sequence variant peptides using the methods of the present invention. The sequence variant peptides have a longer retention time than the corresponding wild type peptide, but the same retention time as the corresponding heavy peptide standard. Specific amino acid sequences are identified using MS as shown in Figure 8B. 2 Check after fragmentation.

[0073] Using the methods of the invention, one can reliably distinguish between amino acid sequences featuring leucine or isoleucine (which may be difficult to distinguish with other methods because these amino acids have identical masses). Figure 9A shows an example of a peptide where the retention time of the sequence variant peptide is smaller than the retention time of the heavy peptide standard, representing a change to leucine. However, the MS peaks from the sequence variant peptide and the heavy peptide standard are 2 The signals are consistent, indicating that the unknown amino acid likely has the same mass as leucine, as shown in Figure 9B. Comparison of the heavy peptide standards representing the leucine variants to the isoleucine variants indicates that the isoleucine variants are expected to have a shorter retention time, as shown in Figure 9C. Direct comparison of the heavy peptide standards representing the isoleucine variants to the sequence variant peptides confirms that the sequence variants are characterized by a change to isoleucine, as shown in Figure 9D.

[0074] The methods of the present invention can also be used to exclude false positive identification of sequence variants. Figure 10A shows LC and MS signals from potential sequence variants with sizes corresponding to alanine substitutions. MS 2 The signal was insufficient to unambiguously identify the amino acid sequence of this peptide, as shown in FIG. 10B. To confirm the sequence variant content, heavy peptide standards featuring the predicted amino acid sequence were used. The corresponding heavy peptide standards did not share the same retention time as the putative sequence variant peptide, as shown in FIG. 10C. MS comparison with heavy peptide standards 2 Analysis revealed that the putative sequence variants were in fact products of non-specific cleavage, as shown in Figures 10D and 10E. Heavy peptide standards can be used to confirm or disprove the identity of the putative sequence variants, allowing for confident identification in comparison to existing methods.

[0075] Figure 11 shows a list of putative sequence variants identified from NISTmAb or experimental antibodies, and whether the sequence variant identification was confirmed or refuted using the methods of the invention. Using the methods of the invention, it was possible to confirm true positive identifications, exclude false positive identifications, and identify the specific amino acid sequences of isoleucine / leucine variants.

[0076] Example 3. Optimization of liquid chromatography gradients Sample sizes for sequence variant analysis can be very large, especially in design of experiments (DOE). To optimize the workflow of the method of the present invention, gradients of different speeds or durations were tested and compared for their effectiveness in identifying sequence variants.

[0077] An exemplary chromatography system useful in the present invention is the Evosep One™. Samples can be loaded onto an Evotip™ (a disposable trap column) and a gradient from two pumps elutes the sample peptides. During this elution, a secondary gradient from a separate, second pump offsets the first gradient to focus the sample peptides once they are introduced onto the analytical column. A high pressure pump pushes the preformed gradient and the pre-separated peptides onto the analytical column.

[0078] A total of five gradients were tested: an 11 (or 11.5) minute gradient, a 21 minute gradient, a 44 minute gradient, and an 88 minute gradient using Evosep One™, and a 95 minute gradient using ultra-performance liquid chromatography (UPLC®) (e.g., Waters® UPLC®). Each run was benchmarked and manually inspected against high-confidence sequence variants identified in the NISTmAb case study using 140 and 150 minute gradients, as well as additional variants detected by data-dependent acquisition (DDA) at 5% false discovery rate (FDR), as shown in FIG. 12. The NISTmAb sequence variants are those identified by two laboratories in Chapter 2 of State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. In addition to identifying sequence variants, the gradients were compared for retention time reproducibility, quantification, and spectral quality.

[0079] The retention time standard deviations for the gradients and peptides tested are shown in Figure 13. The retention time standard deviation for the 44 minute gradient using Evosep One™ is similar to the 95 minute UPLC run.

[0080] The gradients tested and the sequence variant content of the peptides are shown in Figure 14. The 88 and 44 min gradients using Evosep One™ identified more sequence variants than the 95 min UPLC run, including many novel sequence variants. However, even with the 11 min gradient, the target MS 2 sequence variants could be verified using

[0081] MS 2The spectral scores were higher for each Evosep One™ run than for the UPLC® run, as shown in Figure 16. Moderate overlap in quantitative values ​​was observed for all gradients tested, as shown in Figure 17. Peptides shown include extracted ion chromatogram (XIC) extracts of sequence variants not identified by DDA.

[0082] As shown above, the new heavy peptide standard method allows for convenient and robust validation of sequence variants for a protein of interest. This method reduces ambiguity in sequence variant analysis, increases the reliability of the results, and can be performed with relatively high throughput using short liquid chromatography gradients. The method of the present invention is not limited to sequence variant analysis, but can be generally used to identify and / or confirm the amino acid sequence of any peptide (e.g., any digested peptide of a protein of interest).

Claims

1. A method for identifying an amino acid sequence of a digested peptide of a target protein, comprising the steps of: (a) combining a peptide digest having digested peptides of a protein of interest with at least one heavy peptide standard to form a mixture, the at least one heavy peptide standard comprising a heavy isotope at or near each peptide terminus, and an amino acid sequence of the at least one heavy peptide standard being a predicted amino acid sequence of a digested peptide of the protein of interest; (b) subjecting the mixture to analysis using liquid chromatography-mass spectrometry; (c) comparing the retention time and / or at least one mass spectrum of said at least one heavy peptide standard with the retention time and / or at least one mass spectrum of said digested peptides; (d) using the comparison of (c) to identify the amino acid sequence of a digested peptide of the target protein.

2. The method of claim 1 , wherein the amino acid sequence is a sequence variant.

3. The method of claim 2 , wherein the sequence variant is an important quality characteristic.

4. 2. The method of claim 1, wherein the target protein is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, a host cell protein, or a protein pharmaceutical product.

5. 2. The method of claim 1, wherein the chromatography step comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

6. 2. The method of claim 1, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system.

7. 2. The method of claim 1, wherein the comparing step comprises determining whether the retention time of the at least one heavy peptide standard matches the retention time of the digested peptides.

8. The step of comparing comprises: １ The spectrum peaks correspond to the MS １ 2. The method of claim 1, comprising comparing the spectral peaks to determine whether they are shifted by the additional mass of a heavy isotope.

9. The step of comparing comprises: ２ The spectrum peaks correspond to the MS ２ 2. The method of claim 1, comprising determining whether the spectral peak is shifted by the additional mass of one of the heavy isotopes compared to the spectral peak.

10. 2. The method of claim 1, wherein the molar ratio of the peptide digest to the heavy peptide standard is from about 1:50 to about 1:200, or about 1:100.

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