Mass spectrometry-based strategies for determining product-associated variants of biologics

Abstract

The present invention relates to the field of protein characterization, and in particular to a method for identifying key quality attributes of therapeutic proteins expressed in host cells by performing a workflow involving competitive binding assays with poor antigens followed by the use of SCX-MS.

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 / 221,436, filed July 13, 2021, which is incorporated herein by reference.

[0002] Field The present invention relates generally to methods for determining product-associated variants important for maintaining the structure and function of biologics using a competitive binding mass spectrometry workflow. [Background technology]

[0003] background Biologics have emerged as important drugs for the treatment of cancer, autoimmune diseases, infectious diseases, and cardiometabolic disorders, and they represent one of the fastest growing product segments in the pharmaceutical industry. Biologics must meet very high purity standards. Therefore, it can be important to monitor impurities at different stages of drug development, production, storage, and handling. It is often difficult to fully assess the impact of numerous quality attributes that may be related to safety and efficacy. The impact of manufacturing process parameters and material properties on product quality variability is also difficult to fully characterize.

[0004] For robust manufacturing operations, it is critical to develop and improve an integrated control strategy over time, based on systematic process characterization, along with implementing appropriate risk assessment and mitigation measures throughout the product lifecycle. Thus, the need for quality by design exists. The United Nations World Health Organization (WHO) recommends quality by design as the standard, since it is difficult (and virtually impossible) to implement effective quality control by only testing the product after the fact. Critical quality attributes (CQAs) serve as benchmarks around which most quality by design implementations revolve. CQAs are physical, chemical, biological, or microbiological properties or characteristics that should be within appropriate limits, ranges, or distributions to ensure the desired product quality. CQAs are generally associated with drug substances, excipients, intermediates (materials in process), and drug products. For biologics, CQAs can be product- or process-related impurities. Product-related impurities can include size variants (aggregates or fragments), variants with post-translational modifications, or charge variants. Process-related impurities are inherent parts of the process such as host cell DNA or host cell proteins (HCPs), leachables (such as Protein A), and viruses. The presence of these impurities in the final drug product can affect product purity, product potency, and stability.

[0005] Therefore, identifying the CQAs of a biologic can be a complex process. Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS), electrospray ionization-mass spectrometry (ESI-MS), fractionation, or variant identification can be used for physicochemical characterization of intact or digested biologics. Activity characterization can be performed using ELISA-based bioassays, cell-based bioassays, or surface plasmon resonance (SPR) or biolayer interferometry (BLI) for binding activity. In these methods, product-associated CQAs must first be enriched or isolated and then evaluated individually or based on experience or prior knowledge. Such an approach to the workflow can result in reduced throughput.

[0006] Thus, there is a long-felt need in the art for efficient methods for determining such quality control characteristics. Summary of the Invention

[0007] overview The exemplary embodiments disclosed herein fulfill the aforementioned need by providing a method for identifying product-associated CQAs by enriching them.

[0008] The present disclosure provides for characterizing at least one product associated variant, the method comprising: Obtaining a sample comprising a protein of interest and at least one product-associated variant of the protein of interest; exposing the sample to competitive binding conditions containing insufficient target immobilized on beads; washing the beads to collect the flow-through; subjecting the flow-through to liquid chromatography-mass spectrometry analysis to separate the protein of interest and the at least one product-associated variant; characterizing said at least one product-associated variant, comparing the abundance of said at least one product-associated variant to the abundance of said at least one product-associated variant obtained from liquid chromatography-mass spectrometry analysis of a control sample prior to contacting said sample with said competitive binding conditions; Includes.

[0009] In one aspect of this embodiment, the target is an antigen directed to a protein of interest.

[0010] In one aspect of this embodiment, the binding conditions provide an underweight target immobilized on the bead. In the same or another aspect of this embodiment, at least one product-associated variant is impaired in binding to the underweight target.

[0011] In one aspect of this embodiment, the liquid chromatography is cation exchange chromatography. In a particular aspect of this embodiment, the liquid chromatography is strong cation exchange chromatography.

[0012] In one aspect of this embodiment, the mass spectrometer is an electrospray ionization mass spectrometer. In a particular aspect of this embodiment, the mass spectrometer is a nano-electrospray ionization mass spectrometer.

[0013] In one aspect of this embodiment, the beads are magnetic. In another aspect of this embodiment, the beads are non-magnetic. In a further aspect, the beads are agarose beads. In yet another aspect, the beads can be coated with a peptide or protein.

[0014] In one aspect of this embodiment, the flow-through is enriched for the at least one product-associated variant.

[0015] In the same or another aspect of this embodiment, the flow-through is collected by centrifugation.

[0016] In one aspect of this embodiment, the target is biotinylated prior to immobilization on the beads. In the same or other aspects of this embodiment, the beads are coated with streptavidin resin. In a particular aspect of this embodiment, the beads are non-magnetic. In another particular aspect, the beads are magnetic.

[0017] In one aspect of this embodiment, the insufficient target is such that the amount of the target allows for binding of about 30% to about 80% of the protein of interest.

[0018] In another aspect of this embodiment, the sample is incubated for about 1 hour before washing. In the same or other aspects of this embodiment, the sample is incubated at room temperature before washing.

[0019] In one aspect of this embodiment, the method can identify two or more product-associated variants. In certain aspects, the product-associated variants include a size variant. In certain aspects, the size variant is a fragmentation variant of the protein of interest. In certain aspects, the size variant is an aggregation variant of the protein of interest.

[0020] In one aspect of the embodiment, the product-associated variant comprises a charge variant of the protein of interest. In a particular aspect, the product-associated variant comprises a post-translational modification variant of the protein of interest.

[0021] In one aspect of this embodiment, the product associated variant is classified as a critical quality attribute if the abundance of the at least product associated variant is significantly greater than the abundance of the at least product associated variant in the sample prior to contacting the sample with the competitive binding conditions. [Brief description of the drawings]

[0022] [Figure 1] 1 is a depiction of the different possible product-associated variants of antibodies, including size variants, charge variants, and post-translational modifications (PTMs). [Diagram 2] 1 is a depiction of methods routinely used to determine or monitor CQAs during protein drug development. [Figure 3A] 1 illustrates a method for identifying at least one product-associated variant, according to an exemplary embodiment. [Figure 3B] 1 illustrates a method for identifying at least one product-associated variant, according to an exemplary embodiment. [Figure 4] 1 illustrates a method design and workflow of a method for identifying at least one product-associated variant, according to an exemplary embodiment. [Diagram 5] 1 illustrates a method design and workflow for determining antigen-to-antibody ratio according to an exemplary embodiment. [Figure 6] 1 shows titration curves obtained to determine antigen to antibody ratios, according to an exemplary embodiment. [Figure 7] 1 shows a chromatogram of a sample that is not enriched for product-associated variants of mAb1, according to an exemplary embodiment. [Figure 8] 1 shows a comparison of chromatograms of samples enriched for product-related variants of mAb1 with reduced binding affinity according to an exemplary embodiment and a control experiment. [Figure 9] FIG. 1 shows extracted ion chromatograms (XICs) of different product-associated variants of mAb1 enriched for product-associated variants with reduced binding affinity according to an exemplary embodiment, compared to a control experiment. [Figure 10] 1 shows a chart of the relative percentage of product-associated variants of mAb1 identified using methods according to exemplary embodiments and control experiments. [Figure 11] The structure of bsAb1 is shown. [Figure 12] 1 shows a comparison of XICs of samples enriched for product-associated variants of mAb2 with reduced binding affinity according to an exemplary embodiment and a control experiment. [Figure 13] 1 shows a chart of the relative percentage of deamidated variants of bsAb1 identified using methods according to exemplary embodiments and control experiments. [Figure 14] 1 shows a chart of the relative percentage of product-associated variants of bsAb1 identified using methods according to exemplary embodiments and control experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Identification and quantification of product-associated variants in biopharmaceutical products can be of great importance during product production and development. Identification of such variants can be essential to develop a safe and effective product. Therefore, robust methods and / or workflows for characterizing CQAs can be beneficial.

[0024] The ICH Q8 Annex defines CQAs as physical, chemical, biological, or microbiological properties or characteristics that should be within appropriate limits, ranges, or distributions to ensure the quality, safety / immunogenicity, efficacy, and pharmacodynamics / pharmacokinetics of the desired product. (US Food and Drug Administration. Guidance for industry: Q8(R2)pharmaceutical development. www.fda.gov / media / 71535 / download). Thus, CQAs must be within appropriate limits, ranges, or distributions to ensure the quality, safety, and efficacy of the desired product. For example, for monoclonal antibody therapeutics that rely on fractional crystallizable (Fc)-mediated effector functions for clinical activity, the terminal sugars of the Fc glycan have been shown to be important for safety or efficacy. Such CQAs include product-related variants, such as size and charge variants, that may affect the binding of the protein of interest.

[0025] Figure 1 shows non-limiting examples of variants that can affect key quality attributes of a protein. For the antibody shown in Figure 1, product-related impurities can be size variants such as fragmentation products (LMW) and aggregation products (HMW). Other product-related impurities can be charge variants formed by N-terminal blocking, disulfide bond formation, C-terminal clipping, Fc glycan microheterogeneity, or post-translational modifications. These can cause reduced binding of the protein of interest and need to be monitored at various parts of the manufacturing and delivery process.

[0026] One traditional method involves the use of strong cation exchange chromatography (SCX). One such workflow is shown in Figure 2. This involves separation of the protein of interest and its variants by SCX, followed by performing a binding assay of the protein of interest and its variants to identify whether the variants have impaired, i.e., reduced, binding affinity compared to the protein of interest.

[0027] Considering the limitations of existing methods, an effective and efficient method for the identification and quantification of dimeric species was developed.

[0028] 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. All publications mentioned are incorporated herein by reference.

[0029] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation as understood by one of ordinary skill in the art, and endpoints are included when ranges are provided.

[0030] In some exemplary embodiments, the present disclosure provides methods for identifying at least one product-associated variant in a sample containing a protein of interest.

[0031] As used herein, the term "protein" or "protein of interest" includes any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues linked through peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. 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. A protein can contain one or more polypeptides to form a single functional biomolecule. Proteins can include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary embodiment, the protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a review discussing biotherapeutic proteins and their production, see Ghaderi et al. "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," (BIOTECHNOL. GENET. ENG. REV. 147-175 (2012)). In some exemplary embodiments, the proteins include modifications, adducts, 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, etc. Proteins can be classified based on composition and solubility, and thus include simple proteins such as globular proteins, fibrous proteins, complex proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, lipoproteins, and derived proteins such as primary derived proteins, secondary derived proteins, etc.

[0032] In some exemplary embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, or an Fc fusion protein.

[0033] The term "antibody" as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region is made up of three domains: H 1. C H2 , and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L1 ) is included. V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different exemplary embodiments, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. 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 naturally occurring, enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, e.g., from commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical or molecular biology techniques, e.g., to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids.

[0034] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, an 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, Fc fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. Antibody fragments can be produced by various means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody and / or recombinantly from a gene encoding a partial antibody sequence. Alternatively, or additionally, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or additionally, the antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds. The antibody fragment may optionally comprise a multimolecular complex.

[0035] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be 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.

[0036] The term "Fc fusion protein" as used herein includes parts or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not fused in their native state. The preparation of fusion proteins containing certain heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, in Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. A "receptor Fc fusion protein" includes one or more of one or more extracellular domains of a receptor linked to an Fc portion, which in some embodiments includes a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein contains two or more separate receptor chains that bind to one or more ligands. For example, the Fc fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding domain fused to the IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, which are incorporated by reference in their entireties).

[0037] As used herein, the term "target" refers to any molecule that can specifically interact with a therapeutic protein to achieve a pharmacological effect. For example, the target of an antibody may be the antigen to which it is directed, the target of a ligand may be the receptor to which it preferentially binds, or vice versa, the target of an enzyme may be the substrate to which it preferentially binds, and so on. A single therapeutic protein may have more than one target. Various targets are suitable for use in the methods of the invention according to the particular application. The target may be, for example, present on a cell surface, soluble, cytoplasmic, or immobilized on a solid surface. The target may be a recombinant protein. In some exemplary embodiments, the target may be an antigen.

[0038] As used herein, the term "impurities" can include any undesirable proteins present in a protein biologic product. Impurities can include process and product related impurities. Impurities can further be of known structure, partially characterized, or unidentified.

[0039] Process-related impurities can be derived from the manufacturing process and can include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vectors, or total DNA) from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

[0040] Product-related impurities (e.g., precursors, specific degradation products) may be molecular variants arising during production and / or storage that do not have equivalent properties to those of the desired product in terms of activity, efficacy, and safety. Such variants may require significant efforts in isolation and characterization to identify the type of modification. Product-related impurities may include truncated forms, modified forms, and aggregates. Truncation forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modification forms include, but are not limited to, deamidation, isomerization, mismatched S-S linkages, oxidation, or altered conjugated forms (e.g., glycosylation, phosphorylation). Modification forms may also include any post-translational modification forms. Aggregates include dimers and higher multimers of the desired product. (Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products, ICH August 1999, USDept. of Health and Humans Services).

[0041] Some product-related impurities or product-related protein variants have impaired binding affinity. Impaired binding affinity, as used herein, includes reduced binding affinity to a target of a protein of interest in the body or an antigen designed for the protein of interest. Impaired binding affinity can be any affinity that is lower than the affinity of the protein of interest to a target of the protein of interest in the body or an antigen designed for the protein of interest.

[0042] As used herein, the general term "post-translational modification" or "PTM" refers to a covalent modification that a polypeptide undergoes either during (co-translational modification) or after (post-translational modification) ribosomal synthesis. PTMs are generally introduced by specific enzymes or enzymatic pathways. Many are present at the site of specific characteristic protein sequences (signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications consistently affect some aspect of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853). Various post-translational modifications include truncation, N-terminal extension, proteolysis, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (addition of an acetyl group, usually at the N-terminus of a protein), alkylation (addition of an alkyl group, e.g., methyl, ethyl, propyl, usually at a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (hydroxylation of proline and lysine and amidation of the carboxy terminus), vitamin K-dependent modifications (vitamin K catalyzes the carboxylation of glutamic acid residues resulting in the formation of γ-carboxyglutamate (glutamic acid residues)). These include, but are not limited to, covalent attachment of a 4'-phosphopantetheinyl moiety from coenzyme A, such as in the biosynthesis of fatty acids, polyketides, nonribosomal peptides, and leucine, and sulfation (the addition of a sulfate group, usually to a tyrosine residue).Post-translational modifications that change the chemical properties of amino acids include, but are not limited to, citrullination (conversion of arginine to citrulline by deimination) and deamidation (conversion of glutamine to glutamic acid or asparagine to aspartic acid). Post-translational modifications that involve structural changes include, but are not limited to, the formation of disulfide bridges (covalent linkage of two cysteine ​​amino acids) and protein cleavage (cutting of a protein at a peptide bond). Certain post-translational modifications involve the addition of other proteins or peptides, such as ISGylation (covalent linkage to ISG15 protein (interferon-activating gene)), SUMOylation (covalent linkage to SUMO protein (small ubiquitin-related modifier)), and ubiquitinylation (covalent linkage of ubiquitin to a protein). For a more detailed controlled vocabulary of PTMs curated by UniProt, see European Bioinformatics Institute Protein Information Resource, SIB Swiss Bioinformatics Institute, European Bioinformatics Institute Drs - Drosomycin precursor - Drosophila melanogaster - Drs genes and proteins, http: / / www.uniprot.org / docs / ptmlist (last visited: 15 January 2019).

[0043] As used herein, the term "chromatography" refers to a process in which a liquid or gas-borne chemical mixture can be separated into multiple components as a result of differences in the distribution of chemicals as they flow around or over a stationary liquid or solid phase. Non-limiting examples of chromatography include traditional reversed-phase (RP) chromatography, ion-exchange (IEX) chromatography, mixed-mode chromatography, and normal-phase (NP) chromatography.

[0044] As used herein, the term "cation exchange chromatography" refers to a chromatographic method using a "cation exchange chromatography material". Furthermore, depending on the nature of the charged group, the "cation exchange chromatography material" refers to, for example, a cation exchange chromatography material having a sulfonic acid group (S) or a carboxymethyl group (CM). Depending on the chemical nature of the charged group, the "cation exchange chromatography material" can be further classified as a strong ion exchange chromatography material or a weak ion exchange chromatography material depending on the strength of the covalently attached charged substituent. For example, a strong cation exchange chromatography material has a sulfonic acid group as a chromatographic functional group.

[0045] For example, "cation exchange chromatography material" includes Bio-Rex, Macro-Prep CM (available from BioRad Laboratories, Hercules, Calif., USA), weak cation exchanger WCX2 (available from Ciphergen, Fremont, Calif., USA), Dowex MAC-3 (available from Dow chemical companies, Midland, Mich., USA), Mustang C (available from Pall Corporation, East Hills, NY, USA), cellulose CM-23, CM-32, CM-52, hyper-D, and partisphere (available from Whatman plc, Brentford, UK), Amberlite IRC76, IRC747, IRC748, GT73 (available from Tosoh Bioscience GmbH, Stuttgart, Germany), CM1500, CM3000 (BioChrom Labs, Terre cation exchange resins are available from a number of companies under various names such as CM-Sepharose Fast Flow (available from GE Healthcare, Life Sciences, Germany), and CM-Sepharose Fast Flow (available from GE Healthcare, Life Sciences, Germany). In addition, commercially available cation exchange resins further include carboxymethylcellulose, Bakerbond ABX, sulfopropyl (SP) immobilized on agarose (e.g., SP-Sepharose Fast Flow or SP-Sepharose High Performance, available from GE Healthcare-Amersham Biosciences Europe GmbH, Freiburg, Germany), and sulfonyl immobilized on agarose (e.g., S-Sepharose Fast Flow, available from GE Healthcare, Life Sciences, Germany).

[0046] The term "cation exchange chromatography material" includes mixed-mode chromatography materials that combine ion exchange and hydrophobic interaction techniques (e.g., Capto adhere, Capto MMC, MEP HyperCell, Eshmuno HCX, etc.), mixed-mode chromatography materials that combine anion exchange and cation exchange techniques (e.g., hydroxyapatite, ceramic hydroxyapatite, etc.). Cation exchange chromatography materials that can be used for cation exchange chromatography in the present invention include, but are not limited to, all commercially available cation exchange chromatography materials described above. In one embodiment of the present invention, a YMC BioPro SP-F column was used as the cation exchange chromatography material.

[0047] As used herein, the term "mass spectrometer" includes devices that can recognize and measure the exact mass of specific molecular species. The term is meant to include any molecular detector into which a polypeptide or peptide can be eluted for detection and / or characterization. A mass spectrometer includes three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to generate gas phase ions. Analyte atoms, molecules, clusters are transferred to the gas phase and simultaneously ionized (as in electrospray ionization). The choice of ion source is highly dependent on the application.

[0048] In some embodiments, the mass spectrometer can be an electrospray mass spectrometer.

[0049] As used herein, the term "electrospray ionization" or "ESI" refers to a spray ionization process in which positive or negative ions in a solution are transferred to the gas phase by desolvation through the formation at atmospheric pressure of a stream of highly charged droplets generated by applying a potential difference between the tip of an electrospray needle containing the solution and a counter electrode. There are generally three main steps in producing gas phase ions from electrolyte ions in solution. These are (a) generating charged droplets at the ES injection tip, (b) shrinkage of the charged droplets by solvent evaporation and repeated droplet collapse to generate small highly charged droplets that can generate gas phase ions, and (c) generating gas phase ions from the very small, highly charged droplets by a mechanism. Steps (a)-(c) typically occur in the atmospheric pressure region of the device.

[0050] As used herein, the term "electrospray injection setup" refers to an electrospray ionization system that is compatible with a mass spectrometer used for mass analysis of proteins. In electrospray ionization, an electrospray needle is placed with its orifice near the entrance orifice of the spectrometer. A sample containing the protein of interest can be pumped through a syringe needle. An electric potential between the syringe needle orifice and an orifice leading to the mass analyzer forms a spray of the solution ("electrospray"). Electrospray can be performed at atmospheric pressure and provides highly charged droplets of the solution. The electrospray injection setup can include an electrospray emitter, a nebulizing gas, and / or an ESI power supply. The setup can be automated to perform sample aspiration, sample dispensing, sample delivery, and / or to spray the sample, as needed.

[0051] In some exemplary embodiments, the electrospray ionization mass spectrometer can be a nano-electrospray ionization mass spectrometer.

[0052] The term "nanoelectrospray" or "nanospray" as used herein refers to electrospray ionization at very low solvent flow rates, typically hundreds of nanoliters / minute or less, of a sample solution, 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 continuous analysis of a small amount of sample (analyte) solution over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separation on a mixture prior to analysis by a mass spectrometer.

[0053] As used herein, the term "mass analyzer" includes devices capable of separating species, i.e. atoms, molecules, or clusters, according to mass. Non-limiting examples of mass spectrometers that can be used for fast protein sequencing are time-of-flight (TOF), magnetic / electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR) and accelerator mass spectrometry (AMS) techniques.

[0054] In some exemplary embodiments, mass spectrometry can be performed under native conditions.

[0055] As used herein, the term "native conditions" or "native MS" or "native ESI-MS" 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 Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 Protein Science 1176-1192 (2015). Some of the differences between native ESI and regular ESI are illustrated in Table 1 and Figure 1 (Hao Zhang et al., Native mass spectrometry of photosynthetic pigment-protein complexes, 587 FEBS Letters 1012-1020 (2013)).

[0056] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer.

[0057] As used herein, the term "tandem mass spectrometry" includes techniques that use multiple stages of mass selection and mass separation to obtain structural information of sample molecules. The prerequisite is that the sample molecules can be transferred to the gas phase and ionized intact, and that these molecules can be induced to disintegrate in some predictable and controllable manner after the first mass selection step. Multi-stage MS / MS or MS n The first step is to select and isolate precursor ions (MS spectrometry) as long as meaningful information is obtained or fragment ion signals are detectable. 2 ), fragmentation, and isolation of the primary fragment ions (MS 3 ), fragmentation, and isolating the secondary fragment ions (MS 4), etc. Tandem MS has been successfully performed with a variety of analyzer combinations. The choice of which analyzer to combine for a particular application depends on many different factors, including sensitivity, selectivity, speed, but also size, cost, availability, etc. The two main categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids in which a tandem-in-time analyzer is combined in space or with a tandem-in-space analyzer. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function can be designed such that ions are selected in one section of the instrument, dissociated in an intermediate region, and then the product ions are sent to another analyzer for m / z separation and data acquisition. In tandem-in-time, mass analyzer ions generated in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0058] The 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 generated from possible peptides in protein sequence databases. Characterization can include, but is not limited to, determining the amino acid sequence of protein fragments, determining protein sequence determination, determining protein de novo sequence determination, determining the location of post-translational modifications, or identifying post-translational modifications or comparability analysis, or a combination thereof.

[0059] 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 be derived from the cDNA sequences of the species being studied. Public databases that may be used to search for relevant protein sequences included, for example, databases hosted 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 and provide as output interpreted (annotated) MS / MS spectra. Non-limiting examples of such tools are 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).

[0060] In some embodiments, the method for identifying at least one product-associated variant can include using a competitive binding assay with a poor antigen immobilized on a solid surface.

[0061] As used herein, the term "solid surface" can include any surface capable of binding an antigen. Non-limiting examples of solid surfaces can include affinity resins, beads, and coated plates with immobilized proteins such as avidin, streptavidin, or neutral avidin.

[0062] In some embodiments, a sample containing the protein of interest can be digested after the competitive binding assay but before it is evaluated via SCX-MS.

[0063] In some embodiments, a sample containing a protein of interest can be treated by adding a reducing agent to the sample.

[0064] As used herein, the term "reducing" refers to the reduction of disulfide bridges in a protein. Non-limiting examples of reducing agents used to reduce proteins are dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof. In certain embodiments, the treatment can further include alkylation. In certain other exemplary embodiments, the treatment can include alkylation of sulfhydryl groups on the protein.

[0065] As used herein, the terms "treating" or "isotopically labeling" can refer to chemically labeling a protein. Non-limiting examples of methods for chemically labeling a protein include isobaric tags for relative and absolute quantification using reagents such as 4-plex, 6-plex, and 8-plex (iTRAQ), reductive demethylation of amines, carbamylation of amines, isotopically labeling of the C-terminus of a protein, and isotopically labeling of the C-terminus of a protein. 18 This includes O-labeling, or any amine or sulfhydryl group on the protein for labeling amine or sulfhydryl groups.

[0066] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several techniques (e.g., enzymatic or non-enzymatic digestion) for carrying out the digestion of proteins in a sample using an appropriate hydrolytic agent.

[0067] As used herein, the term "hydrolysis agent" refers to any one or combination of a number of different agents capable of performing protein digestion. Non-limiting examples of hydrolysis agents capable of performing enzymatic digestion include trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), immunoglobulin degrading enzyme (IdeS) of Streptococcus pyogenes, chymotrypsin, pepsin, thermolysin, papain, pronase, and protease of Aspergillus Saitoi. Non-limiting examples of hydrolysis agents capable of performing non-enzymatic digestion include high temperature, microwave, ultrasound, high pressure, infrared, solvents (non-limiting examples include ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzyme, and on-chip immobilized enzyme. For a recent review discussing available techniques for protein digestion, see Switazar et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (J. Proteome Research 2013, 12, 1067-1077). One or a combination of hydrolytic agents can cleave peptide bonds in a protein or polypeptide in a sequence-specific manner to generate a predictable collection of shorter peptides.

[0068] Exemplary embodiments Embodiments disclosed herein provide methods for identifying at least one product-associated variant in a sample containing a protein of interest.

[0069] In some exemplary embodiments, the disclosure provides a method for identifying at least one product-associated variant in a sample comprising a protein of interest, the method comprising: contacting a sample comprising the protein of interest and at least one product-associated variant with competitive binding conditions, the binding conditions providing a deficient antigen immobilized on a bead, such that the at least one product-associated variant is impaired in binding to the deficient antigen; incubating the sample with the deficient antigen; After incubation, collecting the flow-through from the wash; identifying at least one product-related critical quality attribute in the flow-through using liquid chromatography-mass spectrometry; Includes.

[0070] In some exemplary embodiments, the product-associated variants are one or more of a truncated form, a modified form, and an aggregate of the protein of interest.

[0071] In some exemplary embodiments, the product-associated variant is deamidation, isomerization, mismatched S-S bonds, oxidation, and / or altered conjugated forms (e.g., glycosylation, phosphorylation) of the protein of interest.

[0072] In some exemplary embodiments, the product-associated variant is a post-translationally modified form.

[0073] In some exemplary embodiments, the product-associated variants have an impaired binding affinity, where the impaired binding affinity is about 90% of the binding affinity of the protein of interest, about 80% of the binding affinity of the protein of interest, about 70% of the binding affinity of the protein of interest, about 60% of the binding affinity of the protein of interest, about 50% of the binding affinity of the protein of interest, about 40% of the binding affinity of the protein of interest, about 30% of the binding affinity of the protein of interest, about 20% of the binding affinity of the protein of interest, or about 10% of the binding affinity of the protein of interest.

[0074] In some exemplary embodiments, the electrospray ionization mass spectrometer can be a nano-electrospray ionization mass spectrometer.

[0075] In some exemplary embodiments, the electrospray ionization mass spectrometer may be run under native conditions.

[0076] It is to be understood that the methods are not limited to any of the aforementioned proteins, impurities, and columns, and that the methods for identification or quantification may be carried out by any suitable means.

[0077] Exemplary embodiments are illustrated in Figures 3A and 3B. Beads with immobilized antigens can be added to a sample containing the protein and possibly its variants, if desired. The amount of beads with immobilized antigens is such that not all of the protein of interest (native mAb) and its variants can bind to it. Any variants with reduced binding affinity to the antigen will be less likely to bind due to the limited amount of antigen present. The flow-through (unbound fraction) can be collected and analyzed using SCX-MS or peptide mapping. The control (i.e., sample without the immobilized antigen binding assay step) can also be analyzed using SCX-MS or peptide mapping. A comparative study between the flow-through and the control results in a chromatogram as illustrated in Figure 3B. Any variants with reduced binding affinity will be more abundant in the flow-through. Comparing the amounts of the variants so identified, it can be seen that the relative proportion of the variants is greater in the flow-through than in the control due to their reduced binding affinity.

[0078] Such an experiment can be devised using the workflow depicted in FIGS.

[0079] In the present invention, the ratio of antigen to protein of interest is very important. The amount of antigen added can be such that about 25% to about 75% of the protein of interest can bind to the antigen. In some embodiments, the amount of antigen added can be such that about 50% of the protein of interest can bind to the antigen.

[0080] The sequential labeling of method steps with numbers and / or letters provided herein is not meant to limit the method, or any embodiment thereof, to the particular order indicated.

[0081] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers and journal articles, are cited throughout this specification. Each of these cited references is incorporated herein by reference in its entirety and for all purposes.

[0082] The present disclosure will be more fully understood by reference to the following examples, which are provided to more fully illustrate the present disclosure and are intended to be illustrative and should not be construed as limiting the scope of the disclosure. EXAMPLES

[0083] Materials. Deionized water was provided by a MilliQ integrated water purification system equipped with a MilliPak Express 20 filter (Millipore Sigma, Burlington, MA). mAb1, mAb2, mAb1 antigen, and mAb2 antigen were produced in-house at Regeneron (Tarrytown, NY).

[0084] Online nSCX-UV / MS analysis Strong cation exchange chromatography was performed using a YMC BioPro SP-F (YMC, Japan). The mobile phases used to separate the samples were 20 mM ammonium acetate, pH 5.6 (mobile phase A) and 150 mM ammonium acetate, pH 7.4 (mobile phase B). A linear pH gradient was used to elute the charge variants of mAb1 and detected at 280 nm.

[0085] Prior to sample injection, the column compartment temperature was set to 45° C. and a strong cation exchange column (100 mm 4.6 mm, 5 μm) (YMC, Japan) was preconditioned with mobile phase A (20 mM ammonium acetate adjusted to pH 5.6 with 20 mM acetic acid) at a flow rate of 0.4 mL / min. Upon injection of an aliquot (10 μg) of protein sample, the gradient was held at 100% mobile phase A for 2 min, followed by a linear increase to 100% mobile phase B (150 mM ammonium acetate, pH 7.4) in 16 min. The gradient was held at 100% mobile phase B for 4 min, then switched back to 100% mobile phase A to recondition the column for 7 min before the next injection. Peaks with relative retention times earlier or later than the main peak were identified using online MS.

[0086] For mass analysis, the resolution was set to 17,500, the capillary spray voltage was set to 1.5 kV, the in-source fragmentation energy was set to 100, the collision energy was set to 10, the capillary temperature was set to 350 °C, the S-lens RF level was set to 200, and the HCD trapping gas pressure was set to 3. Mass spectra were acquired with an m / z range window of 2000–15000.

[0087] Data analysis. Protein Metrics Intact Mass software was used for deconvolution of raw data. Thermo Xcalibur Qual Browser was used for extracted ion chromatogram analysis.

[0088] Example 1. 1.1 Optimization of competitive binding assays To distinguish between variants of a protein of interest with impaired binding, a competitive binding assay was developed. mAb1 was used as an exemplary protein of interest.

[0089] The mAb1 antigen was biotinylated using a biotinylation reagent (NHS-biotin, 30 min at room temperature). The biotinylated mAb1 was loaded onto a bed of streptavidin resin (7 nmol biotin binding capacity, Pierce) in a tube (micro BioSpin, Bio-Rad). After 5 min, filtration was performed by centrifugation, and the gel bed was washed with 100 mM Tris (pH 7.5) (incubate for about 1 min and spin), then washed six times with purified water (Milli-Q, Millipore) to obtain the antigen-immobilized resin.

[0090] A series of tubes containing 11 different increasing amounts of antigen-immobilized resin (1-40 μL) were suspended in binding assay buffer (binding assay buffer can be anything from Tris to PBS buffer). Purified mAb1 was added and the tubes were incubated for 1 hour at 4°C. Binding to the total amount of mAb1 was analyzed by centrifugation (i.e., spinning down at 800×g for 5 minutes at 4°C), removing the supernatant, and measuring the protein concentration of the flow-through at 280 nm using a NanoDrop UV-Vis spectrophotometer. Figure 6 shows an exemplary embodiment of the resulting titration curve. The volume of antigen-immobilized resin was insufficient to capture all of the mAb1 sample, thus providing a flow-through enriched for any binding-impaired variants of mAb1. The resin volume required to obtain 50% binding of mAb1 was selected for further competitive binding assays.

[0091] 1.2 Competitive binding nSCX-UV / MS analysis. mAb1 was subjected to competitive binding assay as described above.

[0092] As shown in Figure 7, SCX-UV analysis of mAb1 indicates that it features substantial glycosylation variants. As shown in Table 1, the specific glycosylation site was identified as lysine (K) 98 of the heavy chain (HC). This glycosylation was previously thought to be involved in antigen binding, although its exact effect was unknown.

[0093] [Table 1]

[0094] To determine whether key variants of mAb1, such as glycosylation, affect antigen binding, the mAb1 flow-through from the competitive binding assay was compared to a control sample of mAb1 using SCX-UV analysis. The control experiment included the use of SCX-UV / MS on mAb1 obtained from the stability study without the enrichment step. A comparison of the two chromatograms is shown in Figure 8. Figure 8 clearly shows the enrichment of the glycated peak of mAb1 in the flow-through from the competitive binding assay compared to the control mAb1. This demonstrates that glycosylation modification indeed impairs binding of mAb1 to the mAb1 antigen and is therefore a CQA that should be considered in product development.

[0095] 1.3 Evaluation of multiple critical quality attributes using competitive binding SCX-MS. Samples from Example 1.2 were further subjected to mass spectrometry analysis. Figure 9 shows the extracted ion chromatogram (XIC) from the mAb1 control sample and the mAb1 flow-through of the competitive binding assay. Several protein variants are identifiable in the compared XIC, demonstrating that the method of the present invention can simultaneously identify several CQAs that adversely affect protein binding. At the same time, PTMs with no change in relative abundance between samples may be ignored as CQAs.

[0096] Figure 10 shows the statistical analysis of enrichment of PTMs in the flow-through of the competitive binding assay compared to the control sample. From the comparison using the competitive binding assay experiment, it is clear that some modifications (e.g., HC K98 glycosylation, HC K98 carboxymethylation (CML), and HC K98 glucuronylation) were enriched and identified as CQAs of mAb1, whereas other modifications (Q at the N-terminus and terminal galactosylation of the Fc glycan) were not enriched. Thus, the method was successful in identifying critical quality attributes and product-associated variants that cause reduced binding of mAb1 to the mAb1 antigen, and distinguishing them from modifications that do not affect binding and therefore can be ignored in product development.

[0097] Example 2. 2.1 Competitive binding SCX-MS analysis of bispecific antibodies The effectiveness of the method of the present invention was further demonstrated by analysis of the bispecific antibody, bsAb1. The structure of bsAb1 is shown in Figure 11. Figure 11 shows that mAb2 contains two separate HC regions (HC and HC*).

[0098] Previous nSCX-MS analysis of bsAb1 lots has shown that deamidation variants feature prominently. As shown in Table 2, previous peptide mapping analysis identified the major variant caused by deamidation at HC N56.

[0099] [Table 2]

[0100] HC N56 is located in the complementarity determining region (CDR) of bsAb1, raising the possibility that it may adversely affect the binding of bsAb1 to its target. To determine any potential effects of bsAb1 variants on binding, bsAb1 was subjected to competitive binding SCX-MS analysis.

[0101] Antigen immobilization resin was optimized and prepared as described in Example 1.1. bsAb1 was subjected to a competitive binding assay and the flow-through from the competitive binding assay was compared to a bsAb1 control sample using SCX-UV / MS. A comparison of the two UV chromatograms is shown in Figure 12. Figure 12 clearly shows the enrichment of deamidated variants of bsAb1 in the flow-through from the competitive binding assay, which is quantified as shown in Figure 13. The enrichment of deamidated variants of bsAb1 in the flow-through of the competitive binding assay demonstrates that deamidation is a CQA in the production of bsAb1.

[0102] 2.2 Assessment of multiple critical quality attributes using competitive binding SCX-MS. The bsAb1 was subjected to further analysis using competitive binding SCX-peptide mapping MS. The extracted ion chromatograms (XIC) from the control experiment and the flow-through of the competitive binding assay showed several different PTMs. A comparative analysis of the amount of variants obtained using the control and competitive binding assay experiments is shown in FIG. 14. From the comparison of the variants, it is clear that using the competitive binding assay experiment, only the N56 deamidated variant was enriched and therefore this was likely the only identified critical quality attribute or product-associated variant of bsAb1 with reduced binding affinity.

Claims

1. A method for characterizing at least one product-related variant, comprising: a. obtaining a sample comprising a protein of interest and at least one product-related variant of the protein of interest; b. contacting the sample with a competitive binding condition comprising a suboptimal target immobilized on beads, wherein binding of the at least one product-related variant to the suboptimal target is impaired; c. washing the beads and recovering the flow-through; d. subjecting the flow-through to liquid chromatography-mass spectrometry analysis to separate the protein of interest and the at least one product-related variant; e. characterizing the at least one product-related variant by comparing the abundance of the at least one product-related variant from (d) with the abundance of the at least one product-related variant obtained from liquid chromatography-mass spectrometry analysis of a control sample of (a). A method comprising the above steps.

2. The method according to claim 1, wherein the liquid chromatography is strong cation exchange chromatography.

3. The method according to claim 1, wherein the beads are agarose beads or magnetic beads.

4. The method according to claim 1, wherein the flow-through is enriched for the at least one product-related variant.

5. The method according to claim 1, wherein the flow-through of (c) is recovered by centrifugation.

6. The method according to claim 1, further comprising subjecting the flow-through of (c) to digestion conditions prior to liquid chromatography-mass spectrometry analysis.

7. The method according to claim 1, wherein the beads are coated with streptavidin resin.

8. The method according to claim 1, wherein the suboptimal target comprises an amount of the target capable of binding from about 30% to about 80% of the protein of interest.

9. The method according to claim 1, wherein the sample of (b) is incubated for about 1 hour.

10. The method according to claim 1, wherein the sample of (b) is incubated at approximately room temperature.

11. The method according to claim 1, wherein the product-related variant comprises a size variant.

12. The method according to claim 11, wherein the size variant is a fragmentation variant of the protein of interest.

13. The method according to claim 11, wherein the size variant is an aggregation variant of the protein of interest.

14. The method according to claim 1, wherein the product-related variant comprises a charge variant of the protein of interest.

15. The method according to claim 1, wherein the product-related variant comprises a post-translational modification variant of the protein of interest.

16. The method according to claim 1, wherein the target is an antigen directed against the protein of interest.

17. The method according to claim 1, wherein the product-related variant is characterized as an important quality attribute when the abundance of the at least one product-related variant from (d) is significantly higher than the abundance of the at least one product-related variant obtained from the liquid chromatography - mass spectrometry analysis of the control sample in (a).

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