Maximizing hydrophobic peptide recovery using mass spectrometry compatible surfactants

Abstract

The present invention relates generally to methods for characterizing peptides or proteins of interest. In particular, the present invention relates to the use of the non-ionic surfactant DDM to improve the recovery of hydrophobic peptides in pharmacokinetic, reduced peptide mapping, or other biotherapeutic protein analyses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 312,225, filed February 21, 2022, which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present application relates to compositions and methods for the characterization of peptides and proteins. [Background technology]

[0003] background LC-MS analysis of a protein or proteomics sample of interest can involve several steps, including protein extraction, enrichment, fractionation, digestion, and sample dehydration. Sample loss can occur at many steps in the protein analysis workflow, which can affect both qualitative and quantitative results. For digested peptides, especially hydrophobic peptides, nonspecific adsorption on container surfaces such as sample tubes, autosampler vials, and pipette tips is a common cause of peptide loss.

[0004] Adsorption losses can be significant for samples with limited analytes, such as single or near single cell samples. Peptide losses due to non-specific adsorption can also adversely affect assays involved in biotherapeutic protein drug development. For example, in pharmaceutical bioanalysis, non-specific surface adsorption of candidate peptides can cause non-linearity in calibration curves, which, if not properly addressed, can lead to underestimation of drug concentrations and inaccurate pharmacokinetic evaluation of investigational drugs. Additionally, when performing peptide mapping analysis, adsorption of digested peptides to the inner surface of containers can result in reproducibility and solution stability criteria not being met during method qualification.

[0005] Several strategies have been developed to minimize peptide adsorption losses. One common strategy is to add organic solvents such as dimethyl sulfoxide (DMSO) or acetonitrile (ACN) to the sample solution when reconstituting or diluting peptide samples, which may increase the recovery of hydrophobic peptides but may also negatively affect the recovery of hydrophilic peptides. Another strategy to prevent peptide losses is to add detergents during protein sample preparation. However, commonly used detergents are not compatible with LC-MS analysis and must be removed before injection into the LC-MS system, which results in further sample loss.

[0006] Thus, a need exists for compositions and methods for improving the recovery of hydrophobic peptides in LC-MS analysis. Summary of the Invention

[0007] overview Methods have been developed to improve the recovery of hydrophobic peptides in LC-MS analysis, for example, to improve detection of peptide standards in pharmacokinetic studies, to select suitable target peptides in pharmacokinetic studies, to improve sequence coverage in peptide mapping analysis, or to improve sequence coverage from dried protein samples. The methods take advantage of the properties of DDM, a non-ionic surfactant, to prevent sample loss, particularly loss of hydrophobic peptides, due to surface adsorption. For example, DDM may be added to peptide samples to improve detection of peptides in LC-MS analysis. DDM may also be used in protein samples prior to enzymatic digestion to improve peptide detection in reduced or non-reduced peptide mapping. Additionally, dried peptide samples, for example samples dried by rapid vacuum, may be reconstituted using DDM prior to LC-MS analysis to improve peptide recovery.

[0008] The present disclosure provides a method for preparing a peptide standard for pharmacokinetic studies for liquid chromatography-mass spectrometry analysis. In some exemplary embodiments, the method includes (a) contacting a peptide standard for pharmacokinetic studies with N-dodecyl-β-D-maltoside (DDM) to form a mixture, and (b) subjecting the mixture to liquid chromatography-mass spectrometry analysis.

[0009] In one embodiment, the peptide standard comprises the amino acid sequence of a target peptide for pharmacokinetic studies, hi a particular embodiment, the amino acid sequence of the target peptide is selected from a complementarity determining region of an antibody.

[0010] In one embodiment, the final concentration of the peptide standard in the mixture is about 10 fmol / uL. In another embodiment, the final concentration of DDM in the mixture is about 0.015%.

[0011] In one aspect, the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0012] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system. In another aspect, the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

[0013] The present disclosure also provides methods for selecting target peptides for pharmacokinetic studies. In some exemplary embodiments, the method includes: (a) obtaining at least two candidate peptide standards that include or have amino acid sequences of at least two candidate target peptides for a pharmacokinetic study; (b) contacting each of the at least two candidate peptide standards with N-dodecyl-β-D-maltoside (DDM) to form at least two mixtures; (c) subjecting each of the at least two mixtures to liquid chromatography-mass spectrometry analysis to generate a mass spectrum for each of the at least two candidate peptide standards; (d) comparing each of the mass spectra with a mass spectrum generated by the liquid chromatography-mass spectrometry analysis of a control mixture to generate an average peak area ratio for each of the at least two candidate peptide standards; (e) comparing the average peak area ratio between each of the at least two candidate peptide standards; and (f) using the comparison of (e) to select a target peptide for a pharmacokinetic study, wherein the control mixture includes the candidate peptide standards and does not include DDM.

[0014] In one embodiment, the amino acid sequence of each of the at least two candidate target peptides is selected from the complementarity determining region of an antibody. In another embodiment, the final concentration of each of the at least two candidate peptide standards in each of the at least two mixtures is about 10 fmol / uL. In a further embodiment, the final concentration of DDM in each of the at least two mixtures is about 0.015%.

[0015] In one aspect, the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0016] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system. In another aspect, the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

[0017] In one embodiment, the target peptide is selected based on having an amino acid sequence of a candidate peptide standard that has a smaller ratio of average peak areas compared to at least one other candidate peptide standard.

[0018] In one embodiment, the control mixture comprises formic acid, hi a particular embodiment, the final concentration of formic acid in the control mixture is 0.1%.

[0019] The present disclosure further provides a method for preparing a protein of interest for peptide mapping analysis. In some exemplary embodiments, the method includes: (a) subjecting a sample containing a protein of interest to conditions suitable for denaturation to form a denatured sample; (b) contacting the denatured sample with at least one alkylating agent under conditions suitable for alkylation to form an alkylated sample; (c) contacting the alkylated sample with N-dodecyl-β-D-maltoside (DDM) to form a mixture; (d) contacting the mixture with at least one digestive enzyme under conditions suitable for digestion to form a peptide digest; and (e) subjecting the peptide digest to liquid chromatography-mass spectrometry for peptide mapping analysis.

[0020] In one embodiment, the peptide mapping analysis is a reduced peptide mapping analysis or a non-reduced peptide mapping analysis.

[0021] In one aspect, the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, a host cell protein, or a protein pharmaceutical product.

[0022] In one embodiment, conditions suitable for denaturation include heating the sample, optionally at about 80° C. for about 10 minutes.

[0023] In one embodiment, the method further comprises subjecting the sample to conditions suitable for reduction. In a particular embodiment, the conditions suitable for reduction comprise contacting the sample with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), optionally wherein the concentration of TCEP-HCl is about 5 mM.

[0024] In one embodiment, the at least one alkylating agent comprises iodoacetamide, hi another embodiment, the final concentration of DDM in the mixture is about 0.1%.

[0025] In one embodiment, the at least one digestive enzyme comprises trypsin, optionally wherein the trypsin is present at an enzyme to substrate ratio of about 1:10 (w / w).

[0026] In one aspect, the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0027] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system. In another aspect, the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

[0028] The present disclosure also provides a method for preparing a protein sample of interest for liquid chromatography-mass spectrometry analysis. In some exemplary embodiments, the method includes: (a) drying a protein sample of interest using a high-speed vacuum to form a dried sample; (b) dissolving the dried sample using a solution containing N-dodecyl-β-D-maltoside (DDM) to form a reconstituted sample; and (c) subjecting the reconstituted sample to liquid chromatography-mass spectrometry analysis.

[0029] In one embodiment, the concentration of DDM in the solution is about 0.015%. In one embodiment, the protein sample of interest is a protein digest. In a further embodiment, the protein sample of interest is a cell digest.

[0030] In one aspect, the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0031] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system. In another aspect, the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

[0032] These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not by way of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief description of the drawings]

[0033] [Figure 1A]1 shows a comparison of unique peptides identified using LC-MS / MS with and without DDM using 0.2 ng of HeLa cell lysate tryptic digest (single cell protein amount), according to an exemplary embodiment. [Figure 1B] 1 shows a comparison of proteins identified using LC-MS / MS with and without DDM using 0.2 ng of HeLa cell lysate tryptic digest (single cell protein amount), according to an exemplary embodiment. [Figure 1C] 1 shows the overlap of total unique peptides identified using LC-MS / MS with and without DDM using 0.2 ng of HeLa cell lysate tryptic digest (single cell protein amount) according to an exemplary embodiment. [Figure 1D] 1 shows a comparison of peptide hydrophobicity between peptides identified using LC-MS / MS with and without DDM compared to peptides identified with DDM only, according to an exemplary embodiment. [Diagram 2] 1 illustrates an application in which sample loss due to surface adsorption can be an issue, according to an exemplary embodiment. [Diagram 3] 1 shows a UPLC-MS analysis demonstrating that DDM eluted after peptide analytes, according to an exemplary embodiment. [Figure 4] Figure 4A shows the correlation between peptide retention time and the ratio of peak areas between DDM and control samples from a synthetic mAb peptide standard, according to an exemplary embodiment. Figure 4B shows the correlation between peptide hydrophobicity and the ratio of peak areas between DDM and control samples from a synthetic mAb peptide standard, according to an exemplary embodiment. [Figure 5A] 1 shows an overlay of UV chromatograms from non-reduced peptide mapping of mAb1 with and without DDM, according to an exemplary embodiment. [Figure 5B] 1 shows an overlay of UV chromatograms from reduced peptide mapping of mAb2 with and without DDM, according to an exemplary embodiment. [Figure 6]Figure 6A shows the UV signal of mAb1 peptide prepared with and without DDM according to an exemplary embodiment, Figure 6B shows the UV signal of mAb2 peptide prepared with and without DDM according to an exemplary embodiment, and Figure 6C shows a comparison of the UV signals of mAb1 and mAb2 peptides prepared with and without DDM according to an exemplary embodiment. [Figure 7] Figure 7A shows the UV signal of the peptide over time in a control sample, Figure 7B shows the UV signal of the peptide over time in a DDM-containing sample, and Figure 7C shows a comparison of the relative standard deviation (RSD) of the UV signal of mAb1 hydrophobic peptide prepared with and without DDM after 48 hours, according to an exemplary embodiment. [Figure 8] 1 shows the experimental design of an assay for comparing peptide samples reconstituted with FA or DDM, according to an exemplary embodiment. [Figure 9A] 13 shows a comparison of peptide peak signals in a BSA tryptic peptide sample reconstituted with DDM compared to FA, according to an exemplary embodiment. [Figure 9B] 1 shows the ratio of peak areas for DDM-lysed BSA tryptic peptide samples compared to FA-lysed BSA tryptic peptide samples for 10 peptides ordered by retention time according to an exemplary embodiment. [Figure 9C] 13 shows the correlation between peak area ratio and retention time for a DDM-lysed BSA tryptic peptide sample compared to a FA-lysed BSA tryptic peptide sample, according to an exemplary embodiment. [Figure 10] 1 shows base peak chromatograms of dried HeLa digest dissolved in FA or DDM solution according to an exemplary embodiment. [Figure 11] Figure 11A shows the total number of unique peptides identified from dried HeLa digest samples reconstituted in DDM or FA solutions according to an exemplary embodiment. Figure 11B shows the total number of protein groups identified from dried HeLa digest samples reconstituted in DDM or FA solutions according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description Proteomics sample preparation may involve several steps, including protein extraction, enrichment, fractionation, digestion, and sample dehydration prior to LC-MS analysis. Sample loss can occur at many steps in the protein analysis workflow, which can affect both qualitative and quantitative results. For digested peptides, especially hydrophobic peptides, nonspecific adsorption on container surfaces such as sample tubes, autosampler vials, and pipette tips is a common cause of peptide loss (Stejskal et al., J Proteome Res 2013, 12, 3057-3062; Kawashima et al., Proteomics 2013, 13, 751-755; Zhou et al., Anal Chem 2015, 87, 9802-9809; Shi et al., Commun Biol 2018, 1, 103; Li et al., J Chromatogr B Analyt Technol Biomed Life Sci 2010, 878, 583-589). With the widespread application of highly sensitive LC-MS systems that can detect peptides at the attomole level, these adsorption losses have attracted more attention. This is especially true for samples with limited analytes, such as single or near-single cell samples, where losses due to contact surface adsorption can be significant and a key factor in obtaining reliable data (Zhang et al., Anal Chem 2019, 91, 1441-1451; Tsai et al., Commun Biol 2021, 4, 265; Tsai et al., Mol Cell Proteomics 2020, 19, 828-838; Dou et al., Anal Chem 2019, 91, 13119-13127; Zhu et al., Anal Chem 2018, 90, 11756-11759; Zhu et al., Nat Commun 2018, 9, 882; Balasubramanian et al., Curr Protoc 2021, 1, e153).

[0035] Peptide loss due to nonspecific adsorption is also a common problem in certain assays during the development of biotherapeutic protein drugs (Palmgren et al., Eur J Pharm Biopharm 2006, 64, 369-378; Chen et al., J Chromatogr B Analyt Technol Biomed Life Sci 2009, 877, 943-947; Groff et al., J Chromatogr B Analyt Technol Biomed Life Sci 2006, 842, 122-130; Duggan, Bioanalysis 2019, 11, 797-814). For example, in pharmaceutical bioanalysis, nonspecific surface adsorption of candidate peptides can cause nonlinearity in calibration curves, which, if not properly addressed, can lead to underestimation of drug concentrations and inaccurate pharmacokinetic evaluation of investigational drugs (Li et al. 2010, Duggan).

[0036] Furthermore, peptide mapping is an important analytical method for investigating the primary structure of biotherapeutic proteins (Bongers et al., J Pharm Biomed Anal 2000, 21, 1099-1128; Mouchahoir and Schiel, Anal Bioanal Chem 2018, 410, 2111-2126). However, in these analyses, some peak intensities may decrease over time due to peptide adsorption to the inner surface of the sample tube or autosampler vial, which may lead to reproducibility and solution stability criteria during method qualification not being met (Bongers et al.). This adsorption may occur in a relatively short time (less than 15 min) based on previous reports (Mouchahoir and Schiel).

[0037] To minimize peptide adsorption losses, several strategies have been developed. One common strategy is to add organic solvents such as dimethyl sulfoxide (DMSO) or acetonitrile (ACN) to the sample solution when reconstituting or diluting peptide samples (van Midwoud et al., J Proteome Res 2007, 6, 781-791). This method can increase the recovery of hydrophobic peptides, but may affect hydrophilic peptides binding to the column (van Midwoud et al., Vatansever et al., J Sep Sci 2010, 33, 2478-2488). Another common strategy to prevent peptide loss is to add detergents such as sodium dodecyl sulfate (SDS) (Gan et al., Mol Cell Proteomics 2021, 20, 100051), CHAPS (Li et al. 2010), polysorbate 20 / 80 (Li et al. 2010), and PEG (Stejskal et al., Kawashima et al.) during protein sample preparation. However, these detergents are not compatible with LC-MS analysis and must be removed before injection into the LC-MS system. Sample loss is inevitable during these extra detergent removal steps. Currently, several commercially available LC-MS compatible surfactants, such as RapiGest SF (Waters), PPS Silent Surfactant, and sodium deoxycholate (SDC) surfactant, are used to increase protein digestion efficiency and prevent peptide loss (Scheerlinck et al., Anal Biochem 2015, 490, 14-19; Waas et al., Anal Chem 2014, 86, 1551-1559). Unfortunately, these surfactants must be removed prior to LC-MS analysis by lowering the pH of the sample, and one report found that some tryptic digested peptides were lost during the removal process because they co-precipitated with the detergent cleaved at low pH during centrifugation and precipitation (Lin, et al., Electrophoresis 2010, 31, 2705-2713).

[0038] Finally, to minimize peptide loss, various low-retention tubes or microplates have been applied to sensitive proteomics protocols (Bark et al., Weikart et al., Future Sci OA 2019, 5, FSO367). However, peptide loss was still observed, especially for samples containing limited analytes.

[0039] Single mammalian cells contain sub-ng levels of protein (0.1-0.5ng) (Dou et al., Zhu et al., Angew Chem Int Ed Engl 2018,57,12370-12374). Surface adsorption loss is a major issue in current MS-based bottom-up mammalian single-cell proteomics analysis (Shi et al., Zhang et al. 2019, Tsai et al. 2021, Zhu et al. 2018b). In recent years, great progress has been made to improve processing recovery from small numbers of cells. One approach is to reduce the sample volume to less than 200 nL, thereby reducing the sample surface contact area on special chips (nanoPOTS) (Dou et al., Zhu et al. 2018b, Zhu et al. 2018c, Li et al., Anal Chem 2018, 90, 5430-5438, Cong et al., Chem Sci 2020, 12, 1001-1006). The second approach is to add an excess amount of exogenous carrier protein such as BSA to minimize surface adsorption of low-abundance proteins and peptides in targeted proteomics quantitative analysis (Shi et al., Zhang et al. 2019). A third approach is to combine tandem mass tag (TMT)-labeled samples with more than 100 cells in one channel as a carrier channel and another channel of single-cell samples to increase the sample starting amount (Dou et al., Budnik et al., Genome Biol 2018,19,161). However, these approaches have drawbacks that limit their widespread application. For example, special equipment is required for nanoPOTS to process samples in such small volumes, the addition of excess exogenous carriers is only suitable for targeted proteomic analysis, and low reproducibility was reported for TMT carrier methods (Shi et al., Zhu et al. 2018b, Budnik et al.).Recently, an easily adaptable detergent-assisted one-pot MS (SOP-MS) method was developed to analyze single-cell proteomics samples by using DDM, an MS-compatible non-ionic detergent, in low-binding PCR tubes or microplates (Tsai et al. 2021). In this study, over 300 proteins and approximately 1000 unique peptides were identified from a single mammalian cell. Other reports have demonstrated that DDM can improve membrane recovery and identification of glycoproteins (Liu et al., Anal Chem 2015, 87, 2054-2057; Zhang et al., Mol Cell Proteomics 2015, 14, 2441-2453). Based on these reports, DDM is compatible with nanoLC-MS and relatively easy to implement, making it potentially applicable to many different fields where prevention of sample loss is important.

[0040] The disclosure herein provides a solution to reduce sample loss and improve peptide signal in the analysis of biologics. In the examples shown below, HeLa single cell proteome was analyzed using different sample preparation methods. Significantly more proteins and unique peptides were identified in samples containing DDM compared to control samples. Further data analysis found that the unique peptides identified only in samples prepared with DDM have significantly higher hydrophobicity compared to the unique peptides common to all samples.

[0041] By applying DDM for the first time to different assays in monoclonal antibody (mAb) drug development, we demonstrate that hydrophobic CDR peptide signals can be significantly enhanced in clinical PK studies by minimizing losses from peptide adsorption. The fold change in intensity increase of the target peptide has a strong correlation with its hydrophobicity and retention time, which may provide guidance for the selection of surrogate peptides and handling of peptide standards in clinical PK studies. In peptide mapping assays, DDM increased the signal intensity of several hydrophobic peptides and improved the stability of peptide solutions over 48 hours with an autosampler set at 4 °C. In addition, we demonstrate that improving or maximizing the recovery of hydrophobic peptides from samples dried in vacuum can be achieved by dissolving the dried samples in a solution containing DDM, which resulted in much higher signals of later eluting peaks and an overall higher proteome coverage in complex samples.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are now described.

[0043] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations as understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" are meant to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0044] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains generally known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, linked through peptide bonds. A "synthetic peptide or polypeptide" refers to a peptide or polypeptide that does not occur in nature. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those of skill in the art. A protein may include one or more polypeptides to form a single functional biomolecule. In another exemplary embodiment, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. A protein of interest may include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and any of bispecific antibodies. Proteins may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as 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” (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, 2014). 147-176 (2012), the entire teachings of which are incorporated herein by reference. In some exemplary embodiments, the proteins include 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, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels and other dyes, and the like. Proteins can be classified based on composition and solubility, and thus include simple proteins, such as globular proteins, fibrous proteins, complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, lipoproteins, and derived proteins, such as primary derived proteins, secondary derived proteins, and the like.

[0045] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried in a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., IgG1), and optionally, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0046] The term "antibody" as used herein includes immunoglobulin molecules, which include four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion techniques or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domains and, optionally, the constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized.The DNA can be sequenced and manipulated using chemical or molecular biology techniques, for example to place one or more variable and / or constant domains in a suitable configuration, to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids.

[0047] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, an antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. In some exemplary embodiments, an antibody fragment comprises sufficient amino acid sequence of a parent antibody that the fragment binds to the same antigen as the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Optionally, or additionally, antibody fragments can be wholly or partially synthetically produced. Antibody fragments can optionally include single chain antibody fragments. Optionally, or additionally, antibody fragments can include multiple chains linked together, for example, by disulfide bonds. Antibody fragments can optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, more typically include at least about 200 amino acids.

[0048] The term "bispecific antibody" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally include two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind to 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 orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.

[0049] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain and a CH3 domain, and an immunoglobulin light chain that does not confer antigen binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind one or more epitopes bound by the heavy chain antigen binding region, or can associate with each heavy chain and bind one or two epitopes. BsAbs can be divided into two main classes, those with an Fc region (IgG-like) and those that lack an Fc region, the latter being usually smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs include various formats such as, but are not limited to, triomabs, knobs into holes IgG (kih IgG), crossMabs, orth-Fab IgG, dual variable domain Igs (DVD-Igs), two-in-one or dual acting Fabs (DAFs), IgG single chain Fvs (IgG-scFvs), or κλ bodies. Various non-IgG-like formats include tandem scFv, diabody formats, single chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Mueller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the teachings of which are incorporated herein in their entirety). Methods for producing bsAbs include, but are not limited to, quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation with chemical crosslinkers, and genetic approaches using recombinant DNA technology.

[0050] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Such molecules typically bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecifics, can also be addressed by the systems and methods disclosed herein.

[0051] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0052] As used herein, a "sample" may be obtained from any step of a bioprocess such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in downstream processing, drug substance (DS), or formulation (DP), including the final formulated product. In some specific exemplary embodiments, the sample may be selected from any step of downstream processing of clarification, chromatographic purification, or filtration.

[0053] In some exemplary embodiments, a sample containing a protein of interest may be prepared prior to LC-MS analysis. Preparation steps may include denaturation, alkylation, dilution, and digestion.

[0054] As used herein, the term "protein alkylating agent" or "alkylating agent" refers to an agent used to alkylate specific free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.

[0055] As used herein, "protein denaturation" or "denaturation" may refer to a process that changes the three-dimensional shape of a molecule from its native state. Protein denaturation can be performed using a protein denaturant. Non-limiting examples of protein denaturants include heat, high or low pH, reducing agents such as DTT, or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of a system by disrupting intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and salts thereof.

[0056] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to perform digestion (e.g., enzymatic or non-enzymatic digestion) of proteins in a sample using an appropriate hydrolysis agent. Digestion of proteins into constituent peptides can produce "peptide digests" that can be further analyzed using peptide mapping analysis.

[0057] As used herein, the term "digestive enzyme" refers to any of a number of different agents capable of carrying out the digestion of proteins.Non-limiting examples of hydrolytic agents capable of carrying out enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, Lys-N protease (Lys-N), Lys-C endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or biologically active fragments or homologues thereof, or combinations thereof. For a recent review discussing available techniques for protein digestion, see Switazar et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzer, Martin Giera & Wilfried MANiessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077(2013)).

[0058] As used herein, the term "protein reducing agent" or "reducing agent" refers to an agent used for the reduction of disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins are dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof. Reduced peptide mapping, a traditional method of protein analysis, involves protein reduction prior to LC-MS analysis. In contrast, non-reduced peptide mapping omits the sample preparation step of reduction in order to preserve endogenous disulfide bonds.

[0059] As used herein, the term "pharmacokinetics" (PK) refers to the field of study that deals with the properties of drugs after administration to a subject. Exemplary components of a pharmacokinetic analysis include the release of a drug from a pharmaceutical formulation, the absorption of a drug into the blood circulation, the distribution of a drug throughout the body, the metabolism of a drug into metabolites (also called biotransformation), and the excretion of a drug from the body. The pharmacokinetics of a drug is an important feature for the evaluation of biotherapeutic candidates. Specifically, pharmacokinetic studies can be performed to evaluate how the levels of a drug and its altered forms and metabolites change over time after administration to a subject. Biotherapeutic proteins can be evaluated through the analysis of representative peptides or "target peptides" or "surrogate peptides" using liquid chromatography-mass spectrometry. A peptide can be a suitable target peptide if it is unique or strongly representative of a protein (e.g., the complementarity determining region of an antibody) and if it can be reliably recovered and measured.

[0060] The examples shown in this disclosure show that the suitability of candidate target peptides may depend on their hydrophobicity, and more hydrophobic peptides are more vulnerable to sample loss due to surface adsorption. The method of the present invention can be used to evaluate the vulnerability of candidate target peptides to sample loss by comparing the recovery rates of synthetic peptide standards with the same amino acid sequence as the candidate target peptides prepared with or without DDM using LC-MS analysis. Peptides with a large ratio of average peak area between DDM-containing samples and control samples may be more vulnerable to sample loss due to surface adsorption, and therefore may be considered less suitable for selection as target peptides.

[0061] Synthetic peptide standards are useful in pharmacokinetic studies to generate standard curves, which can be used to determine the mathematical relationship between LC-MS signal and peptide concentration and then calculate the concentration of the target peptide. Measurement of peptide standards, and therefore the overall pharmacokinetic analysis, can be adversely affected by surface adsorption of the peptide standards to the container. The method of the present invention can be used to improve retention of peptide standards for pharmacokinetic studies by using DDM in preparing the peptide standards to prevent surface adsorption and sample loss.

[0062] As used herein, the term "liquid chromatography" refers to a process in which a biological / chemical mixture carried by a liquid can be separated into components as a result of differences in the distribution of the components as they flow (or are) through a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some embodiments, a sample containing at least one protein or peptide digest of interest can be subjected to any one or combination of the above-mentioned chromatographic methods. Analytes separated using chromatography are characterized by unique retention times that reflect the rate at which the analytes pass through a chromatographic column. Analytes can be compared using chromatograms that plot retention times on one axis and measured signals on another axis, where the measured signals can be generated, for example, from UV or fluorescence detection.

[0063] As used herein, the term "mass spectrometer" includes an instrument capable of recognizing and measuring the exact mass of a specific molecular species. The term is meant to include any molecular detector capable of characterizing a polypeptide or peptide. A mass spectrometer includes three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to generate gas phase ions. Analyte atoms, molecules, or clusters are transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or through a separate process. The choice of ion source depends on the application.

[0064] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques that use multiple stages of mass selection and mass separation to obtain structural information of sample molecules. The prerequisite is that after an initial mass selection step, the sample molecules are transferred to the gas phase and ionized such that fragments are formed in a predictable and controllable manner. MS / MS, or MS2 First, the precursor ion (MS 1 ) and fragment it to obtain meaningful information. Tandem MS is successfully performed with a variety of analyzer combinations. Which analyzer to combine for a particular application can be determined by many different factors, such as size, cost, and availability, as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids where a tandem-in-time analyzer is connected in space or with a tandem-in-space analyzer. A tandem-in-space mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function can be designed such that ions are selected in one section of the instrument, dissociated in an intermediate region, and the product ions are sent to another analyzer for m / z separation and data collection. In tandem-in-time, mass analyzer ions generated in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0065] The peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins, their abundance, their post-translational modifications, or other modifications. They can be used to characterize proteins by correlating experimental and theoretical MS / MS data, the latter generated from potential peptides in protein sequence databases. This characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequence determination, protein de novo sequencing, localization of post-translational modifications or sequence variants, or identification of post-translational modifications or sequence variants, or comparability analysis, or a combination thereof.

[0066] In some exemplary aspects, the mass spectrometer may operate with nanoelectrospray or nanospray. The term "nanoelectrospray" or "nanospray" as used herein refers to electrospray ionization at very low solvent flow rates, often without external solvent delivery, typically hundreds of nanoliters of sample solution per minute or less. The electrospray injection setup that forms the nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small amounts of sample (analyte) solution over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separation of a mixture prior to analysis by a mass spectrometer.

[0067] In some exemplary embodiments, mass spectrometry may be performed under native conditions. As used herein, the term "native conditions" may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see review: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0068] As used herein, the term "database" refers to a compiled collection of protein sequences that may be present in a sample, for example in the form of FASTA format files. The relevant protein sequences may be derived from the cDNA sequences of the species being studied. Public databases that may be used to search for relevant protein sequences included, for example, databases hosted by Uniprot or Swiss-prot. The databases may be searched using databases referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra against all possible sequences in the database(s) and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0069] The present disclosure provides a method for preparing a peptide standard for pharmacokinetic studies for liquid chromatography-mass spectrometry analysis. In some exemplary embodiments, the method includes (a) contacting a peptide standard for pharmacokinetic studies with N-dodecyl-β-D-maltoside (DDM) to form a mixture, and (b) subjecting the mixture to liquid chromatography-mass spectrometry analysis.

[0070] In some exemplary embodiments, the peptide standard comprises or has the amino acid sequence of a target peptide for pharmacokinetic studies. In further exemplary embodiments, the amino acid sequence of the target peptide may be selected from a complementarity determining region of an antigen binding protein. The antigen binding protein may be selected from, for example, a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a fusion protein.

[0071] In some exemplary embodiments, the final concentration of the peptide standard in the mixture is about 0.1 fmol / μL to about 100 fmol / μL, about 1 fmol / μL to about 20 fmol / μL, about 5 fmol / μL to about 15 fmol / μL, about 1 fmol / μL, about 2 fmol / μL, about 3 fmol / μL, about 4 fmol / μL, about 5 fmol / μL, about 6 fmol / μL, about 7 fmol / μL, about 8 fmol / μL, about 9fmol / μL, approximately 10fmol / μL, approximately 11fmol / μL, approximately 12fmol / μL, approximately 13fmol / μL, approximately 14fmol / μL, approximately 15fmol / μL, approximately 16fmol / μL, approximately 17fm ol / μL, about 18 fmol / μL, about 19 fmol / μL, about 20 fmol / μL, about 30 fmol / μL, about 40 fmol / μL, about 50 fmol / μL, or about 100 fmol / μL.

[0072] In some exemplary embodiments, the final concentration of DDM in the mixture can be about 0.001% to about 0.05%, about 0.01% to about 0.02%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.011%, about 0.012%, about 0.013%, about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.1%.

[0073] The present disclosure also provides methods for selecting target peptides for pharmacokinetic studies. In some exemplary embodiments, the method includes: (a) obtaining at least two candidate peptide standards that include or have amino acid sequences of at least two candidate target peptides for a pharmacokinetic study; (b) contacting each of the at least two candidate peptide standards with N-dodecyl-β-D-maltoside (DDM) to form at least two mixtures; (c) subjecting each of the at least two mixtures to liquid chromatography-mass spectrometry analysis to generate a mass spectrum for each of the at least two candidate peptide standards; (d) comparing each of the mass spectra with a mass spectrum generated by the liquid chromatography-mass spectrometry analysis of a control mixture to generate an average peak area ratio for each of the at least two candidate peptide standards; (e) comparing the average peak area ratio between each of the at least two candidate peptide standards; and (f) using the comparison of (e) to select a target peptide for a pharmacokinetic study, wherein the control mixture includes the candidate peptide standards and does not include DDM.

[0074] In some exemplary embodiments, the amino acid sequence of each of the at least two candidate target peptides may be selected from a complementarity determining region of an antigen binding protein.

[0075] In some exemplary embodiments, the final concentration of each of the at least two candidate peptide standards in each of the at least two mixtures is about 0.1 fmol / μL to about 100 fmol / μL, about 1 fmol / μL to about 20 fmol / μL, about 5 fmol / μL to about 15 fmol / μL, about 1 fmol / μL, about 2 fmol / μL, about 3 fmol / μL, about 4 fmol / μL, about 5 fmol / μL, about 6 fmol / μL, about 7 fmol / μL, or about 10 fmol / μL. L, about 8 fmol / μL, about 9 fmol / μL, about 10 fmol / μL, about 11 fmol / μL, about 12 fmol / μL, about 13 fmol / μL, about 14 fmol / μL, about 15 fmol / μL, about 16 fmol / μ L, about 17 fmol / μL, about 18 fmol / μL, about 19 fmol / μL, about 20 fmol / μL, about 30 fmol / μL, about 40 fmol / μL, about 50 fmol / μL, or about 100 fmol / μL.

[0076] In some exemplary embodiments, the final concentration of DDM in each of the at least two mixtures can be about 0.001% to about 0.05%, about 0.01% to about 0.02%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.011%, about 0.012%, about 0.013%, about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.1%.

[0077] In some exemplary embodiments, the control mixture may include formic acid, which may be present in the control mixture at a concentration of about 0.01% to about 0.5%, about 0.05% to about 0.2%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.2%, about 0.3%, about 0.4%, or about 0.5%.

[0078] The present disclosure further provides a method for preparing a protein of interest for peptide mapping analysis. In some exemplary embodiments, the method includes: (a) subjecting a sample containing a protein of interest to conditions suitable for denaturation to form a denatured sample; (b) contacting the denatured sample with at least one alkylating agent under conditions suitable for alkylation to form an alkylated sample; (c) contacting the alkylated sample with N-dodecyl-β-D-maltoside (DDM) to form a mixture; (d) contacting the mixture with at least one digestive enzyme under conditions suitable for digestion to form a peptide digest; and (e) subjecting the peptide digest to liquid chromatography-mass spectrometry for peptide mapping analysis.

[0079] In some exemplary embodiments, conditions suitable for denaturation may include heating the sample. The sample may be heated to about 40° C. to about 100° C., about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C., about 90° C., about 95° C., or about 100° C. The sample may be heated for about 5 minutes to about 20 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes.

[0080] In some exemplary embodiments, the method further includes subjecting the sample to conditions suitable for reduction. In some exemplary embodiments, the conditions suitable for reduction may include contacting the sample with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), where the concentration of TCEP-HCl may be about 1 mM to about 20 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM.

[0081] In some exemplary embodiments, the final concentration of DDM in the mixture can be about 0.01% to about 0.5%, about 0.05% to about 0.2%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.2%, about 0.3%, about 0.4%, or about 0.5%.

[0082] The present disclosure also provides a method for preparing a protein sample of interest for liquid chromatography-mass spectrometry analysis. In some exemplary embodiments, the method includes: (a) drying a protein sample of interest using a high-speed vacuum to form a dried sample; (b) dissolving the dried sample using a solution containing N-dodecyl-β-D-maltoside (DDM) to form a reconstituted sample; and (c) subjecting the reconstituted sample to liquid chromatography-mass spectrometry analysis.

[0083] In some exemplary embodiments, the concentration of DDM in the solution can be about 0.001% to about 0.05%, about 0.01% to about 0.02%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.011%, about 0.012%, about 0.013%, about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, or about 0.1%.

[0084] The present invention is not limited to any of the above-mentioned peptide(s), protein(s), protein(s), protein of interest, antibody(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s), and may be used in conjunction with any peptide. It is understood that the peptide(s), protein(s), protein(s) of interest, antibody(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s) may be selected by any suitable means.

[0085] The present invention may be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0086] Materials and Methods 1. Chemicals and Reagents Formic acid (FA), trifluoroacetic acid (TFA), and LC-MS grade acetonitrile were purchased from Thermo Fisher Scientific (Rockford, IL). N-dodecyl-β-D-maltoside (DDM), iodoacetamide (IAM), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO). Sequencing grade modified trypsin with resuspension buffer was purchased from Promega (Madison, WI), and ultrapure 1 M Tris-HCl buffer (pH 7.5) was obtained from Invitrogen (Carlsbad, CA). Purified monoclonal antibodies (mAbs) were produced in-house by Regeneron (Tarrytown, NY). AQUA™ grade custom synthesized standard mAb peptides, HeLa cell lysate protein digests, and BSA digests were ordered from Thermo Fisher Scientific (Rockford, IL). NANOSEP 30K OMEGA centrifugal filters were obtained from Pall Life Sciences (Port Washington, NY). Protein LoBind tubes were purchased from Eppendorf (Enfield, CT), and glass insert sample vials were obtained from Waters (Milford, MA).

[0087] 2. Sample Preparation HeLa cell lysate protein digests were diluted to 0.05ng / μL with 0.1% FA or 0.015% DDM solution, and 0.2ng (4μL) was injected into the LC-MS to simulate single cell sample analysis. Sixteen synthetic peptides from the constant regions and CDRs of mAbs were diluted and mixed to a final concentration of 10fmol / μL with 0.1% FA, 0.015% DDM solution, or a combined cell digest matrix. For reconstitution experiments, 20μL of 500fmol / μL BSA digest and 200ng / μL HeLa digest were aliquoted into vials and dried in vacuum. The dried samples were reconstituted with 0.1% FA or 0.015% DDM solution. 1pmol of BSA digest and 100ng of HeLa digest were loaded onto the column for nanoLC-MS / MS analysis.

[0088] 3. Preparation of mAb Peptide Mapping Samples A 550 μg aliquot of each mAb sample diluted to approximately 10 μg / μL with H2O was centrifuged through a NANOSEP 30K filter. After reconstitution with 5 mM acetic acid, protein concentration was measured using a NanoDrop 2000 (Thermo Scientific, MA) UV-Vis spectrophotometer. For reduced peptide mapping (RPM) samples, a 100 μg aliquot of each sample was denatured and reduced in 42 μL of a solution containing 5 mM acetic acid and 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) by heating at 80° C. for 10 min. For alkylation, 24 μL of 8 M urea and 2 μL of 0.2 M iodoacetamide were then added to each sample and incubated in the dark for 30 min. Each sample was then diluted with 100 mM Tris-HCl (pH 7.5) and DDM solution to a final concentration of 0.1% DDM and digested with trypsin at an enzyme-to-substrate ratio of 1:10 (w / w) for 3 h at 37°C. Digestion was quenched by adding TFA to a final concentration of 0.3%. For non-reduced peptide mapping (NRPM) samples, 100 μg aliquots of each sample were denatured by heating at 80°C for 10 min. After cooling to room temperature, the denatured samples were alkylated by adding 2 μL of 0.2 M iodoacetamide and incubated in the dark at room temperature for 30 min. All subsequent steps were identical to the preparation of reduced peptide mapping samples.

[0089] 4.NanoLC-MS analysis Single cell and reconstituted samples were analyzed using an UltiMate 3000 RSLCnano system (Thermo Scientific) interfaced to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). A 30 μm ID×25 cm C18 column was used for single cell sample analysis, and a 75 μm ID×25 cm C18 column was used for all other sample analysis (CoAnn Technologies, 360 μm OD). Mobile phase A contained 0.1% FA in water, and mobile phase B contained 0.1% FA in 80% acetonitrile / 20% water. All samples were loaded onto an Acclaim PepMap 100 with a 75 μm×2 cm precolumn (Thermo Scientific) at a flow rate of 5 μL / min for 5 min. For single cell samples, a linear LC gradient was set up at a flow rate of 60 nL / min as follows: 5% B at 0 min, 8% B at 5 min, 40% B at 80 min, and 95–120 min 95% B. For other samples, a flow rate of 0.25 μl / min was used and the LC gradient was as follows: 5% B at 0 min, 8% B at 3 min, 36% B at 95 min, and 95% B from 120 to 130 min.

[0090] Mass spectrometry data acquisition was performed using Xcalibur v4.3 (Thermo Fisher Scientific, CA). For single cell sample analysis, the nanoESI spray voltage was set to 2100 V. In conventional data-dependent acquisition mode (DDA), the top 10 peaks were selected for MS / MS fragmentation. MS full scans were acquired from m / z 375–1575 at 120K resolution (m / z 200) with a standard automatic gain control (AGC) of 400%, and a maximum injection time of 100 ms. MS / MS fragmentation was performed using HCD with a normalized collision energy of 30%, at a resolution of 30K (m / z 200), a standard AGC of 100%, and a maximum injection time of 500 ms. The dynamic exclusion time was set to 60 s with a single repeat count, and only precursors with charge states between +2 and +7 were selected.

[0091] For other sample analysis, survey scans were performed in the Orbitrap with a cycle time of 1.5 seconds using a spray voltage of 2200 V. MS full scans were acquired from m / z 400-1800 at a resolution of 60K (m / z 200) with standard automatic gain control (AGC) of 300% and a maximum injection time of 20 ms. MS / MS fragmentation was performed using HCD at a normalized collision energy of 30%, at a resolution of 15K (m / z 200) and a maximum injection time of 50 ms. All other MS parameters were identical to the analysis of single cell samples.

[0092] 5. Target Quantification by Parallel Reaction Monitoring (PRM) PRM target quantification was performed on an Orbitrap Exploris 480 mass spectrometer equipped with an UltiMate 3000 RSLCnano system and a 75 μm ID × 25 cm C18 column (CoAnn Technologies, 360 μm OD). A short gradient with a flow rate of 0.3 μL / min was set as follows: 5% B at 0 min, 8% B at 3 min, 40% B at 20 min, and 95% B at 30–35 min. The m / z of the most intense surrogate peptide peak was selected for PRM analysis using a 5 min window. MS / MS spectra were acquired using HCD at 30% normalized collision energy with a resolution of 30K (m / z 200) and a maximum injection time of 100 ms.

[0093] 6.UPLC / UV-MS analysis Peptide mapping samples were analyzed using a Waters ACQUITY UPLC I-Class system interfaced with a Thermo Scientific Q Exactive Plus mass spectrometer. Tryptic digest peptide mixtures were separated by a Waters ACQUITY UPLC BEH® 130 C18 column (1.7 μm, 2.1 mm×150 mm) at a flow rate of 0.25 mL / min. Mobile phase A (MPA) was 0.05% TFA in water, and mobile phase B (MPB) was 0.045% TFA in acetonitrile. The gradient was maintained at 0.1% B for the first 5 min, then increased to 35% B for 75 min, followed by a further increase to 90% B for 5 min. The column was equilibrated with 99.9% mobile phase A prior to sample injection, while the column temperature was maintained at 40° C. MS data were acquired on a Thermo Scientific Q Exactive Plus mass spectrometer at 70 K resolution (at m / z 400) from m / z 300 to 2000, followed by five data-dependent MS / MS scans at a resolution of 17.5 K. MS full scans were performed at 1 × 10 6 Automatic gain control (AGC) and a maximum injection time of 50 ms were set. MS / MS fragmentation was 1 × 10 5 High energy C-trap dissociation (HCD) was performed with AGC and a normalized collision energy of 28%, with a maximum injection time of 100 ms. The dynamic exclusion time was set to 15 seconds with a single repeat count.

[0094] 7. Data Analysis Database searches for protein identification were performed using SEQUEST and Mascot embedded in Proteome Discoverer 2.2 (Thermo Fisher Scientific). A human UniProt target-decoy protein database containing common contaminants was used for the HeLa sample database search. Precursor ion mass tolerance was set to 20 ppm and fragment ion mass tolerance was set to 0.02 Da. During the database search, trypsin was specified as the digestion enzyme and one missed cleavage was allowed. Oxidation of methionine (+16 Da) was selected as the variable modification and carbamidomethylation of cysteine ​​was selected as the fixed modification for the regular (alkylation) digest. The false discovery rate (FDR) was set to 1% for peptide identification and 5% for protein identification. Peptide mapping and BSA sample data were analyzed using Protein Metrics Byonic™ (version 3.11.3) by searching raw files against the protein sequences of mAbs or BSA. All peptide quantification was performed using MS of all detected charge states. 1 Analysis was performed using Skyline Daily software (University of Washington, WA) by extracting peak areas. The three most intense fragment peaks without interference were selected for PRM targeted quantification. Peak areas of UV chromatograms were extracted using Empower (Milford, MA). Peptide hydrophobicity was calculated using the peptide synthesis and proteotypic peptide analysis tools from ThermoFisher Scientific (Waltham, MA). GraphPad Prism 9.0.2 (San Diego, CA) was used for correlation analysis and p-value calculation.

[0095] Example 1. Achieving maximum hydrophobic peptide recovery in single-cell proteomics analysis with DDM To demonstrate the effectiveness of DDM in improving or maximizing peptide recovery in single-cell proteomics, 0.2 ng of HeLa digests were prepared in either DDM or formic acid (FA) solutions and analyzed in triplicate by LC-MS. As shown in Figure 1A and Figure 1B, compared to the control sample, the number of identified unique peptides (363 vs. 964) and protein groups (168 vs. 368) were significantly increased in the HeLa digest samples prepared with DDM. The results showed that DDM can maximize peptide recovery in single-cell proteomics by significantly reducing peptide loss due to surface adsorption. This is also consistent with previous reports (Tsai et al. 2021).

[0096] Further analysis found that the majority of unique peptides identified in the control samples (591 out of 656) overlapped with unique peptides identified in the DDM-containing samples. Meanwhile, the majority of the total unique peptides identified (947 / 1603) were identified only in the DDM-containing samples, as shown in Figure 1C. To better understand the difference between the overlapping unique peptides and the peptides identified only in the DDM-containing samples, peptide hydrophobicity index values ​​were calculated. These results, as shown in Figure 1D, showed a significant difference (p<0.0001) between the two unique peptide groups, indicating that the peptides identified only in the DDM-containing samples were much more hydrophobic than the overlapping peptides.

[0097] This analysis suggests that the recovery of hydrophobic peptides in single-cell samples was maximized by significantly reducing surface adsorption losses during sample preparation with DDM. However, as shown in Figure 2, the loss of hydrophobic peptides is not unique to single-cell proteomics but is a common problem during protein sample preparation in general. These findings indicate that DDM may also improve various assays during protein drug development in the biopharmaceutical field. Thus, a novel method was developed to improve peptide and protein characterization in biopharmaceutical assays using DDM.

[0098] Example 2. DDM is a surfactant compatible with mAb drug characterization assays. It has been previously demonstrated that DDM is a surfactant compatible with nanoLC-MS (Tsai et al. 2021, Liu et al.). Although DDM should not typically interfere with peptide mapping UV spectra monitored simultaneously with UPLC-MS analysis, it is still necessary to test the suitability of DDM in mAb drug development assays. Peptide mapping characterization of mAbs was performed with different concentrations of DDM (0.01% and 0.1%). As shown in Figure 3, the DDM compound elutes after all peptides at approximately 48% MPB and does not affect peptide separation or MS detection. Additionally, there was no carryover of DDM in subsequent runs, regardless of the DDM concentration used. Finally, highly comparable UV chromatograms were obtained for the DDM-containing samples and controls.

[0099] These results demonstrate that DDM is a surfactant that is compatible with mAb drug development assays, including those that require simultaneous UV and MS detection or that use different solvents than typical nanoLC-MS analyses.

[0100] Example 3. Improvement of target peptide signals in PK studies by applying DDM to synthetic peptide standard preparations In mAb PK studies, CDR peptides representing mAb drug proteins must be selected to monitor the concentration changes in the serum of patients at different times after drug administration. However, CDR peptides are usually very hydrophobic and therefore prone to loss by surface adsorption, especially when dealing with synthetic peptide standards (Perchiacca et al., Protein Eng Des Sel 2012, 25, 591-601).

[0101] To optimize the method to characterize peptides for PK studies, 16 peptides from different mAb molecules were selected and synthesized, including 13 CDR peptides and 3 constant region peptides. Table 1 shows the information of the synthetic peptide standards, including stock concentration, dilution, hydrophobicity index value, and retention time. All synthetic peptide standards were diluted and mixed as a single peptide mixture with a final concentration of 10 fmol / μl using either DDM solution, formic acid solution (control), or combined HeLa cell lysate digest matrix. PRM acquisition was then used to quantify the peak areas of these 16 target peptides and compare the results between different samples. Table 2 lists the detailed PRM setting information, including the m / z value and charge state of each target peptide. Each sample was analyzed in triplicate, and the average peak area of ​​each peptide from the DDM-prepared and matrix-prepared samples was compared with the control sample. As shown in Table 1, the hydrophobic target peptides showed a significant increase in MS signal in the DDM samples, ranging from a 2-fold change to a 38-fold change. However, other less hydrophobic and hydrophilic peptides showed no substantial differences between the DDM and control samples. These results indicate that DDM can prevent the loss of hydrophobic peptides and maximize their recovery when handling synthetic peptide standards used in PK studies.

[0102] Table 1. Changes in peak area of ​​synthetic mAb peptides for different sample preparation conditions TIFF2025508765000001.tif163170

[0103] Table 2. Synthetic peptide standards used for PRM target quantification TIFF2025508765000002.tif105163

[0104] Typically, complex tissue / cell sample digestion matrices are used to help prevent loss of target peptides during preparation of peptide standards. These results show that all peptides with significant improvement in DDM-containing samples also significantly increased MS signal in matrix-prepared samples, indicating that both DDM and matrix solutions can prevent loss of hydrophobic peptides during preparation of peptide standards. However, complex matrix solutions may introduce interferences or affect quantification accuracy, so careful optimization and transition selection are required for development of targeted quantification methods when using matrix solutions. Conversely, there is no interference when handling peptide standard samples using DDM.

[0105] The data for the DDM-containing and control samples were further analyzed by comparing the change in peak area with the peptide hydrophobicity index value and peptide retention time, as shown in Figure 4B. As shown in Figure 4B, there was a strong correlation (R 2 The correlation between the peptide peak area ratio and retention time was weak, but still positive (R 2 As shown in Figure 4A, all peptides eluting after 25 min showed a significant increase in peak area.

[0106] These results may provide guidance for the selection of target peptides and the preparation of synthetic peptide standards in future PK studies. For hydrophobic peptides or peptides with long RTs, special care should be taken to avoid peptide loss due to surface adsorption.

[0107] Example 4. Improved peptide mapping characterization with DDM Peptide mapping is an important analytical method to study the primary structure of biotherapeutic proteins (Bongers et al., Mouchahoir and Schiel). However, loss of hydrophobic peptides due to adsorption often occurs in peptide mapping analysis, which can impair sequence coverage or lead to failure of stability assessment in method qualification (Bongers et al.). Here, non-reduced peptide mapping (NRPM) and reduced peptide mapping (RPM) experiments were performed for mAb1 and mAb2, respectively. For each molecule, six individual samples were prepared with or without DDM. Overall, high reproducibility was obtained from the six individual sample preparations for both molecules, as shown in Figure 5A and Figure 5B. However, it was observed that the intensities of three hydrophobic peptides from mAb1 and one hydrophobic peptide from mAb2 were significantly different between samples with and without DDM, as indicated by the arrows in Figure 5A and Figure 5B. These four variable hydrophobic peptides are described in more detail in Figure 6C. In all four cases, enhanced peptide UV signals were observed in the DDM-containing samples.

[0108] Two representative peptide peaks, HC128-144 from mAb1 and HC101-120 from mAb2, are shown in Figure 6A and Figure 6B, respectively. Peak areas were extracted using Empower, and the average from three individual samples was used to calculate the shift and RSD. For both peptides, enhanced peptide UV signals were observed in the DDM-containing samples. As shown in Figure 6C, the peak intensities of the four hydrophobic peptides increased by approximately 19%-64% in the DDM-containing samples, and their RSDs significantly decreased from 73% in the control sample to 7% in the DDM-containing samples.

[0109] Another potential negative impact of hydrophobic peptide loss on peptide mapping characterization is increased peptide signal variability that may result in solution stability test criteria not being met during method qualification. Non-reduced peptide mapping samples from mAb1 were injected into the UPLC-MS and analyzed repeatedly every 12 h. In total, five runs were analyzed over 48 h to test the stability of peptide solutions in the autosampler at 4 °C. Except for the three hydrophobic peptides mentioned above, all peptides were found to have excellent reproducibility. Although a gradually decreasing peptide intensity was observed for one peptide (HC128-144) in the control sample, the intensity of this peptide was stable in the DDM-containing sample, as shown in Figure 7A and Figure 7B. On further analysis, the RSD of all three hydrophobic peptides was found to be significantly decreased in the DDM-containing sample compared to the control sample, as shown in Figure 7C.

[0110] These results demonstrate that by adding DDM, greater solution stability can be achieved for mAb peptide mapping, improving the robustness of the method and its transferability to different sites during drug development and manufacturing.

[0111] Example 5. Maximizing peptide recovery in dried samples by resuspension in DDM solution Loss of proteolytic peptides after a fast vacuum drying step prior to LC-MS analysis is a common problem in proteomics sample analysis due to the low solubility of hydrophobic peptides in dried samples using 0.1% TFA or formic acid (Weikart et al.). This example demonstrates the use of DDM solution to improve the recovery of hydrophobic peptides from dried samples.

[0112] As illustrated in FIG. 8, six aliquots of BSA tryptic digests were dried in vacuum and then dissolved by DDM or FA solution. As shown in FIG. 9A, high coverage of BSA was achieved in both samples, but the peak area of ​​peptides dissolved in DDM was significantly increased compared to that of 0.1% FA. This difference was especially noticeable for peptides with later retention times, which increased in intensity by about 3-fold, as indicated by the box in FIG. 9A. To quantify the changes in peptide peaks, 10 landmark peaks (indicated by * in FIG. 9A) from different retention times were selected and their peak areas in DDM-dissolved samples and 0.1% FA-dissolved samples were calculated, as shown in FIG. 9B. For early-eluting peptides, the peak area was slightly increased in DDM-dissolved samples. However, up to 10-fold enhancement was observed for later-eluting peptides when dissolved using DDM.

[0113] In addition, the retention times of all BSA peptides identified with high confidence were compared with the ratio of peak area changes between the DDM- and FA-dissolved samples. As shown in Figure 9C, there was a good correlation (R 2 A value of 0.68 was obtained, which is consistent with the demonstrated effect of DDM on the recovery of hydrophobic peptides, since the retention time in reversed-phase LC generally correlates with the hydrophobicity of the peptide. These results indicate that solubilization of peptides with DDM significantly improved the recovery of especially hydrophobic peptides.

[0114] This finding was further substantiated by the analysis of the combined HeLa digest samples dissolved in DDM or FA solutions. As shown in FIG. 10, significantly enhanced peptide peak signals were observed in the LC-MS chromatograms for peptide peaks eluting later from 82 to 120 min. Based on the database search, more than 15% of the identified unique peptides were found only in the DDM-dissolved HeLa digest samples compared to the FA-dissolved samples, as shown in FIG. 11A. As shown in FIG. 11B, more proteins were also observed in the DDM-dissolved digest than in the control digest. T-test analysis was performed to measure the differences in identified peptides and proteins between samples dissolved in different solvents. Significant p-values ​​(<0.005) were obtained for both the identified unique peptides and protein groups. These results indicate that dried samples dissolved in DDM-containing solutions can enhance proteome coverage in combined digest samples by maximizing the recovery of hydrophobic peptides.

[0115] The above examples demonstrate that the non-ionic surfactant, DDM, improves the recovery of hydrophobic peptides in different samples and sample preparations by preventing their loss due to surface adsorption. Additionally, it is shown that DDM does not adversely affect the recovery of hydrophilic peptides. These results indicate that DDM is a fully compatible surfactant with different mAb drug development assays without the need for purification steps to remove it.

[0116] When DDM was incorporated, no effect on peptide separation was observed in UPLC-MS analysis, and no change in UV absorbance signal was observed in UV / LC-MS analysis. In PK studies, significantly improved MS signal was observed for hydrophobic target peptides. Strong correlations were found between changes in peptide peak area and peptide retention time or peptide hydrophobicity, which can provide guidance for the selection of target peptides and the preparation of synthetic peptide standards.

[0117] In peptide mapping assays, the addition of DDM during sample preparation improved the signal and solution stability of hydrophobic peptides. This may be useful for method qualification and transferring standard operating procedures (SOPs) during protein drug development and manufacturing. In addition, DDM was found to help maximize the recovery of hydrophobic peptides in dried samples. These findings may help reduce sample loss during complex sample cleanup and improve proteome coverage in complex sample analysis.

Claims

1. 1. A method for preparing peptide standards for pharmacokinetic studies for liquid chromatography-mass spectrometry analysis, comprising: (a) contacting a peptide standard for a pharmacokinetic study with N-dodecyl-β-D-maltoside (DDM) to form a mixture; (b) subjecting the mixture to liquid chromatography-mass spectrometry analysis; The method comprising:

2. The method of claim 1 , wherein the peptide standard comprises the amino acid sequence of a target peptide for pharmacokinetic studies.

3. The method of claim 2 , wherein the amino acid sequence of the target peptide is selected from a complementarity determining region of an antibody.

4. 2. The method of claim 1, wherein the final concentration of the peptide standard in the mixture is about 10 fmol / uL.

5. 2. The method of claim 1, wherein the final concentration of DDM in the mixture is about 0.015%.

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

7. The method of claim 1 , wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

8. The method of claim 1 , wherein the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

9. 1. A method for selecting a target peptide for a pharmacokinetic study, comprising: (a) obtaining at least two candidate peptide standards having amino acid sequences of at least two candidate target peptides for a pharmacokinetic study; (b) contacting each of said at least two candidate peptide standards with N-dodecyl-β-D-maltoside (DDM) to form at least two mixtures; (c) subjecting each of said at least two mixtures to liquid chromatography-mass spectrometry analysis to generate a mass spectrum for each of said at least two candidate peptide standards; (d) comparing each of said mass spectra to a mass spectrum generated by liquid chromatography-mass spectrometry analysis of a control mixture to generate a ratio of average peak areas for each of said at least two candidate peptide standards; (e) comparing the ratio of the average peak areas between each of the at least two candidate peptide standards; and (f) using said comparison of (e) to select target peptides for pharmacokinetic studies. Including, the control mixture comprises the candidate peptide standard and no DDM; The method.

10. The method of claim 9 , wherein the amino acid sequence of each of the at least two candidate target peptides is selected from a complementarity determining region of an antibody.

11. 10. The method of claim 9, wherein the final concentration of each of the at least two candidate peptide standards in each of the at least two mixtures is about 10 fmol / uL.

12. 10. The method of claim 9, wherein the final concentration of DDM in each of the at least two mixtures is about 0.015%.

13. 10. The method of claim 9, wherein the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

14. 10. The method of claim 9, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

15. The method of claim 9 , wherein the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

16. 10. The method of claim 9, wherein the target peptide is selected based on having an amino acid sequence of a candidate peptide standard that has a smaller ratio of average peak areas compared to at least one other candidate peptide standard.

17. 10. The method of claim 9, wherein the control mixture comprises formic acid.

18. 18. The method of claim 17, wherein the final concentration of formic acid in the control mixture is 0.1%.

19. 1. A method for preparing a protein of interest for peptide mapping analysis, comprising: (a) subjecting a sample containing a protein of interest to conditions suitable for denaturation to form a denatured sample; (b) contacting the denatured sample with at least one alkylating agent under conditions suitable for alkylation to form an alkylated sample; (c) contacting the alkylated sample with N-dodecyl-β-D-maltoside (DDM) to form a mixture; (d) contacting said mixture with at least one digestive enzyme under conditions suitable for digestion to form a peptide digest; (e) subjecting the peptide digests to liquid chromatography-mass spectrometry for peptide mapping analysis; The method comprising:

20. 20. The method of claim 19, wherein the peptide mapping analysis is reduced peptide mapping analysis or non-reduced peptide mapping analysis.

21. 20. The method of claim 19, wherein the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, a host cell protein, or a protein pharmaceutical product.

22. 20. The method of claim 19, wherein the conditions suitable for denaturation comprise heating the sample, optionally wherein the heating is at about 80° C. for about 10 minutes.

23. 20. The method of claim 19, further comprising subjecting the sample to conditions suitable for reduction.

24. 24. The method of claim 23, wherein the conditions suitable for reduction comprise contacting the sample with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), optionally wherein the concentration of the TCEP-HCl is about 5 mM.

25. 20. The method of claim 19, wherein the at least one alkylating agent comprises iodoacetamide.

26. 20. The method of claim 19, wherein the final concentration of DDM in the mixture is about 0.1%.

27. 20. The method of claim 19, wherein the at least one digestive enzyme comprises trypsin, and optionally the trypsin is present in an enzyme to substrate ratio of about 1:10 (w / w).

28. 20. The method of claim 19, wherein the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

29. 20. The method of claim 19, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

30. 20. The method of claim 19, wherein the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

31. 1. A method for preparing a protein sample of interest for liquid chromatography-mass spectrometry analysis, comprising: (a) drying a sample of a protein of interest using a high velocity vacuum to form a dried sample; (b) dissolving the dried sample using a solution containing N-dodecyl-β-D-maltoside (DDM) to form a reconstituted sample; (c) subjecting the reconstituted sample to liquid chromatography-mass spectrometry analysis; and The method comprising:

32. 32. The method of claim 31 , wherein the concentration of DDM in the solution is about 0.015%.

33. 32. The method of claim 31 , wherein the protein sample of interest is a protein digest.

34. 32. The method of claim 31 , wherein the protein sample of interest is a cell digest.

35. 32. The method of claim 31, wherein the liquid chromatography system comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

36. 32. The method of claim 31, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

37. 32. The method of claim 31 , wherein the liquid chromatography-mass spectrometry analysis comprises a parallel reaction monitoring analysis.

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