Methods for characterizing proteins in serum samples

Abstract

Described herein are methods for enriching, identifying, quantifying, and / or characterizing a protein of interest in a complex biological sample, such as a serum sample. The protein of interest may be enriched using a library of bead-based peptide ligands. The enriched protein of interest may subsequently be subjected to denaturation, reduction, and / or enzymatic digestion, to generate peptides which can be detected using liquid chromatography-mass spectrometry (LC-MS) techniques, and which are uniquely identifying of the protein of interest.

Description

METHODS FOR CHARACTERIZING PROTEINS IN SERUM SAMPLESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 570,726, filed March 27 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] This application includes a sequence listing, submitted electronically in XML format and incorporated by reference in its entirety. The XML copy of the sequence listing was created on March 24. 2025, is named “Sequence Listing. xml”, and is 20.231 bytes in size.TECHNICAL FIELD

[0003] This disclosure is directed to methods for quantifying and characterizing proteins in complex biological samples.INTRODUCTION

[0004] Therapeutic proteins have emerged as important drugs for the treatment of cancer, autoimmune disease, infection and cardiometabolic disorders, and they represent one of the fastest growing product segments of the pharmaceutical industry. Protein bioanalysis plays an important role in understanding the mechanisms of the absorption, distribution, metabolism, and elimination of therapeutic proteins, by providing accurate and precise protein quantification of these agents. During early drug development, the pharmacokinetic (PK) properties of tire therapeutic proteins are critical for the evaluation of drug candidates. Liquid chromatography / mass spectrometry (LC / MS) methods for characterizing PK properties of therapeutic proteins offer a number of advantages over traditional ligand binding assays, such as ELISA. Liquid chromatography coupled to tandem mass spectrometry (LC- MS / MS) methods offer even further advantages, such as sensitivity and specificity. Sensitive and specific identification of a surrogate peptide from a complex matrix using LC-MS / MS may require either extensive method development and optimization when using a direct digestion method, or assay -specific reagents when using an immunoprecipitation method, assuming such reagents are available or affordable. Thus, while LC-MS / MS methods are available, they may not be universally applicable.

[0005] Therefore, it will be appreciated that a need exists for generalizable and rapid methods to sensitively identify, quantify and characterize a protein of interest through the quantification of unique peptides in serum.SUMMARY OF THE DISCLOSURE

[0006] Described herein are methods for quantifying an antibody in a serum sample. The method may comprise contacting a serum sample to a solid support, wherein the serum sample comprises an antibody and the solid support is attached to peptide ligands capable of binding to the antibody; contacting an elution buffer to the solid support, thereby producing an enriched antibody sample; subjecting the enriched antibody same to denaturing conditions, thereby producing a denatured antibody sample; contacting a reducing agent to the denatured antibody sample to produce a reduced antibody sample; contacting an enzyme to the reduced antibody sample to produce a peptide digest, wherein atleast one peptide of the peptide digest is a uniquely identifying peptide for the antibody; contacting the peptide digest to a liquid-chromatography-tandem mass spectrometry (LC-MS / MS) system to determine a quantity of the uniquely identifying peptide; and determining a quantity of tire antibody based on the quantity of the uniquely identifying peptide. The method may further comprise, after contacting the serum sample to the solid support and prior to contacting the elution buffer to the solid support, washing the solid support to remove unbound material.

[0007] The peptide ligands may include a library of combinatorial ligands. The library of combinatorial ligands may be supported on beads.

[0008] The antibody may include a monoclonal antibody, a bispecific antibody, an antibody-drug conjugate, a single chain variable fragment, or a fusion protein. The antibody may be IgGl antibody or an IgG4 antibody.

[0009] The uniquely identifying peptide may have an amino acid sequence that is identical to, or derived from, a fragment of an amino acid sequence of a variable region of the antibody. The uniquely identifying peptide has an amino acid sequence that is identical to, or derived from, a fragment of an amino acid sequence of a complementary’ determining region of the antibody. The uniquely identifying peptide may have a peptide chain length of 6 amino acid residues to 22 amino acid residues.

[0010] The elution buffer may include SLS / SDS. The elution buffer may comprise 12 mM SLS / SDC, 10 mM TCEP, and 40 mM CAA.

[0011] The denaturing conditions may include heat, high pH, low pH, at least one reducing agent, at least one chaotropic agent, or any combination thereof.

[0012] The reducing agent may include di thiothrei tol (DTT), B-mercaptoethanol, Elhnan’s reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1), or any combination thereof.

[0013] The enzyme may include pepsin, trypsin, Tryp-N. chymotrypsin. Lys-N, Lys-C, Asp-N. Arg-C, Glu-C. papain, IdeS, a variant thereof, or any combination thereof.

[0014] Determining a quantity of the uniquely identifying peptide may include comparing a peak area for the uniquely identifying peptide to a peak area for an internal standard; and / or comparing a peak area for the uniquely identifying peptide to a standard curve.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various examples and together with the description, serve to explain the principles of the disclosed examples and aspects.

[0016] Aspects of the disclosure may be implemented in connection with aspects illustrated in the attached drawings. These drawings show different aspects of the present disclosure and, where appropriate, reference numerals illustrating like structures, components, materials, and / or elements in different figures are labeled similarly. It is understood that various combinations of the structures, components, and / or elements, other than those specifically shown, are contemplated and are within the scope of the present disclosure.

[0017] Moreover, there are many aspects and examples described and illustrated herein. The present disclosure is not limited to any single aspect nor embodiment thereof, nor to any combinations and / or permutations of such aspects and / or embodiments. Moreover, each of the aspects of the present disclosure, and / or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and / or embodiments thereof. For the sake of brevity, certain permutations and combinations are not discussed and / or illustrated separately herein. Notably, an aspect or implementation described herein as “exemplary” is not to be construed as preferred or advantageous, for example, over other aspects or implementations; rather, it is intended reflect or indicate the aspect(s) is / are “example” aspect(s).

[0018] FIG. 1 is a block diagram of an exemplary method for quantifying a protein in a sample, according to aspects of the present disclosure.

[0019] FIGS. 2A-2D are schematics illustrating steps of an exemplary method for quantify ing a protein in a sample, according to aspects of the present disclosure. FIG. 2A illustrates an exemplary sample and solid support. FIG. 2B illustrates exemplary optional washing. FIG. 2C illustrates an exemplary enriched sample. FIG. 2D illustrates an exemplary peptide digest.

[0020] FIGS. 3A-3E arc graphs showing results of an exemplary method for protein enrichment and characterization, according to aspects of the present disclosure. FIG. 3A shows peak area of a peptide of interest with transitions for a mAb-1 sample. FIG. 3B shows retention times of the mAb-1 sample, for transitions to 1659.7344 and 1388.5812. FIG. 3C shows retention time of a mouse serum sample, for transitions to 1659.7344 and 1388.5812. FIG. 3D shows retention time of the mAb-1 sample, for transition to 359.2037. FIG. 3E shows retention time of a guinea pig serum sample, for transition to 359.2037.

[0021] FIG. 4 is a bar graph of peak area of a peptide of interest in water or various buffers measured by an exemplary method, according to aspects of the present disclosure.

[0022] FIG. 5 is a bar graph of peak area of a peptide of interest in guinea pig serum samples with water or various buffers measured by an exemplary method, according to aspects of the present disclosure.

[0023] FIG. 6 is a bar graph of amount of protein detected in mouse and guinea pig sera as a function of amount of enrichment beads used in an exemplary method, according to aspects of the present disclosure.

[0024] FIGS. 7A-7B are bar graphs of peak area of a peptide of interest as a function of amount of enrichment beads used in an exemplary method, according to aspects of the present disclosure. FIG. 7A shows peak area in a method using nano LC-PRM. FIG. 7B shows peak area in a method using regular flow LC-MRM.

[0025] FIGS. 8A-8D are bar graphs of peak area of a peptide of interest with transition in human and animal sera samples measured by an exemplary method using nano LC-PRM, according to aspects of the present disclosure. FIG. 8A shows peak area for a first mAb-1 surrogate CDR peptide. FIG. 8B showspeak area for a second mAb-1 surrogate CDR peptide. FIG. 8C shows peak area for a third mAb-1 surrogate CDR peptide. FIG. 8D shows peak area for a mAb-2 surrogate CDR peptide.

[0026] FIGS. 9A-9B are MS / MS spectra of peptides of interest as measured by an exemplary method using trypsin digest, according to aspects of the present disclosure. FIG. 9A shows a mAb-1 surrogate CDR peptide, with precursor m / z 680.3497, z = 3. FIG. 9B shows a mAb-2 surrogate CDR peptide, with precursor m / z 659.7903. z = 2.

[0027] FIG. 10 is a scatter plot of peak area of three peptides of interest as a function of injection amount measured by an exemplary method, according to aspects of the present disclosure.

[0028] FIG. 11 is a bar graph of peak area of a peptide of interest measured by an exemplary method using varying reagents for elution, according to aspects of the present disclosure.

[0029] FIG. 12 is a bar graph of peak areas for several peptides of interest in varying volumes of serum samples measured by an exemplary method, according to aspects of the present disclosure.

[0030] FIG. 13 is a bar graph of peak area for several peptides of interest in var ing ty pes of serum samples measured by an exemplary method, according to aspects of the present disclosure.

[0031] FIG. 14 is a bar graph of peak area for several peptides of interest in varying ty pes of serum samples measured by an exemplary method using nano LC-PRM, according to aspects of the present disclosure.

[0032] FIGS. 15A-15C are stacked bar graphs of MS2 product ion distributions of common IgGl peptides of interest across varying serum types measured by an exemplary' method, according to aspects of the present disclosure. FIG. 15A shows the graph of a first common IgGl peptide with precursor ion m / z 904.5069, z = 2. FIG. 15B shows the graph of a second common IgGl peptide with precursor ion m / z 973.5171. z = 2. FIG. 15C shows the graph of a third common IgGl peptide with precursor ion m / z 899.4513, z = 2.

[0033] FIGS. 16A-16C are stacked bar graphs of MS2 product ion distributions of mAb-1 CDR peptides of interest across varying serum types measured by an exemplary method, according to aspects of the present disclosure. FIG. 16A shows the graph of a first mAb-1 CDR peptide with precursor ion m / z 831.4647. z = 2. FIG. 16B shows the graph of a second mAb-1 CDR peptide with precursor ion m / z 1111.0107, z = 2. FIG. 16C shows a graph of a third mAb-1 CDR peptide with precursor ion m / z 680.3497, z = 3.

[0034] FIG. 17 is a stacked bar graph of peak area distributions of common IgGl peptides of interest across varying serum ty pes measured by an exemplary method, according to aspects of the present disclosure.

[0035] FIGS. 18A-18B are stacked bar graphs of MS2 product ion distributions of common IgGl peptides across varying serum types measured by an exemplary method using nano LC-MS, according to aspects of the present disclosure. FIG. 18A shows a distribution of all the tested common IgGl peptides. FIG. 18B shows a distribution without the ALAPAPIEK peptide.

[0036] FIG. 19 is a bar graph of peak areas of common IgGl peptides of interest detected in samples of four IgGl antibodies enriched in mouse serum by an exemplary method, according to aspects of the present disclosure.

[0037] FIG. 20 is a bar graph of peak areas of a common IgGl peptide of interest in mouse serum and monkey serum measured by an exemplary method, according to aspects of the present disclosure.

[0038] FIGS. 21A-21B are scatter plots of peak areas of peptides of interest in mouse serum using a spiked-in internal standard measured by an exemplary method, according to aspects of the present disclosure. FIG. 21 A shows peak areas for common IgGl peptides of interest. FIG. 21 B shows peak areas for two labelled internal standard peptides.

[0039] FIGS. 22A-22B are bar graphs of peak areas and distributions of mAb-2 CDR peptides of interest across varying sample types measured by an exemplary method, according to aspects of the present disclosure. FIG. 22A shows a first mAb-2 CDR peptide of interest. FIG. 22B shows a second mAb-2 CDR peptide of interest.

[0040] FIGS. 23A-23D are bar graphs of peak areas and distributions of mAb-1 CDR peptides of interest across varying sample types by an exemplary method, according to aspects of the present disclosure. FIG. 23 A shows a first mAb-1 CDR peptide of interest. FIG. 23B shows a second mAb-1 CDR peptide of interest. FIG. 23C shows a third mAb-1 CDR peptide of interest. FIG. 23D shows a fourth mAb-1 CDR peptide of interest.

[0041] FIGS. 24A-24C are graphs showing results of an exemplary method for characterizing a mAb-1 CDR peptide of interest in mouse serum, according to aspects of tire present disclosure. FIG. 24A shows peak area of mAb-1 as a function of mAb-1 concentration. FIG. 24B is a line graph detecting intensity of the peptide of interest as a function of retention time, for an LLQC condition. FIG. 24C is a line graph depicting intensity as a function of retention time in a blank mouse serum sample.

[0042] FIGS. 25A-25C are graphs showing results of an exemplary method for characterizing a mAb-2 CDR peptide of interest in mouse serum, according to aspects of the present disclosure. FIG. 25 A shows peak area of mAb-2 as a function of mAb-2 concentration. FIG. 25B is a line graph detecting intensity of the peptide of interest as a function of retention time, for an LQC condition. FIG. 25C is a line graph depicting intensity as a function of retention time in a blank mouse serum sample.

[0043] FIGS. 26A-26B are graphs showing results of a validation experiment for exemplary methods described herein, according to aspects of the present disclosure. FIG. 26A shows signal detection of a mAb-1 CDR peptide and correlation between peak area of the mAb-1 CDR peptide and spiked-in mAb-1 concentration. FIG. 26B shows signal detection of a mAb-2 CDR peptide and correlation betw een peak area of the mAb-2 CDR peptide and spiked-in mAb-2 concentration.

[0044] FIGS. 27A-27F are graphs of results characterizing a fusion protein (DS-1) measured by an exemplary method, according to aspects of the present disclosure. FIG. 27A is a mass spectrum of a first DS-1 peptide, using precursor m / z 365.7265, z = 2. FIG. 27B is a mass spectrum of a second DS-1 peptide, using precursor m / z 469.7527, z = 2. FIG. 27C is a chromatogram of the first DS-1 peptide, the second DS-1 peptide, and two other DS-1 peptides. FIG. 27D is a bar graph of peak areas for bothpeptides across varying serum sample types. FIG. 27E is a stacked bar graph of an MS2 product ion distribution for the first DS-1 peptide. FIG. 27F is a stacked bar graph of an MS2 product ion distribution for the second DS-1 peptide.

[0045] FIG. 28 is a calibration curve plot of the fusion protein (DS-1) of FIGS. 27A-27F produced according to an exemplary method for analyzing calibration standards using LC-MRM. according to aspects of the present disclosure.

[0046] Again, there are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to any combinations and / or permutations of such aspects and / or embodiments. Each of the aspects of the present disclosure, and / or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and / or embodiments thereof. For the sake of brevity, many of those combinations and pennutations are not discussed separately herein.DETAILED DESCRIPTION

[0047] Reference will now be made in detail to examples of the present disclosure, which are illustrated in tire accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. In the discussion that follows, relative terms such as “about,” “substantially,” “approximately,” etc. are used to indicate a possible variation of ±10% in a stated numeric value.

[0048] Measurement of a time profile for an antibody drug concentration in a patient’s serum, after administration of the drug, is a component of the pharmacokinetic characterization of protein therapeutics in clinical trials. Traditionally, ligand binding assays (LBAs), such as enzyme-linked immunosorbent assay (ELISA), have been used for quantifying biotherapeutic agents. ELISA has high sensitivity and high throughput. However, the time required to develop an ELISA method usually exceeds six months, and the accuracy and specificity of an ELISA method may be compromised by matrix interference. Therefore, an ELISA method might not be transferrable to different species.

[0049] Unlike a ligand-binding assay, an LC-MS / MS assay does not require highly specific affinity capturing reagents for characterizing proteins or antibodies (e.g.. proteinaceous drug products). In addition to the fast method development process, an LC-MS / MS assay also provides wide dynamic range, good accuracy and precision, and excellent selectivity for quantification of protein-based biopharmaceuticals in serum matrix. See, e.g., van den Broek et al.. “Bioanalytical LC-MS / MS of protein-based biopharmaceuticals.” 2013, Journal of Chromatography B, volume 929, pages 161-179. Recently, LC-MS / MS has become a more frequently adopted bioanalytical strategy for both preclinical and clinical sample analysis due to the high instrument sensitivity and stability. See, e.g.. Jiang et al., “Fully Validated LC-MS / MS Assay for the Simultaneous Quantitation of Coadministered Therapeutic Antibodies in Cynomolgus Monkey Serum,” 2013, Analytical Chemistry, volume 85, pages 9859-9867; Zhang et al., “Generic Automated Method for Liquid Chromatography -Multiple Reaction Monitoring Mass Spectrometry Based Monoclonal Antibody Quantitation for Preclinical Pharmacokinetic Studies,” 2014, Analytical Chemistry, volume 86, issue 17, pages 8776-8784; Li et al., “General LC-MS / MSMethod to Quantify Therapeutic Monoclonal Antibodies Using a Common Whole Antibody Internal Standard with Application to Preclinical Studies,” 2012. Analytical Chemistry , volume 84, issue 3. pages 1267-1273; Cardozo et al.. “Establishing a mass spectrometry -based system for rapid detection of SARS- CoV-2 in large clinical sample cohorts.” 2020, Nature Communications, volume 11. article no. 6201, pages 1-13; Fernandez Ocana et al.. “Clinical Pharmacokinetic Assessment of an Anti-MAdCAM Monoclonal Antibody Therapeutic by LC-MS / MS.” 2012, Analytical Chemistry, volume 84. issue 14, pages 5959-5967; Shen et al.. “Online 2D-LC-MS / MS Assay to Quantify Therapeutic Protein in Human Serum in the Presence of Pre-existing Antidrug Antibodies.” 2015, Analytical Chemistry, volume 87. issue 16. pages 8555-8563.

[0050] LC-MS / MS quantification of antibody drug concentration in human serum samples can be based on measurement of ion intensities of the surrogate peptides derived from the variable complementarity determining regions (CDRs) of the antibody drugs. See, e.g.. Jenkins et al., “Recommendations for Validation of LC-MS / MS Bioanalytical Methods for Protein Biotherapeutics.” 2015, The AAPS Journal, volume 17, pages 1-16. The sample processing is relatively simple, which typically involves protease digestion of a few microliters of serum sample after reduction and alkylation; and the reagents needed for the LC-MS / MS assay, such as stable heavy isotope labeled peptides for internal standards, can be easily acquired. The sensitivity and selectivity of the LC-MS / MS assay can rely on the unique peptides selected for quantification. However, identification of a surrogate peptide from a complex matrix using LC-MS / MS may require either extensive method development and optimization when using a direct digestion method, or assay -specific reagents when using an immunoprecipitation method.

[0051] Endogenous serum proteins, such as albumin and immunoglobulins, can be as many as 10,000 times more concentrated than the protein of interest in serum samples. This high concentration can potentially interfere with the detection and analysis of other proteins in serum. To mitigate this interference and increase sensitivity, sample pretreatment can be performed. One method involves immunocapture with anti-idiotypic antibodies, anti-species Fc antibodies, or Protein A or G. before LC / MS analysis. These techniques selectively extract and concentrate the protein of interest from the serum, thus decreasing the lower limit of quantification (LLOQ). e.g., to as low as 0.02 pg / mL. Another method involves depletion of the most abundant serum proteins before LC / MS analysis, thereby improving the detection and identification of the protein of interest. This approach can achieve a decreased LLOQ, e g., as low as 2 pg / mL.

[0052] Another method to enrich therapeutic proteins utilizes interacting peptide ligands, such as a combinatorial ligand library. When the peptide ligand -conjugated beads are applied to a sample containing various protein species, each protein species can bind to its interacting peptide ligands. Without being limited by theory, antibodies may bind to their interacting peptide ligands by hydrophobic force in combination with some weak interaction forces, such as ionic interaction and hydrogen bonding.

[0053] A protein species that is in high abundance can saturate its interacting peptide ligands due to the presence of excess quantity, since there are limited numbers of interacting peptide ligandscorresponding to each protein species in the combinatorial ligand library. The limited numbers of corresponding interacting peptide ligands can be saturated in the presence of excess quantity of high- abundance proteins. The excess quantity of high-abundance proteins that are unable to bind to the interacting peptide ligands can be washed off from the beads. Since the quantity of low -abundance proteins in the sample is relatively low in comparison to the high-abundance proteins, the low-abundance proteins may not saturate their corresponding interacting peptide ligands. Therefore, the low -abundance proteins can be relatively enriched in comparison to the high-abundance proteins. After enrichment, the broad dynamic range of protein concentrations can be reduced to a level that allows for detection of low abundance proteins.

[0054] A combinatorial hexapeptide library immobilized or supported on beads (e.g.. enrichment beads) can used to enrich low-abundance proteins. Examples of suitable enrichment beads include those sold under the PROTEOMINER™ tradename. A method using enrichment beads can be coupled with LC-MRM to quantitate low -abundant biotherapeutics in serum, including, but not limited to, monoclonal antibodies (mAbs) and gene insertion therapy products. The LLOQ of serum concentrations of drug substance in using enrichment beads is comparable to the LLOQ that can be achieved using immuno- cnrichmcnt, but the sample preparation is based on commercially available reagents rather than assayspecific reagents, and therefore can overcome the bias of the immunocapture approach and time required to develop the assay-specific antibodies. This method meets the traditional assay requirements with regards to precision, accuracy and specificity, and improves and accelerates PK analysis in early and late pharmaceutical development processes.

[0055] Although some methods may assist in mitigating matrix interference, they may introduce issues similar to those in the ELISA method, including the time-intensive production of anti-drug antibodies and potentially inadequate detection specificity. The present application describes methods including pretreating serum samples before LC / MS analysis, through enrichment of biotherapeutic agents with enrichment beads, coupled with liquid chromatography -multiple reaction monitoring mass spectrometry (LC-MRM) analysis. Enrichment beads may concentrate the protein of interest through specific ligand binding, while simultaneously decreasing serum proteins by saturating their specific ligands. Methods described herein eliminate the need to develop specific ligands to capture proteins of interest, thereby saving both time and costs with respect to the immunocapture approach. The LLOQ for serum concentrations of drug substances with enrichment beads can be comparable to immunocapture methods and lower than methods including serum protein depletion, e g., as low as 0.02 pg / mL. The disclosued methods satisfy traditional assay requirements for precision, accuracy, and specificity, and can expedite PK analysis in both early and late stages of pharmaceutical development.

[0056] This disclosure provides methods to satisfy the aforementioned demands by providing methods to accurately and rapidly quantify therapeutic proteins present in serum. Provided herein are methods of identification, quantification and characterization of a protein of interest in complex biological samples.

[0057] Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular methods and materials are now described.

[0058] The term “a” should be understood to mean “at least one” and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art and where ranges are provided, endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-limiting and are understood to mean “comprise.” “comprises,” and “comprising” respectively.

[0059] As used herein, the term “protein” or “protein of interest” can include any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides.” “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. “Synthetic peptide or polypeptide” refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods arc known to those of skill in the art. A protein may comprise one or multiple polypeptides to form a single functioning biomolecule. A “therapeutic protein” includes any of 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.

[0060] A protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest can include any of bio- therapeutic 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. Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g.. Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation”. 2012, Biotechnology and Genetic Engineering Reviews, volume 28. pages 147-176. the entirety of which is herein incorporated by reference. In some exemplary aspects, proteins comprise modifications, adducts, and other covalently linked moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans e.g., N-acetylgalactosamine, galactose, neuraminic acid. N- acetylglucosamine. fucose, mannose, and other monosaccharides), PEG, polyhistidine. FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels and other dyes, and the like. Proteins can be classified on the basis of compositions and solubility and can thus 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.

[0061] As used herein, the term “recombinant protein” may refer to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary aspects, the recombinant protein can be an antibody, for example, a chimeric, humanized, or fully human antibody. In certain exemplary aspects, the recombinant protein can be an antibody of an isotype selected from group consisting of: IgG. IgM, IgAl, IgA2, IgD. or IgE. In certain exemplary aspects the antibody molecule is a full-length antibody (e.g., an IgGl) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

[0062] As used herein, the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different aspects of the present disclosure, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human gennline sequences 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. The term “antibody,” as used herein, also includes antigenbinding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, for example, commercial sources, DNA libraries (including, e.g.. phageantibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0063] As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv fragment, a Fvfragment, a dsFv diabody, a dAb fragment, a Fd’ fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies. tetrabodies, linear antibodies, single-chain antibody molecules, and multi specific antibodies formed from antibody fragments. Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some exemplary aspects, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some exemplary aspects, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and / or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multi-molccular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.

[0001] Antibodies described herein may include a therapeutic antibody. For example, the antibody may be or include Alirocumab, Atoltivimab, Maftivimab, Odesivimab, Odesivimab-ebgn, Casirivimab, Imdevimab, Cemiplimab and Cemiplimab-rwlc (human IgG4 monoclonal antibody that binds to PD-1), Sarilumab, Fasimunab, Nesvacumab, Dupilumab (human monoclonal antibody of the IgG4 subclass that binds to the IL-4R alpha (a) subunit and thereby inhibits Interleukin 4 (IL-4) and Interleukin 13 (IL-13) signaling). Trevogrumab. Evinacumab, Evinacumab-dgnb, Fianlimab, Garetosmab, Itepekimab, Odrononextamab, Pozelimab, Rinucumab, and modifications, truncations, and variations thereof.Additional exemplary antibodies include Ravulizumab-cwvz, Abciximab. Adalimumab, Adalimumab- atto, Ado-trastuzumab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab, Belimumab, Benralizumab, Bevacizumab. Bezlotoxumab, Blinatumomab. Brentuximab vedotin, Brodalumab.Canakinumab. Capromab pendetide, Certolizumab pegol, Cetuximab, Denosumab, Dinutuximab, Durvalumab, Eculizumab, Elotuzumab, Emicizumab-kxwh, Emtansine alirocumab, Evolocumab, Golimumab, Guselkumab, Ibritumomab tiuxetan, Idarucizumab, Infliximab. Infliximab-abda, Infliximab- dyyb. Ipilimumab. Ixekizumab, Mepolizumab, Necitumumab, Nivolumab, Obiltoxaximab,Obinutuzumab, Ocrelizumab, Ofatumumab. Olaratumab, Omalizumab, Panitumumab. Pembrolizumab,Pertuzumab, Ramucirumab, Ranibizumab, Raxibacumab, Reslizumab, Rinucumab, Rituximab, Secukinumab, Siltuximab, Tocilizumab, Trastuzumab, Ustekinumab, Vedolizumab, and modifications, truncations, and variations thereof.

[0064] As used herein, the term “bispecific antibody” (bsAb) includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.

[0065] Some bispecific antibodies have two heavy chains each having three heavy chain CDRs, followed by a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes. Bispecific antibodies can be divided into two major classes, those bearing an Fc region (IgG-like) and those lacking an Fc region, the latter normally being smaller than the IgG and IgG-like bispecific molecules comprising an Fc. The IgG-like bsAbs can have different formats such as, but not limited to, triomab, knobs into holes IgG (kih IgG). crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG-single- chain Fv (IgG-scFv), or K / .-bodics. The non-IgG-like different formats include tandem scFvs, diabody format, single -chain diabody, tandem diabodies (TandAbs), dual-affinity retargeting molecule (DART), DART-Fc. nanobodies, or antibodies produced by the dock-and-lock (DNL) method, such as described in Fan et al., “Bispecific antibodies and their applications.” Journal of Hematology & Oncology, volume 8, issue 130; and in Muller et al., “Bispecific Antibodies,” 2014, Handbook of Therapeutic Antibodies, pages 265-310, the entire contents of which are incorporated by reference herein. The methods of producing bsAbs are not limited to quadroma technology based on the somatic fusion of two different hybridoma cell lines, chemical conjugation, which involves chemical cross-linkers, and genetic approaches utilizing recombinant DNA technology.

[0066] As used herein, the term “multispecific antibody” may refer to an antibody with binding specificities for two or more different antigens. While such molecules normally will only bind twoantigens (e.g., bispecific antibodies), antibodies with additional specificities such as trispecific antibody and KIH Trispecific are also contemplated.

[0067] As used herein, the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody can be derived from a single clone, including any eukary otic, prokary otic, or phage clone, by any means available or known in the art. Monoclonal antibodies 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.

[0068] As used herein, a “protein pharmaceutical product”, "proteinaceous drug product," or “biopharmaceutical product” includes an active ingredient which can be fully or partially biological in nature. In one aspect, the protein pharmaceutical product can comprise a peptide, a protein, a fusion protein, an antibody, an antigen, vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, cells, tissues, or combinations thereof. In another aspect, the protein pharmaceutical product can comprise a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, a fusion protein, an antibody, an antigen, vaccine, a peptide-drug conjugate, an antibody -drug conjugate, a protein-drug conjugate, cells, tissues, or combinations thereof.

[0069] As used herein, a “sample” may refer to a mixture of molecules that is subjected to manipulation in accordance with the methods of the disclosure, including, for example, separating, analyzing, extracting, concentrating, profiling and the like. The sample may be a biological sample obtained from an organism, such as a serum sample. As described above, serum samples include relatively high concentrations of endogenous serum proteins, such as albumin and immunoglobulins. Alternatively, the sample may be a biological sample obtained from a process for manufacturing therapeutic proteins, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), a composition produced during any step (e.g., clarification, chromatographic production, or filtration) in downstream processing, drug substance (DS), or a drug product (DP) comprising the final formulated product. The drug product may be selected from manufactured drug product in the manufacturing facility, stored drug product, packaged drug product, shipped drug product, clinical administered drug product, or other lot of drug product.

[0070] The terms “peptide.” “protein” and “polypeptide” may refer to a polymer of amino acids and / or amino acid analogs that are joined by peptide bonds or peptide bond mimetics.

[0071] As used herein, the terms “pharmacokinetics” or “PK” refer to a field of study related to features or properties of a drug after administration to a subject. Exemplary components of pharmacokinetic analysis include liberation of a drug from a pharmaceutical formulation, absorption of a drug into blood circulation, distribution of a drug throughout the body, metabolism (also called biotransformation) of a drug into metabolites, and excretion of a drug from a body. Pharmacokinetics of a drug are a key’ feature for evaluation of a biotherapeutic candidate. In particular, a pharmacokinetic study’ may be conducted to evaluate how levels of a drug and its modified forms and metabolites change over time after administration to a subject. Biotherapeutic proteins may be evaluated through the analysis of representative peptides, or “target peptides” or “surrogate peptides,” using liquid chromatography -massspectrometry. A peptide may be a suitable target peptide if it is unique to or strongly representative of a protein, for example a complementarity -determining region of an antibody, and if it can be reliably recovered and measured. This disclosure sets forth methods for selecting surrogate peptides for a protein of interest. In some exemplary aspects, a surrogate peptide may be selected based on being representative of a class of proteins of interest, for example IgG molecules, while being undetected in a control sample, for example a non-human animal serum.

[0072] As used herein, the term “database” may refer to a compiled collection of protein sequences that may possibly exist in a sample, for example in the form of a file in a FAST A format. Relevant protein sequences may be derived from cDNA sequences of a species being studied. Public databases that may be used to search for relevant protein sequences included databases hosted by, for example, Uniprot or Swiss-prot. Databases may be searched using what are herein referred to as “bioinfonnatics tools”. Bioinformatics tools provide the capacity to search uninterpreted MS / MS spectra against all possible sequences in the database(s), and provide interpreted (annotated) MS / MS spectra as an output. Nonlimiting examples of such tools are Mascot (matrixscience.com), Spectrum Mill (chem.agilent.com), PLGS (waters.com), PEAKS (bioinformaticssolutions.com), Proteinpilot (download.applicdbiosystcms.com / protcinpilot), Phcnyx (phcnyx-ms.com), Sorcerer (sagenresearch.com), OMSSA (pubchem.ncbi.nlm.nih.gov / omssa / ), X! Tandem (thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (proteinmetrics.com / products / byonic) or Sequest (fields.scripps.edu / sequest).

[0073] FIG. 1 depicts an exemplary method 100 for quantifying a protein in a sample. At step 110, the method 100 may comprise contacting a sample including a protein to a solid support. The sample may be a biological sample, such as a serum sample obtained from an organism (e.g., human, monkey, mouse, rat, rabbit, guinea pig, hamster, dog, cat, or any mammal). The protein may be capable of binding a target protein or molecule, and the protein may include an antibody, an antibody fragment, an antibody-drug conjugate, a single chain variable fragment (ScFv), or a fusion protein. A solid support may include any surface with an ability to bind a protein or peptide. Non-limiting examples of solid supports can include affinity resins, beads, and coated plates or microplates. Solid supports can be attached to molecules capable of binding to a protein or peptide, including affinity reagents, antigen -binding molecules, or interacting peptide ligands. The solid support may comprise beads attached to peptide ligands capable of binding to a protein. For example, the solid support may comprise a library of bead-based peptide ligands that can bind to diverse types of proteins, such as PR0TE0MIN0R™ enrichment beads. Such beads may successfully enrich medium- and low-abundance proteins and remove excess of high -abundance proteins with these peptide ligands possessing diverse affinities to proteins. An illustration of step 110 is shown in FIG. 2 A. A sample 10 including various protein components, including a protein of interest 12. is contacted to a solid support 20 attached to peptide ligands 22 capable of binding to the protein of interest 12 to form a complex 30 of the protein of interest, the solid support 20, and the peptide ligands 22.

[0074] Optionally, as illustrated in FIG. 2B, the method may further comprise washing the complex 30 to remove unbound material 40. Unbound material 40 may include host cell proteins, contaminants, orother undesired material. Washing may be performed by contacting the complex 30 to a washing buffer, such as phosphate buffered saline (PBS) as a pH of 7.4. The unbound material 40 may be collected in and removed with the wash buffer, such that only the complexes 30 remain in the sample 10.

[0075] Returning to FIG. 1. method 100 may comprise contacting an elution buffer to the solid support to produce an enriched sample (e.g.. step 120). An elution buffer may be configured to disrupt interactions between bound molecules and a complex, matrix, or column, thereby inducing release of bound molecules from the complex, matrix, or column. The elution buffer may include phase-transfer surfactant (PTS) buffer, urea buffer, or guanidine buffer. For example, the elution buffer may be a PTS buffer including 12 mM sodium deoxycholate (SDC), 12 mM sodium Wlauroylsarcosinate (SLS), 10 mM Tris-(2-carboxyethyl)phosphine, hydrochloride (TCEP), and 40 mM Schistosoma circulating anodic antigen (CAA). As another example, the elution buffer may be a urea buffer including 8 M urea, 10 mM TCEP, and 40 mM CAA. As another example, the elution buffer may be a guanidine buffer including 6 M guanidine, 10 mM TCEP, and 40 mM CAA. An illustration of step 120 is shown in FIG. 2C. An elution buffer may be contacted to complex 30, thereby disrupting the interactions between the complex 30 and solid support. The elution buffer may induce the release of protein of interest 12 from peptide ligands 22 of solid support 20.

[0076] Prior to LC-MS / MS analysis, the sample may be prepared with reduction, denaturation, alkylation, dilution, digestion, and / or separation (e.g., centrifugation).

[0077] For example, referring to FIG. 1, method 100 may comprise subjecting the enriched sample to denaturing conditions to produce a denatured sample (e.g., step 130). Protein denaturing or denaturation is a process by which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation can be carried out by subjecting the protein to denaturing conditions, such as exposure to high heat, high pH. low pH, or exposure to a denaturing agent (also referred to as a denaturant). For example, a denaturing agent may include a reducing agent or a chaotropic agent. Chaotropic agents increase the entropy of the system by interfering with 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- lauroylsarcosine. urea, and salts thereof.

[0078] Method 100 may comprise contacting a reducing agent to the denatured sample to produce a reduced sample (e.g.. step 140). A reducing agent is a composition capable of reducing disulfide bridges in a protein. Non-limiting examples of reducing agents used to reduce a protein are dithiothreitol (DTT), B-mercaptoethanol. Ellman’s reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2- carboxyetlivl)pliosphine hydrochloride (TCEP-HC1). or combinations thereof. A reducing step may be performed in sequence with or concurrent with other sample preparation steps. For example, a reducing step may be performed after a denaturing step. Alternatively, a reducing step and a denaturing step may be performed simultaneously (by adding a reducing agent while incubating a sample at hightemperatures) so that cysteines exposed to the solvent by denaturing can be accessed by the reducing agent.

[0079] Method 100 may comprise contacting an enzyme to the reduced sample to produce a peptide digest, wherein at least one peptide of the peptide digest is a uniquely identifying peptide for the protein (e.g., step 150). As used herein, the term “digestion” refers to hydrolysis of one or more peptide bonds of a protein. There are several approaches to carrying out digestion of a protein in a sample using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion. Digestion of a protein into constituent peptides can produce a composition including peptide, polypeptides, and other polypeptide fragments (e.g.. a peptide digest). The peptide digest may be further analyzed using peptide mapping analysis. Enzymatic digestion may be performed by an enzyme, such as protease.Exemplary proteases include Aspergillus Saitoi, elastase, subtilisin. protease XIII, pepsin, trypsin, Tryp- N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS). thermolysin, papain, pronase. V8 protease, or biologically active fragments or homologs thereof, or combinations thereof. For a recent review discussing the available techniques for protein digestion see Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments,” 2013, Journal of Proteome Research, volume 12, pages 1067-1077, the entire contents of which are incorporated by reference herein.

[0080] A uniquely identifying peptide is a peptide that is unique to or strongly representative of a protein. A uniquely identify ing peptide may have an amino acid sequence identical to a portion of an amino acid sequence of a protein, and may be unique to that protein or may be unique to a class of proteins. For example, the uniquely identifying peptide may have an amino acid sequence identical to a portion of a variable region of an antibody, such as a complementarity-determining region (CDR) of the variable region of the antibody, such that the uniquely identifying peptide is representative of that antibody. As another example, the uniquely identifying peptide may have an amino acid sequence identical to a portion or region present in all human IgG proteins, such that the uniquely identifying peptide is representative of the class of human IgG proteins.

[0081] An illustration of steps 130, 140, and 150 is shown in FIG. 2D. The protein of interest 12 may be denatured, reduced, and digested to form a peptide digest 50 including a uniquely identifying peptide 14. The uniquely identifying peptide 14 is unique to or strongly representative of protein of interest 12.

[0082] Referring to FIG. 1, method 100 may comprise contacting the peptide digest to a liquid chromatography-tandem mass spectrometry7(LC-MS / MS) system to determine a quantity of the uniquely7identifying peptide (e.g., step 160). During liquid chromatography, a mixture of biological components, carried by a liquid, is separated into components as the mixture flows through (or into) a stationary phase (e.g., a stationary liquid phase or a stationary solid phase). The different components of the mixture may be differentially distributed based on their chemistry and structure. Non-limiting examples of liquidchromatography include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and / or mixed-mode chromatography. During mass spectrometry, specific molecular species and their masses may be detected, identified, measured, or otherw ise characterized. A method using both liquid chromatography and mass spectrometry may be referred to as including liquid-chromatography -mass spectrometry (LC-MS) analysis.

[0083] When mass spectrometry is performed using tandem mass spectrometry, the process may be referred to including as liquid-chromatography -tandem mass spectrometry (LC-MS / MS). Tandem mass spectrometry is a chemical technique in w hich structural information on a sample molecule is obtained by using multiple stages of mass selection and mass separation. Tandem mass spectrometry' may include transforming the sample molecule into a gas phase and ionizing the gaseous sample, so that fragments are formed in a predictable and controllable fashion after the first mass selection step. MS / MS (or MS2) can be performed by first selecting and isolating a precursor ion (MSI), and fragmenting it to obtain meaningful information. The specific analyzers included for a certain MS2 application can be determined by many different factors, such as sensitivity', selectivity', speed, size, cost, and availability. The two major categories of MS / MS methods arc tandcm-in-spacc and tandem-in-time, but there arc also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in- space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two nontrapping mass analyzers. Specific mass-to-charge (m / z) separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m / z separation and data acquisition. In tandem-in-time, mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0084] The mass spectrometer in an LC-MS or LC-MS / MS system may be further coupled to a multiple reaction monitoring (MRM) system or selected reaction monitoring (SRM) system. MRM and SRM allow for the precise quantification of small molecules, peptides, and proteins within complex matrices with high sensitivity, specificity and a wide dynamic range, such as described in Picotti et al., “Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions,” 2012, Nature Methods, volume 9. pages 555-566. MRM may be performed with a triple quadrupole mass spectrometer, wherein a precursor ion corresponding to the selected small molecules / peptides is selected in the first quadrupole, and a fragment ion of the precursor ion is selected for monitoring in the third quadrupole, such as described in Choi et al., “Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates,” 2013. Journal of Chromatography B, volume 930, pages 129-135, the entire content of which is incorporated by reference herein. Selected ion monitoring (SIM) is a form of MRM / SRM in w hich an ion of a particular mass is selected in the first stage of a tandem mass spectrometer, and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection. Examples of triple quadrupole mass spectrometers (TQMS) that can perform SIM include, but are not limited to, QTRAP K 6500 System(Sciex). QTRAP® 5500 System (Sciex), Triple QTriple Quad 6500 System (Sciex). Agilent 6400 Series Triple Quadrupole LC / MS systems, and THERMO SCIENTIFIC™ TSQ™ Triple Quadrupole system.

[0085] Peptides may also be quantified through parallel reaction monitoring (PRM). PRM is an application of SRM with parallel detection of all transitions in a single analysis using a high-resolution mass spectrometer. PRM provides high selectivity, high sensitivity and high-throughput to quantify selected peptides (QI), and quantify proteins. Multiple peptides can be specifically selected for each protein. PRM methodology can use the quadrupole of a mass spectrometer to isolate a target precursor ion, fragment the targeted precursor ion in the collision cell, and then detect the resulting product ions in the Orbitrap mass analyzer. PRM can use a quadrupole time-of-flight (QTOF) or hybrid quadrupoleorbitrap (QOrbitrap) mass spectrometer to carry out the identification of peptides and / or proteins. Examples of QTOF include but are not limited to TRIPLETOF® 6600 System (Sciex), TRIPLETOF® 5600 System (Sciex), X500R QTOF System (Sciex). 6500 Series Accurate-Mass Quadrupole Time-of- Flight (Q-TOF) (Agilent) and Xevo G2-XS QT of Quadrupole Time-of-Flight Mass Spectrometry (Waters). Examples of QObitrap include but are not limited to Q EXACTIVE™ Hybrid Quadrupole- Orbitrap Mass Spectrometer (Thermo Scientific) and ORBITRAP FUSION™ TRIBRID™ (Thermo Scientific). Non-limiting advantages of PRM include: elimination of most interferences; providing more accuracy and attomole-level limits of detection and quantification; enabling confident confirmation of the peptide identity with spectral library matching; reducing assay development time since no target transitions need to be preselected; and ensuring UHPLC -compatible data acquisition speeds with spectrum multiplexing and advanced signal processing.

[0086] As discussed above, a peptide identified by the mass spectrometer may be used as a surrogate representative of the intact protein and its post-translational modifications. It can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter generated from possible peptides in a protein sequence database. The characterization may include identifying the protein, sequencing amino acids of the protein fragments, determining protein sequencing, quantifying the protein, locating post-translational modifications, identifying post translational modifications, comparability analysis, or a combination thereof.

[0087] The methods or systems of the present disclosure may utilize a mass spectrometer. Suitable mass spectrometers include an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, and / or a triple quadrupole mass spectrometer. The mass spectrometer may be coupled to a liquid chromatography system, and the mass spectrometer may be configured to perform LC-MS (liquid chromatography -mass spectrometry) analysis and / or LC-PRM-MS (liquid chromatographyparallel reaction monitoring-mass spectrometry) analysis.

[0088] The mass spectrometer may use nanoelectrospray or nanospray ionization, including electrospray ionization at a low solvent flow rate, e.g., hundreds of nanoliters per minute of sample solution or lower, often without the use of an external solvent delivery. The electrospray infusion setup forming a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs a continuous analysis of small sample (analyte)solution volumes over an extended period of time. A dynamic nanoelectrospray emitter may use a capillary column and a solvent delivery system to perform chromatographic separations on mixtures prior to analysis by the mass spectrometer.

[0089] As described herein, exemplary method 100 may include the use of an LC-MS / MS system to determine the quantity of the uniquely identifying peptide. However, other LC-MS methods described above may be substituted for LC-MS / MS in the methods contemplated in the present disclosure. The LC- MS / MS system may be used to measure and calculate a peak area (i.e., area under the curve) for the uniquely identifying peptide. Peak area for the uniquely identifying peptide may be compared to a peak area for an internal standard, or may be compared to a standard curve.

[0090] Method 100 may comprise determining a quantify of the protein of interest based on the quantify of the uniquely identifying peptide (e.g., step 170). Determining a quantity of the protein interest may comprise comparing the quantity of the uniquely identifying peptide to a calibration curve for the protein of interest and the uniquely identifying peptide. For example, a calibration cun e may be generated before the method is performed, and may be generated by comparing a peak area ratio of the uniquely identifying peptide and the protein of interest across increasing concentrations of the protein of interest.

[0091] In some aspects, the methods described herein may be utilized for quantifying an antibody in a serum sample. The method may comprise contacting a serum sample to a solid support, wherein the serum sample comprises an antibody and the solid support is attached to peptide ligands capable of binding to the antibody; contacting an elution buffer to the solid support, thereby producing an enriched antibody sample; subjecting the enriched antibody same to denaturing conditions, thereby producing a denatured antibody sample; contacting a reducing agent to the denatured antibody sample to produce a reduced antibody sample; contacting an enzyme to the reduced antibody sample to produce a peptide digest, wherein at least one peptide of the peptide digest is a uniquely identifying peptide for the antibody; contacting the peptide digest to a liquid-chromatography -tandem mass spectrometry (LC- MS / MS) system to determine a quantity of the uniquely identifying peptide; and detennining a quantify of the antibody based on the quantity of the uniquely identifying peptide.

[0092] In some aspects, the methods described herein may be utilized for identifying, quantifying, and / or characterizing at least one IgG molecule of interest in a sample. The method may comprise contacting a sample including at least one IgG molecule of interest to a solid support, wherein the solid support is attached to interacting peptide ligands capable of interacting with the at least one IgG molecule of interest to form an immobilized IgG molecule; washing the immobilized IgG molecule to remove unbound material, eluting the bormd IgG molecule to produce an enriched IgG molecule; subjecting the enriched IgG to a denaturant; subjecting the denatured IgG molecule to a reducing agent; subjecting the reduced sample to enzymatic digestion conditions to produce a peptide digest; subjecting the peptide digest to liquid chromatography mass spectrometry / mass spectrometry (LC-MS / MS) analysis to identify, quantify' and / or characterize the at least one common IgG peptide; and using the analysis of the LC- MS / MS to identify, quantify and / or characterize the IgG molecule of interest.

[0093] In some aspects, the methods described herein may be utilized for quantifying at least one therapeutic antibody in a sample. The method may comprise contacting a sample including at least one therapeutic antibody of interest to a solid support, wherein the solid support is attached to a hexapeptide ligand library capable of interacting with the at least one therapeutic antibody of interest to form an immobilized therapeutic antibody; washing the immobilized therapeutic antibody to remove unbound material, eluting the bound therapeutic antibody to produce an enriched therapeutic antibody; subjecting the enriched therapeutic antibody to enzymatic digestion conditions to produce a peptide digest, wherein the peptide digest includes a complementarity determining region peptide that is uniquely derived from the therapeutic antibody; and subjecting the peptide digest to liquid chromatography mass spectrometry / mass spectrometry (LC-MS / MS) analysis to identify, quantify and / or characterize the complementarity determining region peptide to identify, quantity' and / or characterize.

[0094] In some aspects, the methods described herein may be utilized for identifying, quantifying and / or characterizing a protein of interest in a sample. The method may comprise contacting a sample including a protein of interest to enrichment beads; washing the immobilized protein of interest to remove unbound material; eluting the bormd protein of interest to produce an enriched protein; subjecting the enriched protein to a denaturant; subjecting the denatured protein to a reducing agent, subjecting the reduced sample to enzymatic digestion conditions to produce a peptide digest, wherein at least one peptide is a uniquely identifying peptide for the protein of interest; subjecting the peptide digest to liquid chromatography -tandem mass spectrometr / mass spectrometry (LC-MS / MS) analysis to identify, quantify and / or characterize the at least one uniquely identifying peptide; and using the anal sis of the LC-MS / MS to identify, quantify and / or characterize the protein of interest.

[0095] It is understood that the present disclosure is not limited to any of the protein(s), therapeutic protein(s). antibody(s). recombinant protein(s), protein pharmaceutical product(s). sample(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), chromatographic method(s). mass spectrometer(s). database(s), bioinformatics tool(s), pH range(s) or value(s), temperature(s), or concentration(s) described herein. Further, any protein(s), therapeutic protein(s), antibody(s), recombinant protein(s), protein pharmaceutical product / s). sample(s). protein denaturing agent(s), protein reducing agent(s). digestive enzyme(s). chromatographic method(s), mass spectrometer(s), database(s). bioinformatics tool(s), pH, temperature(s). or concentration(s) can be selected by any suitable means for use in or with the methods and systems disclosed herein.

[0096] The following examples are provided for additional clarity on aspects of the methods and systems disclosed herein. The following examples should not be construed as limiting the scope of the present disclosure.EXAMPLES

[0097] Materials. Enrichment beads from the PROTEOMINER™ protein enrichment kit from BioRad (Hercules, CA) were used. Chromatography solvents were of LC-MS grade from Fisher Scientific (Waltham. MA). Sodium deoxy cholate (SDC), sodium lauroyl sarcosinate (SLS), 2-Chloroacetamide (CAA), Tris(2 -carboxy ethyl) phosphine hydrochloride (TCEP), rat serum, mouse serum, rabbit serum,guinea pig serum, SILuMab Trastuzumab stable-isotype. SILuMab Rituximabstable-isotype. SILuMab Adalimumab stable-isotype, and SILuMab Bevacizumabstable-isotype were purchased from Sigma- Aldrich (St. Louis. MO). lodoacetamide (IAM). Dithiothreitol (DTT), Trifluoracetic acid (TFA). UltraPure 1 M Tris-HCl pH 8.0 were purchased from Thermo Fisher. The cynomolgus monkey serum was purchased from Abeam and pooled human serum was purchased from Innovative Research (Novi, MI). mAbs and ScFv were produced by Regeneron (Tarrytown. NY).

[0098] Preparation of Standard Solutions. Stock solutions of biotherapeutic drugs in mouse serum were made by co-spiking mAb-1 and mAb-2 drug substances or ScFv into mouse serum containing 2 pg / mL SILuMab Trastuzumab. Calibration standards (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 pg / mL) were made through a serial dilution of stock solution containing mAb-1 and mAb-2 drug substances or stock solution containing ScFv with SILuMab spiked-in mouse serum. Five quality control (QC) standards, lower limit of quantitation (LLOQ. 0.0192 pg / mL ScFv), low QC (LQC, 0.096 pg / mL ScFv or mAb-1 and mAb-2 combination), mid QC (MQC, 0.48 pg / mL ScFv or mAb-1 and mAb- 2 combination), high QC (HQC, 2.4 pg / mL ScFv or mAb-1 and mAb-2 combination) and upper limit of quantitation (ULOQ, 12 pg / mL ScFv or mAb-1 and mAb-2 combination), were prepared by spiking combination antibodies or ScFv into SILuMab spiked-in mouse scrum and performing serial dilutions.

[0099] Sample preparation using enrichment beads. Biotherapeutic s were enriched from serum by PROTEOMINER™ enrichment beads following similar protocol as described by Chen et al. with modifications for improved antibody enrichment. See Chen et al., “A Highly Sensitive LC-MS / MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics”, 2012, Journal of Pharmaceutical Sciences, volume 110, issue 12, pages 3811-3818, the contents of which are incorporated herein by reference. The protocol was modified by spiking 0.5 pg / mL mAb-1 drug substance into mouse serum. In brief, enrichment beads were washed by wash buffer and water sequentially and resuspended into various incubation buffers at different pH, respectively. Different incubation buffers were prepared, including H2O, lx PBS, pH 7.0, 0.1 M Tris-HCl, pH 8.0 and 0.1 M Tris-HCl, pH 9.0. The serum sample was then mixed with 10 pL of resuspended enrichment beads and 100 pL incubation buffer and incubated for 2.5 hours at room temperature. Volume amounts of 5 pL. 10 pL and 20 pL of serum sample were mixed with 10 pL resuspended enrichment beads and 100 pL incubation buffer (H2O) to determine a serum amount used for analysis, and enrichment beads were resuspended into 500 pL, 200 pL, 100 pL, 50 pL and 20 pL of H2O to determine the a volume of enrichment beads. The slurry of beads were then washed with wash buffer and water, eluted with elution buffer, denatured, reduced and alkylated, and digested by trypsin overnight at 37° C. Three different elution buffers were prepared and compared, including PTS buffer containing 12 mM SDC. 12 mM SLS, 10 mM TCEP and 40 mM CAA, urea buffer containing 8M urea. 10 mM TCEP and 40 mM CAA, and guanidine buffer containing 6M guanidine. 10 mM TCEP and 40 mM CAA. The digested peptides were then acidified by TFA. centrifuged at 14,000 xg and desalted for sequential LC-MS / MS analysis.

[0100] Nano LC-Parallel reaction monitoring mass spectrometry (PRM-MS) analysis. The peptide mixture was resuspended in 0.1% formic acid (FA) in water and peptide mixture concentrationwas measured by nanodrop 2000. Peptide mixture of 1.25 jj.g were loaded onto an nano LC system coupled with a mass spectrometer. Peptides were sequentially loaded onto a trap column (20 cm x 0.075 mm Acclaim PepMap 100 Cl 8) at 5 pL / min and separated with an analytical column with inner diameter of 0.075 mm (30 cm, 1.7 pm, 100 A, CoAnn Technologies) at 0.25 pL / min for desalting and separation. Mobile phase A was 0.1% FA in water, and mobile phase B was 0.1% FA in acetonitrile. The gradient started with 4% mobile phase B for 6 min. was increased to 20% B over 84 min and further increased to 36% B within 35 min, and then was steeply increased to 95% B in 9 min and held for 9 min. Each sample was analyzed with a targeted MS2 scan at 15,000 resolutions, with an isolation winder of 2 m / z, and HCD was set at 30% NCE.

[0101] LC-multiple reaction monitoring mass spectrometry (MRM-MS) analysis. LC-MRM- MS experiments was performed on a UPLC system coupled with a triple quadrupole mass spectrometer. Up to 6 pg of peptide mixtures that resuspended in 0.1% FA in water were loaded and separated on a ACQUITY Premier CSH C18 column (1.7 pm, 2.1 x 100 mm). Ten microliters of a digested serum sample, proportional to approximately 45 nL of the original serum, was loaded onto a C18 column (ACQUITY UPLC BEH300 1.7 pm, 2.1 mm x 100 mm, Waters) and separated by reversed-phase gradient elution using mobile phase A as 0.1% formic acid in water and mobile phase B as 0.1% formic acid in acetonitrile at a flow rate of 0.4 mL / min. Prior to each injection, the sample injection path was sequentially flushed with IPA / ACN / H2O v / v / v (3: 1 : 1), ACN / H2O / FA v / v / v (25:75:0.1), and ACN / H2O / FA v / v / v (5:95:0.1). The LC gradient for MRM experiments using a cocktail of mAb-1 and mAb-2 was set as follows: 0-0.5 min, 5% B; 0.5-15 min, 5-30% B; 15-16 min, 30-40% B; 16-16.5 min, 40-90% B; 16.5-19.5 min, 90% B; 19.5-20.5 min, 90-5% B; and 20.5-25 min, 5% B. The LC gradient for MRM experiments using drug substance-1 (DS-1) was as follows: 0-1 min, 5% B; 1-8 min, 5-30% B: 8-9 min, 30-60% B; 9-9.5 min, 60-95% B; 9.5-11 min, 95%B; 11-11.5 min, 95-5% B; and 11.5-13 min, 5% B. The column temperature was set at 60 °C in both experiments, and the gas temperature for the MS ion source was set as 200° C, with a gas flow rate at 12 L / min, nebulizer gas at 20 psi. and sheath gas temperature at 300° C.

[0102] Data Analysis. Raw MS data were searched against the biotherapeutics sequence with no redundant entries, using Protein Metrics Byonic software. The spectra were searched with 10 ppm mass tolerance for precursor ions and 0.02 Da for fragment ions, and tryptic digestion with a maximum of one missed cleavage sites. Carbamidomethylation of cysteines (+57.0214 Da) was included as a static modification, and the variable modifications included oxidation (+15.9949 Da) on methionine and deamidation (+ 0.984 Da) on asparagine. PRM and MRM data were manually analyzed with Skyline software.

[0103] Example 1: Development of Methods Using IgG and PROTEOMINER™ Protein Enrichment Kit with Nano LC-PRM and LC-MRM

[0104] Identifying, characterizing, and quantifying proteins in complex biological matrices such as serum presents a challenge due to, for example, a high dynamic range of protein concentrations and matrix interference. Samples used were either serum sample, which had a lower limit of quantitation(LLOQ) of about 1 pg / mL to 20 pg / mL, or immunoprecipitated sample, which had a LLOQ of about 100 ng / mL to 1 pg / mL. Samples were subjected to denaturation and / or reduction to form a denatured and / or reduced sample, enzymatic digestion to form a peptide digest, and were subsequently injected into a liquid chromatography (LC) column coupled to a mass spectrometer for MS-MRM analysis.

[0105] The effectiveness of enriching peptides using different incubation buffers and varying amounts of enrichment beads was evaluated. Method development was carried out using either nano LC- PRM or regular flow LC-MRM, and an exemplary peptide from the complementarity -determining region (CDR) of a therapeutic antibody was characterized. To determine if binding efficiency between enrichment beads and biotherapeutics was impacted by the pH of the binding solution, and to evaluate the conditions for antibody enrichment from serum, mouse serum containing 0.5 pg / mL mAb-1 was incubated with resuspended enrichment beads in 100 pL of varying incubation buffers, including Milli-Q H2O, lx PBS at pH 7.0, 0.1 M Tris-HCl, pH 8.0 and 0.1 M Tris-HCl, pH 9.0. The peak area of mAb-1 CDR peptide GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11), with transition 1111.0108 —>1659.7344, was selected for further evaluation since it was not detectable in blank mouse serum using nano LC-PRM-MS (FIGS. 3A-3C). For regular flow LC-MRM-MS, the peak area of the mAb-1 CDR peptide LLIYAASSLQSGVPSR (SEQ ID NO: 10) with transition 831 .46 >359.20 was used to quantify the amount of mAb-1 in guinea pig serum (FIGS. 3D-3E).

[0106] As shown in FIG. 4, a maximum amount of mAb-1 was enriched when incubation was performed in water, while a minimal amount of mAb-1 was enriched when incubation was performed in PBS buffer. To confirm this result, various buffers were applied to different animal serums. The pH evaluation was also performed in guinea pig serum containing 0.5 pg / mL mAb-1, and Milli-Q H2O was still the buffer that allowed the maximum antibody detection (FIG. 5). Therefore, water was used for the rest of the experiments.

[0107] Average protein concentration in animal sera ranged from 60 mg / mL to 80 mg / mL. Thus, it the amount of beads used for protein enrichment was adjusted to remove most proteins in the serum while maintaining enough drug substance (DS) for detection by MS. The LC-MS method was conducted with varying amounts of beads, including one-half (1 / 2) of a PROTEOMINER™ kit. one-fifth (1 / 5) of a PROTEOMINER™ kit, one-tenth (1 / 10) of a PROTEOMINER™ kit, one-twentieth (1 / 20) of a PROTEOMINER™ kit, and one-fiftieth (1 / 50) of a PROTEOMINER™ kit. The varying amounts of beads were incubated with IgG peptides and the signal strength of the peptide of interest was compared using nano LC-PRM. In addition, varying amounts of beads, including 1 / 5 of a PROTEOMINER™ kit. 1 / 20 of a PROTEOMINER™ kit and 1 / 50 of a PROTEOMINER™ kit, were used in combination with LC-MRM for peptide identification. As shown in FIG. 6. the remaining protein amount in the eluate was positively correlated to the amount of beads added for enrichment. Approximately 57 pg, 28 pg, 14 pg. 7 pg and 2.3 pg of protein was collected from 10 pL of mouse serum using 1 / 2 of a PROTEOMINER™ kit, 1 / 5 of a PROTEOMINER™ kit, 1 / 10 of a PROTEOMINER™ kit, 1 / 20 of a PROTEOMINER™ kit and 1 / 50 of a PROTEOMINER™ kit, respectively. Approximately 40 pg. 6 pg, and 3.5 pg of proteinwas collected from 10 gL of guinea pig serum using 1 / 5. 1 / 20, and 1 / 50 of the beads from the PROTEOMINER™ kit, respectively.

[0108] For nano LC, 1 pg of digested peptides was loaded on to the column for PRM-MS analysis, utilizing varying amounts of PROTEOMINER™ beads, including 1 / 2 of a PROTEOMINER™ kit, 1 / 5 of a PROTEOMINER™ kit. 1 / 10 of a PROTEOMINER™ kit, 1 / 20 of a PROTEOMINER™ kit and 1 / 50 of a PROTEOMINER™ kit. For regular flow LC, 6 pg of digested peptides eluted from 1 / 5 of a PROTEOMINER™ kit, 1 / 20 of a PROTEOMINER™ kit and 1 / 50 of a PROTEOMINER™ kit were loaded for MRM-MS analysis.

[0109] As shown in FIGS. 7A and 7B. peak area of mAb-1 CDR peptide GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) with transition 1111.0108 -4659.7344 was highly impacted by the amount of beads applied for enrichment. For nano LC -PRM-MS, 1 / 50 of PROTEOMINER™ kit showed the highest sensitivity7for mAb-1 detection, as ~99.5% of serum proteins had been removed, allowing for more peptides to be loaded on the column without exceeding the column capacity, thus enabling beter detection of mAb-1 with less interference (FIG. 7 A). For regular flow LC- MRM-MS, more proteins can be loaded onto the column, therefore the amount of peptides enriched from 1 / 20 of a PROTEOMINER™ kit loaded arc 2-3 times higher than amount of peptides enriched from 1 / 50 of a PROTEOMINER™ kit (FIG. 7B).

[0110] Consequently, the peak area of the mAb-1 CDR peptide was comparable when with enrichment was performed with 1 / 20 and 1 / 50 of the beads from the kit. Regular flow LC is typically used for PK studies, because of its ability to accommodate many samples with high throughput. For tire remaining examples including regular flow LC -MRM-MS. 1 / 20 of the beads from the kit were used. [01U] mAb-1 and mAb-2 were both IgGl molecules that target nonoverlapping epitopes on virus protein. Therefore, the surrogate peptides for MRM-based drug quantification had to be chosen from the CDR regions of mAb-1 and mAb-2 of the variable domains. To determine suitable surrogate peptides to be used for LC-MRM-MS method, rat, rabbit, mouse, genuine pig, monkey and human sera were enriched and injected into nano LC for PRM-MS analysis against all CDR peptides precursor ions. A cocktail comprising a mixture of mAb-1 and mAb-2 was used to potentially achieve a broader immune response and greater treatment efficacy. This combination is often used in therapeutic or research setings to target different, non-overlapping epitopes on a viral protein. The surrogate peptides for MRM-based drug quantification were selected from the CDRs of the variable domains of mAb-1 and mAb-2. To identify the appropriate surrogate peptides for the LC-MRM-MS method, the following types of serum samples were enriched: rat. rabbit, mouse, guinea pig, monkey, and human.

[0112] These enriched samples were then injected into a nano LC system for PRM-MS analysis, which was conducted against all precursor ions of the CDR peptides, the results of which are shown in FIGS. 8A-8D. The mAb-1 CDR peptides GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) with transition 1111.0108^1659.7344 (FIG. 8A), LLIYAASSLQSGVPSR (SEQ ID NO: 10) with transition 831.46-4017.53 (FIG. 8B), and AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) with transition 680.3497— >848.4625 (FIG. 8C), as well as mAb-2 CDR peptide AEDTAVYYCAR (SEQ ID NO: 14)with transition 659.3497— >732.3134 (FIG. 8D), were not detectable in mouse serum after the enrichment process. Therefore, these peptides served as surrogate peptides for quantification with nano LC-PRM- MS. The MRM transition parameters of these four surrogate peptides were adjusted using regular flow LC-MRM-MS on an Agilent QQQ system. As shown in the CID MS / MS spectra in FIGS. 9A-9B. two peptides were chosen for monitoring mAb-1 and mAb-2 with LC-MRM-MS. For mAb-1, the peptide AGQSISSFLNWYQQKPGK (SEQ ID NO: 12). with a transition from a +3 precursor ion to a y3 product ion, and a collision energy of 22.7. For mAb-2, the peptide AEDTAVYYCAR (SEQ ID NO: 14). with a transition from a +2 precursor ion to a y5 product ion, and a collision energy of 18.5. was selected.

[0113] The linearity of the response for varying injection amounts and varying peptides was also evaluated. FIG. 10 shows the correlation between the injection amount and the peak area detected for three peptides. The response for each peptide showed good linearity, indicating that quantification remained accurate even with small injection amounts. Additional methods using with vary ing reagents to elute the peptides from the beads were characterized. Elution was evaluated by monitoring mAb-1 CDR peptide GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) with transition 1111.0108 ->1659.7344 using mouse serum with 0.5 pg / mL spiked-in mAb-1. mAb-1 was enriched with 1 / 5 PROTEOMINER™ beads in water and eluted from the beads using three solutions: PTS buffer (containing 12 mM SLS / SDC, 10 mM TCEP and 40 mM CAA); 6 M guanidine, 10 mM TCEP and 40 mM CAA; and 8 M urea, 10 mM TCEP and 40 mM CAA, respectively. As shown in FIG. 11, PTS buffer had the highest elution efficiency for mAb-1. Subsequent examples described herein include an elution buffer comprising PTS buffer.

[0114] Methods including a range of sample volumes for incubation with the beads were characterized. As shown in FIG. 12, the highest sample volume of 20 pL yielded the highest peak area of the internal standard peptides evaluated.

[0115] Based on the results described above, a method was selected that includes using water as the incubation buffer, including an internal standard (commercial isotope-labeled antibody) for normalization: elution using 12 mM SLS / SDC, 10 mM tris(2-carboxylethyl)phosphine (TCEP), and 40 mM chloroacetamide (CAA); using 1 / 20 of the PROTEOMINER™ beads from a PROTEOMINER™ bead kit; and injecting 6 pg of protein when using LC-MRM or 1 pg when using nano LC-PRM.

[0116] Example 2: Protein Quantification Using IgG Common Peptides

[0117] The quantification of individual antibodies in preclinical PK studies can be accomplished by quantifying CDR peptides, which are unique to each mAb, or alternatively by quantify ing the common peptides from the constant regions of human IgG molecules, which are not detectable in animal sera. Both methods provide a viable approach to accurately measuring the concentrations of individual antibodies in preclinical PK studies. Two complementary strategies for quantifying a protein of interest, for example a therapeutic antibody, were evaluated. A benefit of the quantification of common peptides from common regions of IgG molecules as a measure of an antibody of interest was that it is generally applicable to any antibody of interest and does not require adjustments based on the target. This strategy may be applied, for example, for quantifying an antibody of interest in animal serum in an early-stage pharmacokinetic (PK) study.

[0118] Benefits of the use of unique peptides from a protein of interest, for example CDR peptides from an antibody of interest, for quantifying the protein include that it may be employed in any type of serum, for example animal serum or human serum, may be applied to specifically quantifying one therapeutic antibody in a co-formulation comprising multiple therapeutic antibodies, and may be applied to diverse drug formats.

[0119] The method developed in Example 1 was used to evaluate the feasibility of the first protein quantification strategy described above. Previous studies have identified MRM transitions from IgG peptides found in various animal sera, and some transitions that were not identified in the animal sera, which therefore could be useful for specifically identifying an administered antibody without interference. However, the transitions of interest were identified in animal sera, and therefore would not be useful for specifically identifying an antibody of interest, as shown in FIG. 13.

[0120] In order to identify peptide transitions that could be used for the first strategy, a survey was conducted of all common IgGl Fc peptides in animal sera and human serum using the present methods. All common peptides from human IgGl constant regions were screened against enriched animal sera (rat, rabbit, mouse, genuine pig, monkey) to identify’ potential background interferences. As shown in FIG. 13, surprisingly, all common peptides from human IgGlwcrc identified in animal sera after enrichment, including those peptides that were previously reported as absent from animal serums. Amino acid sequences of the peptides shown in FIG. 13 are included in Table 1, below.Table 1

[0121] As shown in FIG. 14, the abundance of IgGl common peptides varied up to 5, 000-fold after enrichment in different animal sera, indicating that certain peptides may serve as surrogate peptide in certain animal sera but not in the others. As shown in FIGS. 15 A-15C, the common IgGl peptides VVSVLTVLHQDWLNGK (SEQ ID NO: 8) (FIG. 15A), TVAAPSVFIFPPSDEQLK (SEQ ID NO: 7) (FIG. 15B), and SGTASVVCLLNNFYPR (SEQ ID NO: 3) (FIG. 15C) identified in animal sera were the same as those in human IgGl. By contrast, as shown in FIGS. 16A-16C, the mAb-1 CDR peptides LLIYAASSLQSGVPSR (SEQ ID NO: 10) (FIG. 16A), GLEYVSAISSDGGSTYDADSVK (SEQ IDNO: 11) (FIG. 16B). and AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) (FIG. 16C) were not identified in animal sera, although certain transitions were detected. As controls, blank samples and mAb 3 samples (containing the mAb-1 antibody derived from a different lot) were also tested.

[0122] All common IgG peptides were detected in the tested animal sera, with MS2 transitions similar to mAb peptides. The common peptide distribution is shown in FIG. 17. As shown in FIG. 18A, rabbit and guinea pig sera displayed an even higher distribution of the ALPAPIEK (SEQ ID NO: 1) peptide than human serum. Due to the high distribution of the ALPAPIEK (SEQ ID NO: 1) peptide, a distribution of the common Fc peptides without the ALPAPIEK (SEQ ID NO: 1) peptide is shown in FIG. 18B. Rat. rabbit, mouse, and monkey sera had a relatively low amount of TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5) and STSGGTAALGCLVK (SEQ ID NO: 4) peptides, as compared to other common IgG peptides. Peptide VYACEVTHQGLSSPVTK (SEQ ID NO: 9) was extremely high in monkey serum, as compared to human serum.

[0123] The quantitation estimation of pre-existing peptides was calculated using as an internal standard SILuMab Trastuzumab, which has [13C,15N]-lysine / arginine labeling. PROTEOMINER™ beads enrich antibodies based on their specific peptide ligands and therefore, different antibodies could be enriched in different folds using the PROTEOMINER™ enrichment method. Several low-abundancc common IgGl peptides were quantified in mouse serum according to die calibration curve established with trastuzumab. The abundances of those common IgGl peptides were estimated in rat, rabbit, guinea pig, and monkey sera, through comparison of the normalized peak area of each peptide over their isotope labeled peptide from trastuzumab in other animal sera, with the normalized peak area of each peptide over their isotope labeled peptide from trastuzumab in mouse serum.

[0124] The quantification of pre-existing peptides was based on trastuzumab. PROTEOMINER™ beads enrich antibodies according to their specific peptide ligands, which may result in varying degrees of enrichment with different antibodies. Four IgGl SILuMab antibodies (trastuzumab, rituximab, adalimumab, and bevacizumab) synthesized by Sigma Aldrich were tested in mouse serum samples, and the peak area of a few common isotope-labeled Fc peptides were compared. As shown in FIG. 19, a seven-fold difference in enrichment factor was observed when enrichment beads were applied to the four different antibodies. Thus, abundance of the peptide STSGGTAALGCLVK (SEQ ID NO: 4) in rat, mouse, and monkey sera ranged from 0.02 pg / mL to 0.47 pg / mL. and abundance of the peptide TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5) in rabbit and genuine pig sera ranged from 0.01 pg / mL to 0.23 pg / mL. As shown in FIG. 20, a comparison of enrichment of trastuzumab in mouse serum versus monkey serum demonstrated greater enrichment in monkey serum, indicating a possible effect of serum matrix on antibody enrichment.

[0125] Trastuzumab was also used as an internal standard to correlate peak area to peptide concentration for peptides identified using the method of the present invention. Trastuzumab was spiked in mouse serum and the following common IgGl Fc peptides detected and measured: ALPAPIEK (SEQ ID NO: 1), GPSVFPLAPSSK (SEQ ID NO: 2), SGTASVVCLLNNFYPR (SEQ ID NO: 3), TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5), TVAAPSVFIFPPSDEQLK (SEQ ID NO: 7),VVSVLTVLHQDWLNGK (SEQ ID NO: 8), VYACEVTHQGLSSPVTK (SEQ ID NO: 9). As shown in FIG. 21 A. a linear relationship was observed between increasing concentrations of trastuzumab and peak area for common IgGl Fc peptides. As shown in FIG. 21B, a linear relationship was observed for increasing concentrations for two internal standard peptides in mouse serum: [13C6. 15N4]- SGTASVVCLLNNFYPR* (SEQ ID NO: 3) and [13C6, 15N2]-STSGGTAALGCLVK* (SEQ ID NO:4).

[0126] As shown by the spiked-in experiment using trastuzumab, 0.1 pg / mL in mouse serum was easily used for characterization and quantification. Therefore, the lower limit of quantitation (LLOQ) of IgG molecule detection in animal serum would based on the potential interference peaks from animal serum. Based on the estimation of potential interference peptide concentration in animal serum, an LLOQ lower than 4.7 pg / mL in rat and mouse and lower than 2 pg / mL in monkey serum was achieved after applying enrichment by quantification of IgGl peptide STSGGTAALGCLVK (SEQ ID NO: 4). The LLOQ of 1 pg / mL in rabbit serum and 2 pg / mL in rabbit and genuine pig serum might be achieved by quantitation of IgGl peptide TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5).

[0127] Utilizing the peak area of the peptides obtained in mouse serum, the concentration of peptides in other animal scrums was estimated. Common peptides from animal scrum were enriched, resulting in a LLOQ of above 1 pg / mL. Certain low-abundant common IgGl peptides were quantitated in mouse serum based on the calibration curve established with trastuzumab, and the abundance of those common IgGl peptides were estimated in rat, rabbit, guinea pig serum and monkey serum by comparing the normalized peak area of each peptide over their isotope labelled peptide from trastuzumab in other animal serums with the normalized peak area of each peptide over their isotope labelled peptide from trastuzumab in mouse serum. As shown in Table 2, below, the abundance of peptide STSGGTAALGCLVK (SEQ ID NO: 4) was the lowest in rat, mouse and monkey serum, ranging from 0.06 pg / mL to 0.14 pg / mL, while the abundance of peptide TPEVTCVVVDVSHEDPEVK (SEQ ID NO:5) was the lowest in rabbit and guinea pig serum, at 0.03 pg / mL and 0.07 pg / mL.Table 2

[0128] In conclusion, the sensitivity of methods described herein led to the identification of common IgG peptides in all animal serums tested, which poses an obstacle in the strategy of relying on common IgG peptides for specific identification of an administered antibody in animal serum. Therefore, a second strategy using a uniquely identifying peptide for a protein of interest was explored.

[0129] Example 3: Protein Quantification Using Unique Peptides

[0130] As described in Example 2, one strategy for quantifying a protein of interest was through identifying a unique peptide to use as a surrogate in LC-MRM-MS analysis. The method described in Example 1 was used for CDR peptide screening in various animal sera using two different monoclonal antibodies, mAb-1 and mAb-2. mAb-1 and mAb-2 were both IgGl molecules targeting non-overlapping epitopes on a virus protein. Therefore, the surrogate peptides for MRM-based drug quantification had to be chosen from the CDR regions of the variable domains of mAb-1 and mAb-2. To find suitable surrogate peptides to be used for the LC-MRM-MS method, rat, rabbit, mouse, guinea pig, monkey, and human sera were enriched and injected into nano LC for PRM-MS analysis against all CDR peptides precursor ions.

[0131] Peak area and peptide distribution for mAb-2 CDR peptide AEDTAVYYCAR (SEQ ID NO: 14) in several different animal sera are shown in FIG. 22A. The AEDTAVYYCAR (SEQ ID NO: 14) peptide was observed in all tested sera except mouse serum, although peak area was very low in rabbit and rat sera (FIG. 22A, top panel). Distribution of the AEDTAVYYCAR (SEQ ID NO: 14) peptide of several serum samples was highly reproducible across several replicates (e.g.. series) (FIG. 22A. bottom panel).

[0132] Peak area and peptide distribution for mAb-2 CDR peptide LLIYAASNLETGVPSR (SEQ ID NO: 15) in several different animal sera are shown in FIG. 22B. The LLIYAASNLETGVPSR (SEQ ID NO: 15) peptide was observed in all tested sera (FIG. 22B, top panel). Distribution of the LLIYAASNLETGVPSR (SEQ ID NO: 15) peptide of several serum samples w as highly reproducible across several replicates (e.g., series) (FIG. 22B. bottom panel).

[0133] Peak area and peptide distribution for mAb-1 CDR peptide AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) in several different animal sera are shown in FIG. 23 A. The AGQSISSFLNWYQQKPGK peptide was observed in all tested sera except mouse serum (FIG. 23 A, top panel). MS2 product ion distributions of the AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) peptide are shown in FIG. 23 A, bottom panel.

[0134] Peak area and peptide distribution for mAb-1 CDR peptide GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) are shown in FIG. 23B. The GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) peptide was observed in human, monkey, rabbit, and rat sera, although peak area was very low for monkey and rabbit sera (FIG. 23B, top panel). MS2 product ion distributions of the GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) peptide are shown in FIG. 23B. bottom panel.

[0135] Peak area and peptide distribution for mAb-1 CDR peptide LLIYAASSLQSGVPSR (SEQ ID NO: 10) are shown in FIG. 23C. The LLIYAASSLQSGVPSR (SEQ ID NO: 10) peptide was observed in human, monkey, rabbit, and rat sera, although peak area was very low for rabbit and rat sera (FIG. 23C. top panel). MS2 product ion distributions of the LLIYAASSLQSGVPSR (SEQ ID NO: 10) peptide are shown in FIG. 23C, bottom panel.

[0136] Peak area and peptide distribution for mAb-1 CDR peptide LSCSASGFTFSR (SEQ ID NO: 13) are shown in FIG. 23D. The LSCSASGFTFSR (SEQ ID NO: 13) peptide was observed in all tested sera (FIG. 23D, top panel). Reproducibility of distribution of the LSCSASGFTFSR (SEQ ID NO: 13) peptide across several animal sera across several replicates (e.g., series) is shown in FIG. 23D, bottom panel.

[0137] GLEYVSAISSDGGSTYDADSVK (SEQ ID NO: 11) with transition 1111.0108—1659.7344 (FIG. 23B), LLIYAASSLQSGVPSR (SEQ ID NO: 10) with transition 831.46—1017.53 (FIG. 23C) and AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) (FIG. 23A) from mAb- 1 and AEDTAVYYCAR (SEQ ID NO: 14) (FIG. 22A) from mAb-2 were not detectable in mouse serum after enrichment and therefore could serve as surrogate peptides for quantitation using nano LC-PRM- MS. LC-PRM-MS was conducted using a mass spectrometer. The MRM transition parameters of these four peptides were further adjusted using regular flow LC-MRM-MS on an Agilent QQQ system.

[0138] PROTEOMINER™ beads could successfully enrich low abundant mAbs from endogenous serum proteins and thus improved the detection limit of the assay. However, because variations could be introduced during the enrichment process, as well as during chromatography and MS ionization, an internal standard was introduced to account for and correct the variability. Traditionally, a stable isotope labelled internal standard peptide was commonly used in LC-MRM-MS quantitation to correct for variability in chromatography and MS ionization. However, such an internal standard peptide could not correct the variability from the enrichment step, since peptides might be enriched differently from mAbs. Therefore, as described in the previous example. SILUMAb Trastuzumab, a [13C,15N]-lysine / arginine labelled internal standard that shares the sequence of constant region of human IgGl heavy chain, was introduced at 2 pg / mL into the serum, and the response of mAb peptide was normalized to internal standard peptides for quantitation. This approach enabled mitigated variability across all steps of the process, including enrichment and digestion.

[0139] The linearities of the standard curv es for mAb-1 peptide AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) and mAb-2 peptide AEDTAVYYCAR (SEQ ID NO: 14) were evaluated simultaneously, as an analog of co-formulated drugs. The peak area ratio (PAR) of mAb-1 peptide (FIG. 24 A) or mAb-2peptide (FIG. 25A) over average peak area of internal standard peptides STSGGTAALGCLVK* (SEQ ID NO: 4), TPEVTCVVVDVSHEDPEVK* (SEQ ID NO: 5) and VYACEVTHQGLSSPVTK* (SEQ ID NO: 9) was calculated with Skyline software. Calibration curves generated by correlating PAR of eight nonzero standards of mAb-1 or six nonzero standards of mAb-2 with standard concentrations with linear regression. The correlation coefficients R2for mAb-1 and mAb-2 were both above 0.99. An LLQC of 0.096 pg / mL was achieved for mAb-1 CDR peptide AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) (FIG. 24B), with no interference observed from blank mouse serum (FIG. 24C). An LQC of 0.48 pg / mL was achieved for mAb-2 CDR peptide AEDTAVYYCAR (SEQ ID NO: 14) (FIG. 25B). with no interference observed from blank mouse serum (FIG. 25C).

[0140] The concentration of protein spiked into the sample was compared to the measured value.Quality control (QC) samples were rim in triplicate and the accuracy, standard deviation (Std) and coefficient of variation (CV) were determined for the conditions of lower limit quality control (LLQC), low quality control (LQC), medium quality control (MQC), and high quality control (HQC) for mAb-1, as shown in Table 2A, below. Similarly, Std and CV were determined for LQC, MQC, and HQC for mAb-2, as shown in Table 2B, below.

[0141] As shown in Table 2 A, for mAb-1, the LLQC had an accuracy of -0.82%, a standard deviation of 0.00414 and a %CV of 0.44%. The LQC had an accuracy of 0.71%, a standard deviation of 0.018256 and a %CV of 3.78%. The MQC had an accuracy of -4.33%, a standard deviation of 0.142987 and a %CV of 6.23%. The HQC had an accuracy of 3.63%, a standard deviation of 0.435733 and a %CV of 3.5%. As shown in Table 2B, for mAb-2, the LQC had an accuracy of -9.97%, a standard deviation of 0.0331 and a %CV of 7.66%. The MQC had an accuracy of 1.39%. a standard deviation of 0.2408 and a %CV of 9.90%. The HQC had an accuracy of 0.76%, a standard deviation of 0.1505 and a %CV of 1.24%.

[0142] As shown in Table 3A and Table 3B. the average accuracy of QCs ranged from 95% to 104% for mAb-1 and 90% to 102% for mAb-2, and coefficient of variation (CV%) of measured concentration varied from 0.44% to 9.9% for both mAb-1 and mAb-2, which met the general criteria of ± 20% of the nominal value.Table 3ATable 3B

[0143] Calibration curves were generated for mAb-1 and mAb-2 by spiking in low to high concentrations of the protein. A peak area ratio (PAR) was calculated for each peptide of interest, by comparison to heavy isotope labelled IgGl peptides STSGGTAALGCLVK (SEQ ID NO: 4), TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5), and VYACEVTHQGLSSPVTK (SEQ ID NO: 9). Both peptides of interest were uniquely identifying peptides that were not detected in mouse serum, as described above.

[0144] For mAb-1, the spiked-in protein concentration ranged from 50 ng / mL to 12.8 pg / mL. As shown in FIG. 26A, retention time and signal intensify of detected mAb-1 peptide AGQSISSFLNWYQQKPGK (SEQ ID NO: 12) (top panel) are indicated by the arrow at around 11.4 minutes. PAR of the peptide showed a linear response with increasing protein concentration (bottom panel).

[0145] For mAb-2, the spiked-in protein concentration ranged from 400 ng / mL to 12.8 pg / mL. As shown in FIG. 26B, retention time and signal intensity of detected mAb-2 peptide AEDTAVYYCAR (SEQ ID NO: 12) (top panel) are indicated by the arrow at around 4.1 minutes. PAR of the peptide showed a linear response with increasing protein concentration (bottom panel).

[0146] This example shows drat methods of the present disclosure can be used for accurate, precise, and sensitive identification and quantification of a protein of interest using unique peptides, such as CDR peptides, even when using a complex sample such as serum. The detection limit of IgG in animal sera has ranged between 1 pg / mL to 20 pg / mL by using direct digestion followed by regular flow LC-MRM-MS, however, extensive method development and optimization is usually involved to achieve this LLOQ. By applying the presently described enrichment methods, no additional method development would be required to achieve approximately 1 pg / mL LLOQ in any animal serum. Thus, this approach can be applied when there is an urgent need for method development without extensive optimization. In addition, this approach also had a comparable LLOQ for antibody detection as albumin depletion-coupled LC-MRM-MS analysis, as well as the LLOQ of certain immunocapture-coupled LC-MRM-MS methods, and therefore can be used as a sensitive and general IgGl quantitation method in animal serum that can be applied directly.

[0147] Example 4. Case Study for Unique Peptides of an ScFv

[0148] PROTEOMINER™ beads could enrich any type of drug module from serum and enable the development of sensitivity assays for pharmacokinetics (PK) studies or toxicokinetic (TK) analysis. Drug Substance-1 (DS-1) was a recombinant fusion protein consisting of a human single chain variable fragment (ScFv) against a specific target, and the expected ScFvl level in an animal serum pilot TK study was below 3 pg / mL. The methods described in Example 1 were used to screen for uniquely identifying peptides for DS-1. To select suitable surrogate peptides for MRM quantification, 26 peptides that had a peptide chain length longer than 5 amino acid residues and shorter than 20 amino acid residues and did not contain sites susceptible to post translational modification (PTM) were screened against enriched rat, rabbit, mouse, guinea pig, monkey and human serum samples. This process helped ensure that the selected peptides were suitable for MRM quantification across species.

[0149] As shown in FIGS. 27A-27F, signal intensities for candidate DS-1 peptides DILTLR (SEQ ID NO: 16) (FIG. 27A) and TTPTFFPK (SEQ ID NO: 17) (FIG. 27B) were relatively high, as compared to other candidate peptides (FIG. 27C). As shown in FIG. 27D. both peptides were tested in various animal sera, and both were not observed in rat, mouse, rabbit and guinea pig sera, even after enrichment and monitoring by nano LC-PRM-MS. Therefore, DS-1 peptide DILTLR (SEQ ID NO: 16) with transition from +2 precursor ion to y4 product ion, and DS-1 peptide TTPTFFPK (SEQ ID NO: 17) with transition from +2 precursor ion to y6 product ion, were used for quantification of DS-1 in mouse serum in LC-MRM-MS analysis. Distributions of each DS-1 peptide were compared across monkey serum and human serum, and compared against internal standard control SILuMab in serum (to correct for variabilities introduced during sample preparation and ion detection). The distribution for the DS-1 peptide DILTLR (SEQ ID NO: 1 ) is shown in FIG. 27E, and the distribution for the DS-1 peptide TTPTFFPK (SEQ IS NO: 17) is shown in FIG. 27F.

[0150] As shown in FIG. 28, a calibration curve plot of DS-1 was generated from LC-MRM of calibration standards with individual drug concentration ranging from 0.025 pg / mL to 12.8 pg / mL. PAR was calculated by dividing the average peak areas of DILTLR (SEQ ID NO: 16) and TTPTFFPK (SEQ ID NO: 17) by average peak areas of three peptides STSGGTAALGCLVK (SEQ ID NO: 4), TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 5), and VYACEVTHQGLSSPVTK (SEQ ID NO: 9) from internal standard trastuzumab. The resulting correlation coefficient (R2) exceeded 0.99, validating the accuracy and precision of tire DS-1 peptides for characterizing serum concentration of DS-1.

[0151] QC samples were run in triplicate and the accuracy, standard deviation and coefficient of variation were determined for the lower limit QC (LLQC), LQC, MQC, HQC and UHQC for DS-1, with results shown in Table 4. below. The LLQC sample had an accuracy of -0.04%, a standard deviation of 0.0011 and a %CV value of 5.76%. The LQC sample had an accuracy of -3.09%, a standard deviation of 0.0103 and %CV value of 11.03%. The MQC sample had an accuracy of -13.92%, a standard deviation of 0.0246 and a %CV of 5.45%. The HQC had an accuracy of -0.86%, a standard deviation of 0.0021 and a %CV of 0.09%, while the UHQC sample had an accuracy of 7.35%, a standard deviation of 0.10887 and a %CV of 0.84%. The LLOQ of 0.019 pg / mL was achieved for DS-1, and accuracy and precision were all within 15% and met the general criteria of ± 20% of the nominal value.Table 4

[0152] This example shows that the methods were able to accurately, precisely and sensitively identify and quantify an ScFv protein of interest, proving that the method is not limited to a particular drug format.

Claims

CLAIMS1. A method for quantifying an antibody in a serum sample, the method comprising: contacting a serum sample to a solid support, wherein the serum sample comprises an antibody and the solid support is attached to peptide ligands capable of binding to the antibody; contacting an elution buffer to the solid support, thereby producing an enriched antibody sample; subjecting the enriched antibody same to denaturing conditions, thereby producing a denatured antibody sample; contacting a reducing agent to the denatured antibody sample to produce a reduced antibody sample; contacting an enzyme to the reduced antibody sample to produce a peptide digest, wherein at least one peptide of the peptide digest is a uniquely identifying peptide for the antibody; contacting the peptide digest to a liquid-chromatography -tandem mass spectrometry (LC- MS / MS) system to determine a quantify of the uniquely identifying peptide; and determining a quantify of the antibody based on the quantify of the uniquely identifying peptide.

2. The method of claim 1, further comprising, after contacting the scrum sample to the solid support and prior to contacting the elution buffer to the solid support, washing the solid support to remove unbound material.

3. The method of claim 1, wherein the peptide ligands include a library of combinatorial ligands.

4. The method of claim 3, wherein the library of combinatorial ligands are supported on beads.

5. The method of claim 1, wherein the antibody includes a monoclonal antibody, a bispecific antibody, an antibody -drug conjugate, a single chain variable fragment, or a fusion protein.

6. The method of claim 1, wherein the antibody is an IgGl antibody or an IgG4 antibody.

7. The method of claim 1 , wherein the uniquely identifying peptide has an amino acid sequence that is identical to a fragment of an amino acid sequence of a variable region of the antibody.

8. The method of claim 1 , wherein the uniquely identifying peptide has an amino acid sequence that is identical to a fragment of an amino acid sequence of a complementary determining region of the antibody.

9. The method of claim 1, wherein the uniquely identifying peptide has a peptide chain length of 6 amino acid residues to 22 amino acid residues.

10. The method of claim 1, wherein the elution buffer comprises SLS / SDC.

11. The method of claim 10, wherein the elution buffer comprises 12 mM SLS / SDC, 10 mM TCEP, and 40 mM CAA.

12. The method of claim 1, wherein the denaturing conditions comprise heat, high pH. low pH. at least one reducing agent, at least one chaotropic agent, or any combination thereof.

13. The method of claim 1, wherein the reducing agent comprises dithiothreitol (DTT), B- mercaptoethanol, Ellman’s reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2- carboxyethyl)phosphine hydrochloride (TCEP-HC1). or any combination thereof.

14. The method of claim 1, wherein the enzyme comprises pepsin, trypsin, Tryp-N, chymotrypsin, Lys-N, Lys-C, Asp-N, Arg-C, Glu-C, papain, IdeS, a variant thereof, or any combination thereof.

15. The method of claim 1, wherein determining a quantity of the uniquely identifying peptide comprises: comparing a peak area for tire uniquely identifying peptide to a peak area for an internal standard; and / or comparing a peak area for the uniquely identifying peptide to a standard curve.

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