Combination of fractionation based on isoelectric focusing and mass spectrometry

Abstract

The present invention relates generally to a method for characterizing charge variants of a protein of interest. In particular, the present invention relates to the use of desalting size-exclusion chromatography-reduced peptide mapping mass spectrometry to identify charge variants separated by capillary isoelectric focusing.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 217,125, filed on June 30, 2021, and U.S. Provisional Patent Application No. 63 / 301,350, filed on January 20, 2022, each of which is incorporated herein by reference.

[0002] Field This application relates to methods for characterizing charge variants of therapeutic proteins.

Background Art

[0003] Background Biophysical properties, including domain - specific variants of therapeutic peptides and proteins, can affect their safety, efficacy, and shelf - life. For example, the presence of different charge variants can change the solubility, binding, and stability of proteins.

[0004] Therapeutic peptides or proteins, such as antibodies, can acquire different variants and become heterogeneous due to various post - translational modifications (PTMs), proteolysis, enzymatic modifications, and chemical modifications. These changes to biophysical properties can occur at almost any point during and after the production of peptides and proteins. Since these changes to biophysical characteristics can affect the safety, efficacy, and shelf - life of therapeutic peptides and proteins, it is important to identify different variants for a particular therapeutic peptide or protein and, further, to investigate the modifications that cause charge variants.

[0005] Isoelectric focusing (IEF) has become a common tool for separating components of a sample based on charge (pI), thereby enabling the separation of protein charge variants. IEF analysis can also be combined with mass spectrometry (MS) to obtain further information about the protein associated with each charge variant. However, conventional methods are limited in the techniques that can be used in IEF-MS analysis. To date, it has been impossible to perform high-resolution reduced peptide mapping analysis of narrow IEF fractions. Therefore, it has not been possible to identify specific protein modification sites, such as amino acid residues, associated with specific charge variants.

[0006] Therefore, it will be understood that there is a need for methods and systems to specifically characterize modifications of therapeutic proteins related to charge variants. [Overview of the Initiative]

[0007] overview Methods have been developed for characterizing charge variants of proteins of interest. In exemplary embodiments, a sample containing the protein of interest is subjected to capillary isoelectric focusing. A UV trace of the protein sample is generated, containing UV peaks corresponding to the charge variants. Fractions of the sample are collected after isoelectric focusing. The fractions may be collected in a high-throughput manner to represent the total sample output from the isoelectric focusing step, and may include narrow intervals, e.g., 15-second intervals. In addition to modifying the fraction buffer to be suitable for mass spectrometry, the fractions are further processed using desalting size exclusion chromatography to separate the analytes by size. Finally, the eluate from the desalting size exclusion chromatography can be subjected to mass spectrometry, which can be used to identify specific post-translational modifications corresponding to each fraction and therefore each charge variant.

[0008] This disclosure provides a method for characterizing charge variants of a protein of interest. In some exemplary embodiments, the method includes (a) subjecting a sample containing the protein of interest to capillary isoelectric focusing to separate the charge variants of the protein of interest; (b) collecting a fraction from the capillary isoelectric focusing step; (c) subjecting the fraction to desalting size exclusion chromatography; and (d) subjecting the eluate from step (c) to mass spectrometry to characterize the charge variants of the protein of interest.

[0009] In one embodiment, the protein is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product. In another embodiment, the capillary isoelectric focusing is imaging capillary isoelectric focusing. In yet another embodiment, the desalting size exclusion chromatography system is connected to the mass spectrometer.

[0010] In one embodiment, the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer. In another embodiment, the mass spectrometry includes intact mass spectrometry or reduced peptide mapping analysis. In yet another embodiment, the mass spectrometer can perform multiple reaction monitoring or parallel reaction monitoring.

[0011] In one embodiment, the method further includes the step of contacting the fraction with at least one hydrolyzing agent prior to desalting size exclusion chromatography. In a particular embodiment, the at least one hydrolyzing agent is selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC, and ArgC.

[0012] In one embodiment, the method further includes the step of contacting the fraction with at least one reducing agent prior to desalting size exclusion chromatography. In another embodiment, the desalting size exclusion chromatography is performed under native conditions.

[0013] These and other aspects of the present invention will be better recognized and understood in conjunction with the following description and accompanying drawings. The following description illustrates various embodiments and numerous specific details thereof, but is not limiting and is given for illustrative purposes only. Many substitutions, modifications, additions, or rearrangements can be made within the scope of the present invention. [Brief explanation of the drawing]

[0014] [Figure 1] The workflow of the method of the present invention according to an exemplary embodiment is shown. [Figure 2A] This example illustrates the correlation between peaks from UV traces detected by capillary isoelectric focusing (cIEF) and corresponding peaks from desalting size exclusion chromatography-mass spectrometry (SEC-MS). [Figure 2B] The following illustrates charge variants identified by desalting SEC-MS and corresponding cIEF peaks and fractions according to an exemplary embodiment. [Figure 3A] The deconvoluted mass spectra of charge variants detected by desalting SEC-MS, corresponding to the main UV peaks detected by cIEF, are shown in an exemplary embodiment. [Figure 3B] The deconvoluted mass spectrum of the charge variant detected by desalting SEC-MS, corresponding to the B1 UV peak detected by cIEF, is shown in an exemplary embodiment. [Figure 3C] The deconvoluted mass spectrum of the charge variant detected by desalting SEC-MS, corresponding to the B2 UV peak detected by cIEF, is shown in an exemplary embodiment. [Figure 3D] The deconvoluted mass spectrum of the charge variant detected by desalting SEC-MS, corresponding to the A1 UV peak detected by cIEF, is shown in an exemplary embodiment. [Figure 3E]Shows the deconvoluted mass spectrum of charge variants detected by desalted SEC-MS corresponding to the A2 UV peak detected by cIEF, according to an exemplary embodiment. [Figure 3F] Shows the deconvoluted mass spectrum of charge variants detected by desalted SEC-MS corresponding to the A3 UV peak detected by cIEF, according to an exemplary embodiment. [Figure 4] Shows the reduced peptide mapping spectrum for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5A] Shows the distribution of aspartic acid isomerization at multiple amino acid residues for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5B] Shows the distribution of aspartic acid cyclization at multiple amino acid residues for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5C] Shows the distribution of asparagine deamidation at multiple amino acid residues for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5D] Shows the distribution of asparagine succinimide at multiple amino acid residues for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5E] Shows the distribution of lysine glycation at multiple amino acid residues for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5F] Shows the distribution of C-terminal lysine in each heavy chain for each UV peak detected by cIEF, according to an exemplary embodiment. [Figure 5G] Shows the distribution of N-acetylneuraminic acid for each UV peak detected by cIEF, according to an exemplary embodiment.

Mode for Carrying Out the Invention

[0015] Detailed explanation Therapeutic antibodies produced in mammalian cells (including monoclonal antibodies (mAbs) or bispecific antibodies (bsAbs)) are heterogeneous as a result of post-translational modifications (PTMs), enzymatic modifications, and chemical modifications, which contribute to size and charge variants. These modifications may include, for example, glycosylation, deglycosylation, amidation, deamidation, oxidation, glycation, terminal cyclization, C-terminal lysine mutations, C-terminal arginine mutations, N-terminal pyroglutamate mutations, C-terminal glycinamidation, C-terminal prolineamidation, succinimide formation, sialylation, or desialylation. Furthermore, aggregation, degradation, denaturation, fragmentation, or isomerization of protein products can also introduce charge heterogeneity. Table 1 shows exemplary protein modifications and their effects on changes in the charge of peptides or proteins.

[0016] [Table 1]

[0017] During the manufacture of therapeutic peptides or proteins, such as monoclonal antibodies, charge heterogeneity may be introduced as a result of proteolysis and / or the presence of PTMs. Characterization of the charge variant morphology of proteins within the manufactured active pharmaceutical ingredient is necessary to fully understand the correlation between protein properties, such as potency, and the physical and chemical changes associated with charge variants.

[0018] Several methods exist that enable the separation of protein charge variants, including ion exchange chromatography and isoelectric focusing (IEF). IEF has become a more common technique due to its capacity for high-resolution separation of sample components based on pI, and its ability to consider both surface-exposed and internal amino acids without loss of resolution due to hydrophobic interactions. IEF, particularly capillary IEF (cIEF), can also be combined with mass spectrometry (MS) to obtain further information about protein samples. However, the buffers used for cIEF and MS are not immediately compatible, which creates difficulties when using samples separated by cIEF for MS analysis. To solve this problem, two main approaches are employed: using either an offline or online connection to the MS.

[0019] When using an offline connection, fractions are collected from the cIEF, the buffer is modified to suit MS, and the modified fractions are subjected to MS analysis. However, fraction collection from the cIEF has low throughput, resulting in the collection of a small number of large fractions, which reduces the resolution and specificity of charge variant analysis.

[0020] Alternatively, an online connection may be used to output the separated sample from cIEF to MS without a fraction collection step. This requires an intermediate online step (e.g., preliminary chromatography or dialysis) to change the sample buffer between cIEF analysis and MS analysis. This online connection maintains the high-resolution separation of cIEF but is limited to intact mass spectrometry because it does not allow for further processing of the sample separated by cIEF, such as digestion or reduction of the protein of interest.

[0021] Therefore, there is a need for methods and systems to characterize charge variants of proteins of interest in a flexible and high-resolution manner. In particular, there is a need for methods to identify site-directed protein modifications associated with charge variants.

[0022] This disclosure presents a novel method for identifying site-directed protein modifications associated with charge variants of proteins of interest. This method utilizes cIEF with offline connectivity to MS. Unlike previous methods, cIEF fractions are collected in a comprehensive and high-throughput manner, for example, by collecting all output from the cIEF capillary and separating them into fractions, each representing a 15-second interval. This novel high-throughput fraction collection enables offline processing of cIEF-separated samples without substantial loss of pI resolution. The collected fractions can be further processed, for example, by contacting them with hydrolyzing agents and / or reducing agents to generate reduced peptides for reduced peptide mapping analysis. The collected fractions may be subjected to various processing steps according to user needs, or they may not be subjected to processing steps if, for example, they are used for intact mass spectrometry.

[0023] Next, the collected fractions are individually subjected to desalting size exclusion chromatography (SEC), as described, for example, in Yan et al., 2020, J Am Soc Mass Spectrom, 31:2171-2179. This high-throughput desalting SEC method allows for efficient processing of fractions, further separation of sample components by size, and modification of fraction buffers to suit MS. The desalting SEC system can be connected online to a mass spectrometer.

[0024] The fractions output from desalted SEC are subjected to MS analysis, such as intact mass spectrometry or reduced peptide mapping analysis. MS analysis generates fragments of the analyte and separates them based on the mass-to-charge (m / z) ratio. This separation allows for the identification of modifications to proteins, such as PTMs. In particular, the resolution of reduced peptide mapping analysis allows for site-specific identification of PTMs, e.g., identification of specific chemical changes at specific amino acid residues on the protein of interest. The identified site-specific modifications resulting from known cIEF fractions can then be associated with specific charge variants of the protein of interest. This method enables high-resolution, high-throughput analysis of site-specific protein modifications that give rise to charge variants, allowing for the monitoring and improvement of therapeutic protein production processes to verify and optimize the biophysical characteristics and homogeneity of proteins.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. Any methods and materials similar or equivalent to those described herein may be used in practice or testing, but specific methods and materials are described herein.

[0026] The term “one (a)” should be understood to mean “at least one,” and the terms “about” and “approximately” should be understood to allow for a standard deviation, as understood by those skilled in the art, and to include the endpoint where a range is provided. As used herein, the terms “include,” “includes,” and “including” are understood to be non-limiting, meaning “comprise,” “comprises,” and “comprising,” respectively.

[0027] As used herein, the terms “protein” or “protein of interest” may include any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains commonly known in the art as “polypeptides.” A “polypeptide” is a polymer composed of amino acid residues, associated naturally occurring structural variants, and naturally occurring analogs of its synthesis, linked via peptide bonds. A “synthetic peptide or polypeptide” is a peptide or polypeptide that does not exist naturally. Synthetic peptides or polypeptides can be synthesized, for example, using automated polypeptide synthesizers. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein may comprise one or more polypeptides to form a single functional biomolecule. In another exemplary embodiment, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. A protein of interest may include any of the following: a biotherapeutic protein, a recombinant protein used in research or therapy, a capture protein and other chimeric receptor Fc fusion proteins, a chimeric protein, an antibody, a monoclonal antibody, a polyclonal antibody, a human antibody, and a bispecific antibody. Proteins can be produced using recombinant cell-based production systems, such as insect baculoviruses, yeasts (e.g., species of the genus Pichia (Pichia sp.)), and mammals (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).For recent reviews discussing 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 teaching is incorporated herein). In some exemplary embodiments, the protein includes modifications, adducts, and other covalent moieties. Examples of these modifications, adducts, and parts include avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels, and other dyes. Proteins can be classified based on their composition and solubility, and thus may include simple proteins such as globular proteins and fibrous proteins, conjugated proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins, and derived proteins such as primary-derived proteins and secondary-derived proteins.

[0028] In some exemplary embodiments, the protein of interest may be a recombinant protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a fusion protein, an scFv, or a combination thereof.

[0029] As used herein, the term “recombinant protein” refers to a protein produced as a result of transcription and translation of a gene held on a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an isotype antibody selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule may be a full-length antibody (e.g., IgG1), or the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0030] As used herein, the term “antibody” includes immunoglobulin molecules and their polymers (e.g., IgM), comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain (CL1). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus. In different embodiments of the present invention, the FRs of the anti-big ET-1 antibody (or its antigen-binding moiety) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding moiety” of an antibody and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, 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 obtained from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding an antibody variable domain and optionally a constant domain. Such DNA is publicly known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized.DNA can be sequenced and manipulated using chemical or molecular biological techniques to, for example, position one or more variable and / or constant domains into a preferred configuration, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0031] As used herein, “antibody fragment” includes a portion of an intact antibody (e.g., the 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, 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 the immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the immunoglobulin light and heavy chain variable regions are linked by a peptide linker. In some exemplary embodiments, an antibody fragment includes a sufficient amino acid sequence of a parent antibody, which is a fragment that binds to the same antigen as the parent antibody; 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 may be produced enzymatically or chemically by fragmentation of an intact antibody, and / or by recombination from a gene encoding a partial antibody sequence. Alternatively, antibody fragments may be produced entirely or partially synthetically. Antibody fragments may optionally include single-chain antibody fragments. Alternatively, antibody fragments may also include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, and more typically, at least about 200 amino acids.

[0032] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody typically contains two distinct heavy chains, each specifically binding to a different epitope on either two different molecules (e.g., antigens) or the same molecule (e.g., the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody may be present on the same or different targets (e.g., the same or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable regions that recognize different epitopes of the same antigen can be fused with nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains.

[0033] A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, as well as an immunoglobulin light chain that does not confer antigen-binding specificity but can either associate with each heavy chain, or can associate with each heavy chain and bind to one or more epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain and allow one or both heavy chains to bind to one or both epitopes. bsAbs can be divided into two main classes: those with an Fc region (IgG-like) and those without an Fc region, the latter usually being smaller than IgG and IgG-like bispecific molecules that contain Fc. IgG-like bsAbs may take various forms, including but not limited to triomabs, knob-into-hole IgG (kih IgG), crossMab, orth-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual-action Fab (DAF), IgG-single-stranded Fv (IgG-scFv), or κλ bodies. Non-IgG-like variants include tandem scFv, diabody form, single-stranded diabody, tandem diabody (TandAb), biaffinity retargeting molecule (DART), DART-Fc, nanobody, or antibodies produced by the Dock-and-Lock (DNL) method (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 entire teaching is incorporated herein). Methods for producing bsAb are not limited to genetic techniques utilizing quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical linking including chemical crosslinking agents, and recombinant DNA technology.Examples of bsAb include the following patent applications incorporated herein by reference: U.S. Patent Application No. 12 / 823838 filed June 25, 2010; U.S. Patent Application No. 13 / 488628 filed June 5, 2012; U.S. Patent Application No. 14 / 031075 filed September 19, 2013; U.S. Patent Application No. 14 / 808171 filed July 24, 2015; and U.S. Patent Application No. 1 filed September 22, 2017. Examples include those disclosed in U.S. Patent Application No. 5 / 713574, U.S. Patent Application No. 15 / 713569 filed on 22 September 2017, U.S. Patent Application No. 15 / 386453 filed on 21 December 2016, U.S. Patent Application No. 15 / 386443 filed on 21 December 2016, U.S. Patent Application No. 15 / 22343 filed on 29 July 2016, and U.S. Patent Application No. 15814095 filed on 15 November 2017.

[0034] As used herein, “multispecific antibody” refers to an antibody that has binding specificity to at least two different antigens. Such molecules typically bind to only two antigens (i.e., bispecific antibodies, bsAb), but antibodies with further specificity, such as triplicate antibodies and KIH triplicate antibodies, can also be addressed by the systems and methods disclosed herein.

[0035] As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be obtained from a single clone, including any eukaryote, prokaryote, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in this disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display technologies, or combinations thereof.

[0036] In some exemplary embodiments, the protein of interest may have a pI in the range of about 4.5 to about 9.0. In one exemplary embodiment, the pI may be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some exemplary embodiments, there may be two or more types of the protein of interest in the composition.

[0037] In some exemplary embodiments, the protein of interest may be produced from mammalian cells. Mammalian cells may be of human or non-human origin and may include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells and retinal epithelial cells), established cell lines and their strains (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLaS3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clones M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells. Cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells or their derivatives), fibroblasts derived from any tissue or organ (including, but not limited to, the heart, liver, kidney, colon, intestine, esophagus, stomach, and nervous tissue (brain, spinal cord)), lungs, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), spleen, and fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit551 cells, Detroit510 cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR- 90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M -May include MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, mouse L cell line 2071, mouse L cell line LM, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian mosquito cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells or their derivatives.

[0038] In some exemplary embodiments, a sample containing the protein of interest can be prepared before desalting SEC-MS analysis. The preparation steps may include alkylation, reduction, denaturation, and / or digestion.

[0039] As used herein, the term “protein alkylating agent” refers to a drug used to alkylate specific free amino acid residues in a protein. Non-exclusive examples of protein alkylating agents include iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0040] As used herein, “protein denaturation” may refer to the process by which the three-dimensional shape of a molecule changes from its native state. Protein denaturation can be carried out using protein denaturants. Non-limiting examples of protein denaturants include heat, high or low pH, reducing agents such as DTT (see below), or exposure to chaotropic agents. Multiple 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 bonds, 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 their salts.

[0041] As used herein, the term “protein reducing agent” refers to a drug used to reduce disulfide crosslinks in proteins. Non-exclusive examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Elman’s reagent, hydroxylamine hydrochloride, sodium borohydride cyanohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof.

[0042] As used herein, the term “digestion” refers to the hydrolysis of one or more peptide bonds of a protein. Several methods exist for digesting proteins in a sample using appropriate hydrolyzing agents (e.g., enzymatic or non-enzymatic digestion).

[0043] As used herein, the term “digestive enzyme” refers to any of a number of different drugs capable of digesting proteins. Non-limiting examples of hydrolysants capable of enzymatic digestion include proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin 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), immunoglobulinase (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or their biologically active fragments or homologs, 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 Switzar, Martin Giera & Wilfried MANiessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013)).

[0044] As used herein, the terms “charge variant” or “variant” of a polypeptide refer to a polypeptide containing an amino acid sequence that is at least about 70–99.9% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to the reference or native amino acid sequence of the protein of interest. Sequence comparison can be performed, for example, by the BLAST algorithm, where the algorithm parameters are selected to give the greatest match between each sequence over the full length of each reference sequence (e.g., expected threshold: 10; word size: 3; maximum match within query range: 0; BLOSUM62 matrix; gap cost: present 11, extended 1; conditional composition score matrix adjustment). Polypeptide variants can also refer to polypeptides containing the referenced amino acid sequence, excluding one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) such as missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. The following references relate to the BLAST algorithm, which is often used for sequence analysis. BLAST ALGORITHMS: Altschul et al.(2005)FEBSJ.272(20):5101-5109, Altschul,SF,et al.,(1990)J.Mol.Biol.215:403-410,Gish,W.,et al.,(1993)Nature Genet.3:266-272, Madden,TL,et al.,(1996)Meth.Enzymol.266:131-141,Altschul,SF,et al.,(1997)Nucleic Acids Res.25:3389-3402,Zhang,J.,et al.,(1997)Genome Res.7:649-656, Wootton, JC, et al. al., (1993) Comput. Chem. 17:149-163, Hancock, JMet al., (1994) Comput. Appl. Biosci.10:67-70; Alignment Scoring Systems: Dayhoff, M.O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C., Schwartz, R.M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. “M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C., Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70, Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919, Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; Alignment Statistics: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039, and Altschul, S.F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, NY; all of these teachings are incorporated herein.

[0045] Some variants may be covalent modifications that polypeptides undergo either during (simultaneous translational modification) or after (post-translational modification "PTM") ribosome synthesis. PTMs are generally introduced by specific enzymes or enzymatic pathways. Many are located at specific characteristic protein sequences (e.g., signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some aspect of the protein's structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853, the full text is incorporated herein).

[0046] In certain exemplary embodiments, the protein composition may contain variants of one or more types of the protein of interest. Such variants may include both acidic and basic species. Acidic species are typically variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while basic species are variants that elute later than the main peak from CEX or earlier than the main peak from AEX. In exemplary embodiments, basic species may elute earlier than the main peak from cIEF, and acidic species may elute later than the main peak from cIEF.

[0047] As used herein, the terms “acidic species,” “AS,” “acidic region,” and “AR” refer to protein variants characterized by an overall acidic charge.

[0048] In certain embodiments, the sample may contain more than one type of acidic species variant. For example, but not limited to, the total acidic species can be classified based on the chromatographic retention time of the peaks that appear, or by the UV peaks generated using IEF.

[0049] Among the chemical degradation pathways that result in acidic or basic species, the two most commonly observed covalent modifications in proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan, and tyrosine are some of the most susceptible amino acids to oxidation. Met and Cys are susceptible due to their sulfur atoms, while His, Trp, and Tyr are susceptible due to their aromatic rings.

[0050] As used herein, the terms “oxidized species,” “OS,” or “oxidized variant” refer to protein variants formed by oxidation. Such oxidized species can also be detected by various methods such as ion exchange, e.g., WCX-10 HPLC (weak cation exchange chromatography), or IEF. Oxidized variants may arise from oxidation occurring at histidine, cysteine, methionine, tryptophan, phenylalanine, and / or tyrosine residues.

[0051] As used herein, the terms “basic species,” “basic region,” and “BR” refer to variants of a protein, such as an antibody or its antigen-binding moiety, characterized by an overall basic charge compared to primary charge variant species present within the protein. For example, in recombinant protein preparations, such basic species can be detected by various methods (e.g., ion exchange (e.g., WCX-10 HPLC (weak cation exchange chromatography)) or IEF). Exemplary variants may include, but are not limited to, lysine variants, aspartic acid isomerization, succinimide formation at asparagine, methionine oxidation, amidation, incomplete disulfide bond formation, serine-to-arginine mutation, deglycosylation, fragmentation, and aggregation. Typically, basic species elute later than the main peak in CEX or earlier than the main peak in AEX analysis. (Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs. 2012 Sep 1;4(5):578-585. doi:10.4161 / mabs.21328, the entire instruction is incorporated herein by reference).

[0052] In certain embodiments, a sample may contain two or more types of basic species variants. For example, but not limited to, all basic species may be split based on the chromatographic retention time of the resulting peaks, or based on the UV peaks generated using IEF. Another example of all basic species being split may be based on the type of variant—multiple variants, structural variants, or fragmentation variants.

[0053] As used herein, “Sample” can be obtained from any step in a bioprocess, such as a cell culture fluid (CCF), a harvested cell culture fluid (HCCF), any step in a downstream process, a drug substance (DS), or a formulation (DP) containing the final formulated product. In some other specific exemplary embodiments, the sample may be selected from any step in a downstream process, such as clarification, chromatographic generation, viral inactivation, or filtration. In some specific exemplary embodiments, the formulation may be selected from a manufactured formulation in a clinic, during shipment, storage, or handling.

[0054] In some embodiments, the disclosed method may include subjecting a sample to capillary isoelectric focusing to separate charge variants of the protein of interest.

[0055] As used herein, “isoelectric focusing” or “IEF” (also known simply as electrophoresis) is a technique for separating charged molecules (typically proteins or peptides) based on their isoelectric points (pI) (e.g., the pH at which a molecule has no charge). IEF works because, in an electric field, molecules in a pH gradient move toward their pI. Various techniques exist for performing IEF. For example, in capillary isoelectric focusing (cIEF), the sample moves through a capillary based on an applied electric field. A UV detector can be used at a point along the capillary to detect the time it takes for the analyte (e.g., a protein) to cross that point in the capillary. Since the time it takes to move through the capillary is directly related to the charge (pI) of the analyte, the UV signal from the point in the capillary over time can be represented as a UV trace, which represents the diverse charge (pI) of the sample components. In an exemplary embodiment, the UV trace generated by the cIEF represents the charge variant of the protein of interest, and each UV peak represents a significant charge variant. Variations of the cIEF, such as imaging cIEF (icIEF), can also be used.

[0056] Size exclusion chromatography (SEC) or gel filtration relies on the separation of components as a function of their molecular size. Separation depends on the amount of time a substance spends in a porous stationary phase compared to the time it spends in a fluid. The probability of a molecule being present in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a substance to penetrate the pores is determined by the diffusion mobility of the polymer, which is higher for smaller polymers. Very large polymers may not penetrate the pores of the stationary phase at all, while for very small polymers, the probability of penetration is close to 1. Components with larger molecular sizes pass through the stationary phase more quickly, while components with smaller molecular sizes have longer pathways through the pores of the stationary phase and are therefore retained in the stationary phase for longer.

[0057] Chromatographic materials may include size exclusion materials, which are resins or membranes. The matrix used for size exclusion is preferably an inert gel medium, which may be a complex of crosslinked polysaccharides (e.g., crosslinked agarose and / or dextran in the form of spherical beads). The degree of crosslinking determines the size of the pores present in the swollen gel beads. Molecules larger than a certain size do not enter the gel beads and therefore move through the chromatography bed most quickly. Smaller molecules, such as surfactants, proteins, and DNA, enter the gel beads to varying degrees depending on their size and shape, and their passage through the bed is slower. Therefore, molecules are generally eluted in an order of decreasing molecular size.

[0058] Suitable porous chromatography resins for viral size exclusion chromatography can be made from dextrose, agarose, polyacrylamide, or silica, each possessing different physical properties. Polymer combinations can also be used. The most commonly used is the "SEPHADEX" branded resin, commercially available from Amersham Biosciences. Other size exclusion supports derived from different construction materials are also suitable, such as Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, CA).

[0059] In some exemplary embodiments, the SEC may be operated in “desalting mode” to achieve online buffer exchange before native MS detection. Desalting aims to remove buffer salts from the sample by exchanging them for water (along with the water used to pre-equilibrate the SEC resin). A desalting SEC method suitable for the present invention is described, for example, Yan et al., 2020, J Am Soc Mass Spectrom, 31:2171-2179. Desalting the SEC allows for subsequent MS analysis of the sample eluted from the cIEF, which may otherwise have unsuitable buffer conditions.

[0060] The protein load of a sample containing the protein of interest can be adjusted to a total protein load on the column of approximately 50 g / L to 1000 g / L; approximately 5 g / L to 150 g / L; approximately 10 g / L to 100 g / L; approximately 20 g / L to 80 g / L; approximately 30 g / L to 50 g / L; or approximately 40 g / L to 50 g / L. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to the protein concentration of the material loaded onto the column of approximately 0.5 g / L to 50 g / L; or approximately 1 g / L to 20 g / L.

[0061] As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their precise masses. This term implies the inclusion of any molecular detector capable of characterizing polypeptides or peptides. A mass spectrometer may include three main components: 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 moved to the gas phase and can be ionized simultaneously (as in electrospray ionization) or through a separate process. The choice of ion source depends on the application. In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes a technique in which structural information about a sample molecule is obtained by using multiple steps of mass selection and mass separation. A requirement is that the sample molecule is converted to the gas phase and ionized so that fragments are formed in a predictable and controllable manner after the first mass selection step. Multi-stage MS / MS, or MS n As long as meaningful information can be obtained or fragment ion signals can be detected, the precursor ion (MS) is first detected. 2 ) to select and isolate, fragment it, primary fragment ions (MS 3 ) to isolate, fragment it, secondary fragment (MS 4This can be carried out by isolating the ions, etc. Tandem MS is successfully performed using a wide range of analyzer combinations. The choice of analyzer combination for a particular application can be determined by many different factors, such as size, cost, and availability, as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are tandem-in-space and tandem-in-time, but hybrids also exist in which a tandem-in-time analyzer is coupled with a tandem-in-space analyzer or in space. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-capture mass spectrometers. A particular m / z separation function may be designed so that ions are selected in one section of the instrument, dissociated in an intermediate region, and then the resulting ions are sent to another analyzer for m / z separation and data acquisition. In the case of tandem-in-time, the ions in the mass spectrometer generated in the ion source can be captured, isolated, fragmented, and m / z separated in the same physical device. Peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter being generated from possible peptides in a protein sequence database. Characterization methods include, but are not limited to, amino acid sequencing of protein fragments, protein sequencing determination, protein denovo sequencing determination, locating or identifying post-translational modifications, equivalence / homogeneity analysis, or a combination thereof.

[0062] In some exemplary embodiments, the mass spectrometer may be equipped with and operated by a nanoelectrospray or nanospray.

[0063] As used herein, the terms “nanoelectrospray” or “nanopray” often refer to electrospray ionization of sample solutions at very low solvent flow rates, typically hundreds of nanoliters / minute or less, without the use of external solvent delivery. The electrospray injection setup forming the nanoelectrospray can use either a static or dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small amounts of sample (analyte) solution over extended periods. A dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separation of the mixture before analysis by mass spectrometer.

[0064] In some exemplary embodiments, SEC-MS can be implemented under native conditions.

[0065] As used herein, the term “native conditions” may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see the review by Elisabetta Boeri Erba & Carlo Pe-tosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0066] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer.

[0067] As used herein, the term “tandem mass spectrometry” includes techniques in which structural information about sample molecules is obtained by using multiple steps of mass selection and mass separation. Requirements include that the sample molecules can be moved to the gas phase and ionized intact, and that these molecules can be induced to disintegrate in a somewhat predictable and controllable manner after the first mass selection step. Multi-stage MS / MS, or MS n As long as meaningful information can be obtained or fragment ion signals can be detected, the precursor ion (MS) is first detected. 2 ) to select and isolate, fragment it, primary fragment ions (MS 3 ) to isolate, fragment it, secondary fragment (MS 4 This can be carried out by isolating ions, etc. Tandem MS is successfully performed using a wide range of analyzer combinations. The choice of analyzer combination for a particular application can be determined by many different factors, such as size, cost, and availability, as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are tandem-in-space and tandem-in-time, but hybrids also exist in which a tandem-in-time analyzer is coupled with a tandem-in-space analyzer or in space. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-capture mass spectrometers. A particular m / z separation function may be designed so that ions are selected in one section of the instrument, dissociated in an intermediate region, and then the resulting ions are sent to another analyzer for m / z separation and data acquisition. In the case of tandem-in-time, the mass spectrometer ions generated in the ion source can be captured, isolated, fragmented, and m / z separated in the same physical device.

[0068] Peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter being generated from possible peptides in a protein sequence database. Characterization methods include, but are not limited to, amino acid sequencing of protein fragments, protein sequencing determination, protein denovo sequencing determination, locating or identifying post-translational modifications, equivalence / homogeneity analysis, or a combination thereof.

[0069] As used herein, the term “database” refers to an edited collection of protein sequences that may be present in a sample, for example, in the form of FASTA format files. The relevant protein sequences may originate from the cDNA sequences of the species being studied. Public databases that may be used to search for relevant protein sequences include, for example, databases maintained by Uniprot or Swiss-prot. Databases can be searched using what is referred herein as a “bioinformatics tool.” The bioinformatics tool provides the capacity to search for uninterpreted MS / MS spectra for all possible sequences in the database and provides interpreted (annotated) MS / MS spectra as output. Non-exclusive 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).

[0070] In some exemplary embodiments, the mass spectrometer is connected to a chromatography system, such as a SEC or desalting SEC.

[0071] The present invention is not limited to any of the aforementioned proteins, antibodies, pIs, protein alkylating agents, protein denaturants, protein reducing agents, digestive enzymes, hydrolysants, charge variants, post-translational modifications, samples, IEF systems, SEC systems, mass spectrometers, databases, or bioinformatics tools, and it is understood that any protein, antibody, pI, protein alkylating agent, protein denaturant, protein reducing agent, digestive enzyme, hydrolysant, charge variant, post-translational modifications, samples, IEF systems, SEC systems, mass spectrometers, databases, or bioinformatics tools may be selected by any preferred means.

[0072] The present invention will be better understood by referring to the following embodiments. However, they should not be construed as limiting the scope of the invention. [Examples]

[0073] Example 1. Overview of the present invention method A novel method for characterizing charge variants of a protein of interest is disclosed herein. An exemplary workflow of the method of the present invention is shown in Figure 1. A sample, e.g., from a therapeutic protein product or a protein of interest production step, is subjected to capillary isoelectric focusing (cIEF). cIEF separates components of a protein sample based on charge (pI), including separating charge variants of the protein of interest. UV traces of the sample components are generated across the cIEF capillary, with maximum protein concentrations considered as "peaks," corresponding to protein charge variants. Fractions are collected sequentially from the capillary. Fractions may be collected in a high-throughput manner so that all of the cIEF eluate is collected, and the fractions show narrow intervals (e.g., 15-second intervals). This high-throughput fraction collection allows for the maintenance of high-resolution separation of cIEF without requiring an online connection to a mass spectrometer for subsequent MS analysis.

[0074] Optionally, the fractions may be exposed to hydrolyzing agents, alkylating agents, and / or reducing agents to produce reduced peptide fragments of the protein of interest. The fractions may be recombined before subsequent analyses, depending on the desired concentration and resolution of the analysis.

[0075] Next, the fraction is subjected to desalting size exclusion chromatography (SEC). Desalting SEC serves two purposes: to replace the buffer from the collected cIEF fraction with a buffer suitable for mass spectrometry (MS), and to further separate the fraction components based on size.

[0076] Finally, the eluate from the desalted SEC is subjected to mass spectrometry, such as intact mass spectrometry or reduced peptide mapping analysis. The desalted SEC may be connected online to a mass spectrometer (desalted SEC-MS). In particular, reduced peptide mapping analysis allows for the identification of site-specific protein modifications that may contribute to charge variations. Since the identified protein modifications arise from known fractions, and these fractions coincide with known portions of the cIEF UV trace, a causal relationship can be established between site-specific protein modifications and protein charge variants.

[0077] Example 2. Intact mass spectrometry of protein charge variants Using the method of the present invention, the bispecific antibody bsAb-1 was subjected to the steps of cIEF, fractionation, and desalting SEC-MS. The UV trace generated by cIEF (shown in red) and the MS signal generated by desalting SEC-MS (shown in blue) can be correlated as shown in Figure 2A. Six UV peaks (corresponding to charge variants), namely B2, B1, Main, A1, A2, and A3 (in order from basic to acidic), were designated in this UV trace. Each blue peak represents the desalting SEC-MS analysis of the fraction.

[0078] Desalting SEC-MS analysis of the cIEF fraction allows for the assignment of specific protein modifications to charge variants, as shown in Figure 2B. Since each desalting SEC-MS analysis arises from a known fraction, and each fraction represents a known portion of the UV trace, the protein modifications detected by MS can be directly correlated with the charge variants detected by cIEF. In this way, the specific causes of charge variants in the protein of interest can be revealed.

[0079] Further analysis is shown in Figure 3. Deconvoluted mass spectra for each charge variant of bsAb-1 detected by desalting SEC-MS are shown, each featuring various m / z peaks that can be identified by specific protein modifications. Figure 3A shows the major charge variant and the peak representing the major mAb species. Figure 3B shows the B1 charge variant and the peak representing the species with C-terminal lysine. Figure 3C shows the B2 charge variant and the peak representing the species with two C-terminal lysine. Figure 3D shows the A1 charge variant with one peak representing the species with glycation modification and one peak representing the species with deamidation modification. Figure 3E shows the A2 charge variant with one peak representing the species with glycation / glucuronyl modification, one peak representing the species with deamidation modification, and one peak representing the species with N-acetylneuraminate modification. Figure 3F shows the A3 charge variant, one peak representing a species with glycation / glucuronyl modification, one peak representing a species with deamidation modification, and one peak representing a species with two N-acetylneuraminic acid modifications.

[0080] These experiments demonstrate the capacity of the present invention's method to use desalted SEC-MS to correspond to charge variants detected by cIEF and determine the specific protein modifications that cause them.

[0081] Example 3. Reduced peptide mapping analysis of protein charge variants The method of the present invention can also be used in conjunction with reduced peptide mapping analysis to identify protein modifications at specific residues of a protein of interest and to associate these site-specific modifications with charge variants of the protein. The bispecific antibody bsAb-1 was subjected to cIEF, fractionation, and desalting SEC-MS steps as previously described. After subjecting the fractions to protein reduction and hydrolysis, they were subjected to desalting SEC to produce reduced peptide fragments.

[0082] Exemplary MS signals corresponding to each charge variant detected by cIEF are shown in Figure 4. Peptides were analyzed using peptide mapping to identify specific protein modifications at particular residues, as shown in Figure 5. Using this method, a statistical distribution of charge variants of protein modifications at specific amino acid residues could be established. Figures 5A–5G show exemplary modifications that can result in charge variants, including aspartate isomerization, aspartate cyclization, asparagine deamidation, asparagine succinimide, lysine glycosylation, C-terminal lysine, or N-acetylneuraminic acid. Specific modified residues are identified across the light chain (LC), first heavy chain (HC), or second heavy chain (HC*) of bsAb-1.

[0083] These experiments demonstrate the capacity of the present invention to provide amino acid residue-level resolution for specific protein modifications that can produce specific charge variants of the protein of interest. This information can be used, for example, to monitor and / or modify the process of generating therapeutic proteins to achieve acceptable biophysical properties and product homogeneity.

Claims

1. A method for characterizing the charge variant of a protein of interest, (a) To separate the charge variant of the protein of interest, the sample containing the protein of interest is subjected to capillary isoelectric focusing, (b) Collecting fractions from the capillary isoelectric focusing step, (c) Subject the fraction to desalting size exclusion chromatography, (d) To characterize the charge variant of the protein of interest, the eluate from step (c) is subjected to mass spectrometry. Methods that include...

2. The method according to claim 1, wherein the protein is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

3. The method according to claim 1, wherein the capillary isoelectric focusing electrophoresis is imaging capillary isoelectric focusing electrophoresis.

4. The method according to claim 1, wherein the desalting size exclusion chromatography system is connected to the mass spectrometer.

5. The method according to claim 1, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.

6. The method according to claim 1, wherein the mass spectrometry includes intact mass spectrometry or reduced peptide mapping analysis.

7. The method according to claim 1, wherein the mass spectrometer can perform multiple reaction monitoring or parallel reaction monitoring.

8. Step 1: Before desalting size exclusion chromatography, the fraction is brought into contact with at least one hydrolyzing agent. The method according to claim 1, further comprising:

9. The method according to claim 8, wherein the at least one hydrolyzing agent is selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC, and ArgC.

10. Step 1: Before desalting size exclusion chromatography, the fraction is brought into contact with at least one reducing agent. The method according to claim 1, further comprising:

11. The method according to claim 1, wherein the desalting size exclusion chromatography is performed under native conditions.

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