Materials and methods for differential characterization of molecular conjugates and conjugation

Abstract

The present invention describes a method for analyzing drug polypeptide conjugates using tandem mass spectrometry involving dual tandem mass spectrometry tags (TMT). An integrated method is provided herein that uses TMT to obtain three analytical measurements, called "triple fusion", which enables the identification of drug occupancy, normalization between multiple samples, and triggering of additional MS / MS to identify and locate the conjugation site of the payload. A method for multiplexing conjugation reactions in a single run with TMT tags is also described to enhance throughput capabilities while maintaining the same sensitivity as current mass spectrometry instruments.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 902,958, filed on September 19, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to methods and reagents for characterizing, in particular, molecular conjugates, including, for example, quantification, normalization, detection and / or identification of conjugated molecules. As taught in the present application, the present invention can be used for various types of molecules, including, for example, molecular conjugates, drug conjugates, such as antibody-drug conjugates (ADCs), etc. Background Art

[0003] The conjugation of molecules can be advantageous.For example, drug conjugates (such as antibody-drug conjugates (ADC)) are a growing class of therapies for the treatment of the following diseases: cancer (Polakis, Pharmacological reviews 68, 3-19 (2016)) and immune disorders (McPherson. Methods inmolecular biology 2078, 23-36 (2020)). The general structure of ADC contains a monoclonal antibody (mAb) connected to the drug via a breakable or non-breakable joint. For example, ADC can have humanized / human mAb, such as cytotoxins, steroids and antisense oligonucleotides, and the humanized / human mAb is connected to a bioactive payload via an unbreakable or breakable joint, such as an acid-labile linker, a protease-breakable linker or a disulfide bond joint. The joint can be covalently connected to mAb at the conjugation site via lysine coupling, cysteine ​​alkylation or enzymatic reaction. ADCs bind to their target cell surface antigen receptors, which enables targeted delivery of the drug to the target site and minimizes systemic toxic effects on healthy tissues, resulting in increased selectivity, improved efficacy and safety relative to alternative chemotherapeutic approaches or other first-generation mAbs (see, e.g., Dan et al., 2018, Pharmaceuticals (Basel) 11).

[0004] Complete conjugation is difficult to achieve. An incomplete conjugation process may result in free or non-conjugated drug, drug-linker, or drug-related impurities. Degradation products may occur over time in the formulation and in vivo. Characterization and quantification of ADCs are important for the safety, efficacy, and uniformity of ADCs. Summary of the invention

[0005] The present application provides improved (e.g., more accurate, reliable, sensitive, etc.) materials and methods for the efficient identification and quantification of molecular conjugates, including therapeutic conjugates (e.g., drug conjugates). The present invention teaches, for example, materials and methods for such improved characterization.

[0006] The present application particularly relates to a method for analyzing a conjugate comprising a drug covalently linked to a polypeptide by using a Tandem Mass Tag (TMT).

[0007] In one aspect, the present application relates to a method of analyzing a conjugate comprising a drug covalently linked to a polypeptide, the method comprising:

[0008] (i) contacting a sample comprising the conjugate with a first tandem mass tag (TMT), thereby labeling a polypeptide of the conjugate with the first TMT;

[0009] (ii) digesting the polypeptide of the conjugate labeled with the first TMT to produce a first mixture comprising one or more unlabeled peptides and one or more peptides labeled with the first TMT;

[0010] (iii) contacting the first mixture with a second tandem mass tag (TMT) to obtain a second mixture comprising one or more peptides labeled with at least one of the first TMT and the second TMT, wherein the first TMT and the second TMT do not have the same reporter ion mass;

[0011] (iv) subjecting the second mixture to liquid chromatography (LC) to produce an eluent for LC; and

[0012] (v) subjecting the eluate to tandem mass spectrometry to obtain a mass spectrum of the peptide comprising at least one reporter ion of the first TMT and the second TMT; and

[0013] (vi) detecting a mass-to-charge ratio (m / z) associated with the at least one reporter ion, thereby analyzing the conjugate in the sample.

[0014] In another aspect, a sample comprising a conjugate of a drug covalently linked to a polypeptide is analyzed together with a control sample comprising a polypeptide not covalently linked to a drug. The method comprises:

[0015] (i) contacting a sample comprising the conjugate with a first tandem mass tag (TMT), thereby labeling a polypeptide of the conjugate with the first TMT;

[0016] (ii) digesting the polypeptide of the conjugate labeled with the first TMT to produce a first mixture comprising one or more unlabeled peptides and one or more peptides labeled with the first TMT;

[0017] (iii) contacting the first mixture with a second tandem mass tag (TMT) to obtain a second mixture comprising one or more peptides labeled with at least one of the first TMT and the second TMT, optionally one or more unlabeled peptides;

[0018] (iv) contacting a control sample comprising a polypeptide not covalently linked to the drug with a third TMT, thereby labeling the polypeptide not covalently linked to the third TMT;

[0019] (v) digesting the polypeptide not covalently linked to the drug and labeled with the third TMT to produce a third mixture comprising one or more unlabeled peptides and one or more peptides labeled with the third TMT;

[0020] (vi) contacting the third mixture with a fourth tandem mass tag (TMT), thereby obtaining a fourth mixture comprising one or more peptides labeled with at least one of the third TMT and the fourth TMT, optionally one or more unlabeled peptides;

[0021] (vii) combining the second mixture with the fourth mixture and subjecting the combination to liquid chromatography (LC) to produce an eluent for LC; and

[0022] (viii) subjecting the eluate to tandem mass spectrometry to obtain a mass spectrum of the peptide comprising at least one reporter ion of the first TMT, the second TMT, the third TMT, and the fourth TMT; and

[0023] (ix) detecting a mass-to-charge ratio associated with the at least one reporter ion, thereby analyzing the conjugate in the sample,

[0024] wherein none of the first TMT, the second TMT, the third TMT and the fourth TMT have the same reporter ion mass, the first TMT and the third TMT are selected from a first isobaric group of TMTs, the second TMT and the fourth TMT are selected from a second isobaric group of TMTs, and the first isobaric group of TMTs reacts with an unconjugated amino acid residue capable of forming a covalent bond with a drug, and the second isobaric group of TMTs reacts with a lysine or a free amine at the N-terminus of the peptide.

[0025] In one aspect, the method of the present application is used to determine the occupancy ratio of the conjugation site in the conjugate. For example, the individual site occupancy of the conjugate can be determined by: 1) using the method of the present application to obtain a mass spectrum of a peptide labeled with both the first TMT and the second TMT and a peptide labeled with both the third TMT and the fourth TMT, wherein the mass spectrum includes a reporter ion of the first TMT, a reporter ion of the third TMT, a reporter ion of the second TMT, and a reporter ion of the fourth TMT; 2) detecting the mass-to-charge ratio (m / z) associated with the reporter ion in the mass spectrum; and 3) based on the intensity of the reporter ion of the first TMT in the mass spectrum and the intensity of the reporter ion of the second TMT, or the intensity of the reporter ion of the second TMT and the intensity of the reporter ion of the fourth TMT, determine the occupancy ratio of the conjugation site in the conjugate, preferably determined by the following equation:

[0026] (Intensity of the reporter ion of the third TMT - Intensity of the reporter ion of the first TMT) / intensity of the reporter ion of the third TMT, or

[0027] (Intensity of the reporter ion of the fourth TMT−Intensity of the reporter ion of the second TMT) / Intensity of the reporter ion of the fourth TMT.

[0028] In certain embodiments, the occupancy ratio of the conjugation sites in the conjugate is determined at various time points, wherein additional TMT is used to label the polypeptide at different time points.

[0029] In another embodiment, the method of the present application is used to normalize the conjugate sample with a control sample. For example, the conjugate sample is normalized with a control sample by a method comprising: 1) using the method according to claim 2 to obtain a mass spectrum of a peptide labeled only with a second TMT and a peptide labeled only with a fourth TMT, wherein the mass spectrum includes a reporter ion of the second TMT and a reporter ion of the fourth TMT, but does not include a reporter ion of the first TMT or the third TMT; 2) detecting the mass-to-charge ratio (m / z) associated with the reporter ion; and 3) normalizing the sample with a control sample by the ratio of the intensity of the reporter ion of the second TMT to the intensity of the reporter ion of the fourth TMT.

[0030] In another embodiment, the method of the present application is used to locate the drug conjugation site, for example, the site where the drug is covalently bound to the polypeptide in the conjugate. For example, the drug conjugation site can be located by a method including the following: 1) using the method of the present application to obtain a mass spectrum of a peptide labeled only with the second TMT, wherein the mass spectrum includes only the reporter ion of the second TMT, but does not include the reporter ion of the first TMT, the third TMT, or the fourth TMT; and 2) triggering a second tandem mass spectrometry analysis on a peptide labeled only with the reporter ion of the second TMT, thereby locating the drug conjugation site. In certain embodiments, the peptide labeled with the reporter ion of the second TMT obtained after digestion with the second mixture is completely conjugated to the drug, for example, all amino acid residues on the peptide that can be conjugated to the drug have been covalently linked to the drug. When the peptide contains only one amino acid residue (e.g., Cys or Lys) that can form a covalent bond with the drug, the peptide can be conjugated to only one drug. When the peptide contains more than one amino acid residue (e.g., Cys or Lys) that can form a covalent bond with the drug, the peptide can also be conjugated to more than one drug.

[0031] In certain embodiments, the method for locating the drug conjugation site in the conjugate comprises: 1) using the method of the present application to obtain a mass spectrum of a peptide labeled only with the first TMT and the second TMT, wherein the mass spectrum includes only the reporter ions of the first TMT and the second TMT, but does not include the reporter ions of the third TMT or the fourth TMT; and 2) triggering a second tandem mass spectrometry analysis on the peptide labeled only with the first TMT and the second TMT, thereby locating the drug conjugation site. In certain embodiments, the peptide labeled only with the first TMT and the second TMT obtained after digestion of the second mixture is not completely conjugated to the drug, for example, only some but not all amino acid residues that can be conjugated to the drug have been covalently linked to the drug.

[0032] In one embodiment of the present application, the conjugate is a drug-antibody conjugate (ADC), more preferably the ADC comprises a drug covalently linked to one or more cysteine ​​(Cys) or lysine (Lys) residues of a monoclonal antibody.

[0033] As will be appreciated by those of ordinary skill in the art, in view of the present disclosure, various suitable tandem mass spectrometry can be used in the methods of the present application.See, for example, Friese et al., MAbs 10, 335-345 (2018), a review of tandem mass spectrometry, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, high energy collision induced dissociation tandem mass spectrometry (HCD-MS2) is used to obtain a mass spectrum of a peptide comprising at least one reporter ion of the first TMT, the second TMT, the third TMT, and the fourth TMT. In other embodiments, after one or more TMT reporter ions are detected from the peptide analyzed by HCD-MS2, additional dissociation analysis is triggered to additionally characterize the peptide, for example, to locate the drug conjugation site in the peptide. Additional dissociation can be performed by a second tandem mass spectrometry analysis. Examples of the second tandem mass spectrometry that can be used for such methods include, but are not limited to, electron transfer dissociation tandem mass spectrometry (ETD-MS2) or electron capture dissociation tandem mass spectrometry (ECD-MS2).

[0034] In other embodiments, a higher trigger intensity threshold and / or a narrow separation window are used to improve triggering of the second tandem mass spectrometer.In certain embodiments, simultaneous precursor selection with tribrid technology is applied to tandem mass spectrometry to improve specificity and accuracy of detection and quantification.

[0035] Any suitable TMT can be used according to the disclosure in the present application. For example, see Bachor et al., Molecules 2019, 24, 701, a review of TMT, the contents of which are incorporated herein by reference in their entirety. In some embodiments of the present application, the first isobaric group of TMT includes two or more TMTs reacted to reduced cysteine. Preferably, the first isobaric group of TMT includes two, three, four, five, six or more TMTs reacted to reduced cysteine. In some embodiments, the first isobaric group includes two, three, four, five, six or more isobaric isomers (e.g., same mass and structure), which are activated by iodoacetyl groups for covalent, irreversible labeling of sulfhydryl (— SH) ​​groups. In some embodiments, the first isobaric group includes two, three, four, five or six isobaric isomers of the IodoTMTsixplex isobaric labeling reagent group available from ThermoFisher Scientific (Catalog No. 90101).

[0036] In certain embodiments, each of the first TMT and the third TMT includes a mass reporter, a mass normalizer, and a cysteine ​​reactive group covalently linked to each other. Thus, each of the first TMT and the third TMT labels the reduced cysteine ​​in the polypeptide of the conjugate and can be used to analyze the conjugate containing a drug covalently linked to one or more cysteine ​​residues of the polypeptide. Preferably, each of the first TMT and the third TMT is selected from the isobaric group of IodoTMTsixplex.

[0037] In some embodiments of the present application, the second isobaric group of TMT includes two, three, four, five, six or more primary amines at the N-terminus of the TMT or peptide that react with lysine. In one embodiment of the present application, the second isobaric group of TMT includes two, three, four, five, six, seven, eight, nine, ten or more isobaric compounds with amine-reactive NHS ester groups, spacer arms and mass reporters. For example, the second isobaric group may include TMT10plex TM (ThermoFisher, catalog number 90110), TMTsixplex TM (ThermoFisher, Cat. No. 90061) or TMTpro TM 16plex (ThermoFisher, Cat. No. A44520) labeling reagent sets of two, three, four, five, six or more isobaric isomers.

[0038] In certain embodiments, each of the second TMT and the fourth TMT includes a mass reporter, a mass normalizer, and an amine reactive group covalently linked to each other. Thus, each of the second TMT and the fourth TMT labels a lysine or N-terminus of a polypeptide of the conjugate and can be used together with the first TMT and the third TMT to analyze a conjugate containing a drug covalently linked to one or more cysteine ​​residues of the polypeptide. Preferably, each of the second TMT and the fourth TMT is selected from an isobaric group of TMT6plex, TMT10plex, or TMT pro16plex.

[0039] In some embodiments, the first isobaric group and the second isobaric group of TMTs each independently include two, three, four, five, six or more TMTs that react with lysine or a primary amine at the N-terminus of the peptide. In certain embodiments, each of the first TMT, the second TMT, the third TMT and the fourth TMT includes a mass reporter, a mass normalizer and an amine reactive group covalently linked to each other. TMTs can be used to analyze conjugates containing drugs covalently linked to one or more lysine residues of a polypeptide.

[0040] In some embodiments, multiple samples comprising one or more conjugates are analyzed together. In certain embodiments, the drug conjugation site in the conjugate is characterized by a mass spectral barcode comprising (2n+2) reporter ions, and the conjugate is normalized by a mass spectral barcode comprising n+1 reporter ions, and n is the number of samples analyzed by the method.

[0041] Other aspects of the present application relate to a system for carrying out any of the methods according to claims 1 to 23.

[0042] Yet another aspect of the present application relates to a composition comprising a peptide labeled with at least one of a first TMT and a second TMT, optionally a mixture of one or more unlabeled peptides, wherein the first TMT and the second TMT do not have the same reporter ion mass, and the mixture of peptides comprises at least one peptide conjugated to a drug and labeled with at least one of the first TMT and the second TMT.

[0043] Other aspects, features and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims, which, as one of ordinary skill in the art will understand, are exemplary and non-limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The foregoing summary and the following detailed description of preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0045] This patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication in color will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0046] Unless otherwise indicated, in all figures herein, the reporter ions of IdoTMT are represented by As shown below, the reporter ion of TMT6 is The peptides analyzed are shown below. denoted below, and drugs are denoted by ■ below.

[0047] Figure 1A-Figure 1D The structure of an exemplary tandem mass tag (TMT) that can be used to label cysteine ​​in a polypeptide is shown: Figure 1A -IodoTMTsixplex group, Figure 1B -TMTsixplex group, Figure 1C -TMT10plex group, Figure 1D-TMTpro16plex, including the chemical structure of TMT and the positions of 13C and 15N stable isotopes (*);

[0048] Figure 2 The generation of unique barcodes for sample characterization according to embodiments of the present application is shown, for example, for quantification (e.g., drug occupancy), normalization (e.g., normalization of mimetic versus conjugate), and based on tandem mass spectrometry (MS 2 ) analyzing the intensity and combination of the detected predetermined TMT reporters (patterns) to detect (e.g., locate drug conjugation sites) the sample;

[0049] Figure 3A An embodiment of the present application for generating MS barcodes via triple fusion by sequential double TMT labeling of a conjugate conjugated to a drug (D) (such as an antibody or a fragment thereof conjugated to a drug), and a mimetic (such as an antibody or a fragment thereof not conjugated to a drug) using an analytical scheme that can be quantified, normalized, and localized is shown;

[0050] Figure 3B A process for quantifying drug conjugates using dual TMT according to an embodiment of the present application is shown, wherein one drug (D) is conjugated to a peptide obtained by trypsin digestion;

[0051] Figure 3C 1 shows a process for quantifying drug conjugates using dual TMT according to an embodiment of the present application, wherein up to two drugs (D1 and D2) are conjugated to a peptide obtained by trypsin digestion;

[0052] Figure 3D shows the use of dual TMT to quantify drug conjugates according to an embodiment of the present application, where up to three drugs (D1, D2, and D3) are conjugated to peptides obtained by trypsin digestion;

[0053] Figure 4 Several exemplary experimental ADC systems analyzed by the methods of the present invention are shown, including NIST monoclonal antibody (mAb) cysteine ​​conjugated with iodoacetamide (IAA) and biotin-PEO acetamide conjugate formed by conjugation with biotin PEO iodoacetamide, NIST mAb or HSA peptide containing 1-3 cysteines conjugated with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM-3), and ADC standard MSQC8 (dansyl fluorophore LC-SMCC crosslinker) from Sigma;

[0054] Figure 5A and Figure 5B Each shows the use of dual TMT by performing HCD-MS 2 (Although other MS2 , such as ETD-MS 2 ) to quantify site-specific ADC on NIST mAb, for example, using iodoTMT (io, labeling Cys residues or C) or TMT (tm, labeling Lys residues or N-terminal amine) reporter ion intensity to quantify the occupancy of the drug on a specific residue, and applied to the formula for calculating occupancy %, an embodiment of the present application, wherein the ADC is NIST mAb-biotin-PEO acetamide;

[0055] Figure 5C and Figure 5D HCD-MS showed 2 Corresponding MS of sequenced peptides 2 Spectra (with all backbone product ions);

[0056] Fig. 6A and Figure 6B shows the use of TMT to perform HCD-MS 2 (Although other MS 2 , such as ETD-MS 2 ) to normalize the mixing ratio of NIST mAb across reaction conditions with non-cysteine ​​peptides, and to correct for mixing or digestion bias between samples (Sample) containing ADC (NIST mAb-biotin-PEO acetamide) and unconjugated samples (Mock) using the normalized ratio shown in the formula;

[0057] Figure 7 The method according to the embodiment of the present application and the use of ETD-MS are shown. 2 Analysis of backbone fragmentation of peptides containing the drug as well as fragments corresponding to the fragmentation of the biotin-PEO-acetamide molecule by HCD-MS 2 Product ion mass spectra: Note that small molecule conjugates are prone to fragmentation, resulting in complex spectra, and that the fragment masses from the drug are specific to each drug;

[0058] Fig. 8A TMT-130 reporter triggering for the NIST triple fusion workflow is shown;

[0059] Figure 8B Figure 2 shows the use of TMT-130 characteristic ions (barcodes) to trigger ETD-MS. 2 to locate the conjugated drug using the method according to the embodiments of the present application and the observed total ion chromatogram (TIC);

[0060] Fig. 9A and Fig. 9BFigure 2 shows the localization of biotin PEO acetamide via mass triggered ring of TMT-130 using a method according to an embodiment of the present application: from HCD-MS 2 The TMT-130 is unique and triggering is independent of the type of ADC and ETD-MS 2 Localization of drug without fragmentation; D = Biotin-PEO acetamide

[0061] Fig. 9C and Fig.9D Triggered mass detection of a peptide corresponding to biotin PEO acetamide conjugated at Cys-23 is shown, where TMT130 is less abundant compared to the iminium ion generated by biotin PEO acetamide;

[0062] Fig. 10A and Fig. 10B A method for improving the specificity of the trigger loop TMT-130 according to an embodiment of the present application is shown: co-isolation and co-fragmentation of peptides leads to loss of specificity of TMT-130, and a higher trigger intensity threshold or a narrow isolation window leads to improved mass triggering;

[0063] Fig.11 1. It shows how the Sample Prep Station (SPS-3) is used for an isobaric labeling experiment according to an embodiment of the present application, wherein precursor ions are transferred from an Ion Routing Multipole (IRM) to an ion trap (IT), and simultaneous precursor selection (SPS) is performed in the IT;

[0064] Figure 12A-12C The improved specificity and accuracy of TMT quantification using simultaneous precursor selection according to embodiments of the present application are shown: Fig. 12A The MS2 TMT reporter used for simultaneous precursor ion selection (top 5 fragments) is shown; Fig. 12B SPS-MS3 was shown to improve quantitative sensitivity and accuracy when interferents were co-isolated; and Fig. 12C Reporter quantification is shown where ADC is SigmaMAb ADC mimetic (MSQC8) - a human universal mAb standard conjugated to a dansyl fluorophore, and mimetic is a human universal mAb standard not conjugated to drug (MSQC4, IgG1 mAb);

[0065] FIG. 13A to FIG. 13FShown are experiments and results of dual TMT to quantify site-specific ADC (dansyl fluorophore) on MSQC8 (mAb antibody-drug conjugate mimetic) using methods according to embodiments of the present application, including MS-based barcodes for site-specific conjugation occupancy;

[0066] Figure 14A-Figure 14C It was shown that the MSQC8 broad site ADC quantification using the methods according to the embodiments of the present application correlated with the following known methods: MSQC8 light chain conjugation based on triple fusion quantification at Cys-218 = 60% correlated with a light chain SLIM-IMS DAR0 / DAR1 ratio of 1:2, and occupancy of Cys-266 (max) = 60% and Cys-372 = 15% were assessed, while all other cysteines showed 0% conjugation;

[0067] Fig.15 Identification of dansyl fluorophores in MSQC8 using methods according to embodiments of the present application is shown, which shows that small molecule conjugates are prone to fragmentation, resulting in complex spectra, and that the fragment masses from the drugs are specific to each drug;

[0068] Fig.16A and Fig. 16B shows TMT analyzing the reaction time course using the method according to an embodiment of the present application;

[0069] Fig. 16C shows a triple fusion workflow applied to monitor the response of multiple drugs (D1, D2, D3, D4 and D5) in a multiplexed manner according to an embodiment of the present application;

[0070] Fig.16D Mass triggering via a single TMT reporter ion specific for each drug molecule is shown;

[0071] Fig.17A and Fig. 17B It was shown that the results from TMT analysis correlated with the fluorescence quantification results at high occupancy;

[0072] Fig.18A Described are multiplexing schemes using TMT in various combinations for triple fusion analysis of up to 4 ADC samples using dual TMT according to embodiments of the present application;

[0073] Fig.18B Shown is a multiplexed analysis of 4 ADC samples with 4 different site-specific ADC occupancies according to an embodiment of the present application; TMT using TMT10plex reagent;

[0074] Figure 19A-Figure 19CThe results of using the method of the present invention to analyze at high throughput, for example, analyzing 4 ADCs, which achieve dual TMT quantification of four ADC samples in a single run;

[0075] Fig. 20A Using non-cysteine ​​peptide sequences, now with five TMTs on MS2-HCD 10 The barcodes of the reporter ions (one mock (B) and four conjugate samples (1) to (4)) are used to correct for sample concentrations in multiplexed experiments: The normalization factor for the i-th sample is given by Equation 2, and the corrected occupancy for each ADC sample can be obtained by Equation 3; and

[0076] Fig. 20B Normalized occupancies obtained for four samples in a single acquisition are shown. DETAILED DESCRIPTION

[0077] 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 the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification. All patents, published patent applications and publications cited herein are incorporated herein by reference as if fully set forth herein.

[0078] The discussion of documents, acts, materials, devices, articles and the like included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0079] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0080] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include ± 10% of the value. For example, an amount of about 50ppm or less includes 45ppm or less to 55ppm or less. Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0081] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "include" and "contain" will be understood to imply the inclusion of the stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes", or sometimes when used herein, with the term "having".

[0082] When used herein, "consisting of excludes any element, step, or ingredient not specified in the elements of the claim. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of one aspect or embodiment of the present application, any of the foregoing terms "comprising," "containing," "including," and "having" may be replaced with the terms "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0083] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0084] As used herein, "MS / MS" or "MS 2 " or "MS2" refers to tandem mass spectrometry. Tandem mass spectrometry is a technique in instrumental analysis in which two or more mass analyzers are linked together using additional reaction steps to increase their ability to analyze a sample. The tandem use of mass analysis can be performed with the reaction steps separated in space (spatial tandem) and / or with the reaction steps separated in time (temporal tandem). A common use of tandem mass spectrometry is the analysis of biomolecules such as proteins, peptides, organic and inorganic molecules, lipids, metabolites, and oligonucleotides.

[0085] As used herein, "reporter ion" or "diagnostic ion" refers to the characteristic product ion of a labeled peptide containing an N-terminal tag or label observed in an ETD mass spectrum. Typically, it is the most dominant product ion in the mass spectrum and is used to trigger subsequent MS / MS events to further sequence the labeled peptide.

[0086] As used herein, "3-in-1 technology" refers to a technology that uses a hybrid mass spectrometer with more than a single type of mass analyzer. It enables multiplexing of TMT samples to perform tandem mass spectrometry experiments with great flexibility. The 3-in-1 technology can be used to characterize challenging samples including low-abundance peptides in complex matrices, determine the location of intact proteins and post-translational isoforms, resolution of isobaric metabolites, and protein structural characterization using chemical cross-linking.

[0087] As used herein, "orbital trap" or "OT" refers to an ion trap mass analyzer consisting of two outer electrodes and a central electrode. This setup enables the orbital trap to be used as both an analyzer and a detector. Ions entering the orbital trap are trapped and oscillate around the central electrode and between the two outer electrodes. Different ions oscillate at different frequencies, which causes them to separate. The oscillation frequency caused by the ions on the outer electrodes is measured, and the mass spectrum of the ions is acquired using image current detection.

[0088] As used herein, "isobaric" means having the same nominal molecular weight or formula weight. Preferably, the isobaric TMTs of the present invention suitable for use in this application have the same mass and structure, and they are also referred to as isotopes.

[0089] As used herein, "ion" refers to a molecule having a net charge due to the loss or gain of one or more electrons. Examples of ions may be a, b or y type product ions, which are the result of amide bonds cleaved along the protein backbone by collision induced dissociation (CID).

[0090] As used herein, "sequence ion" refers to an ion (or ions) corresponding to the product of a particular peptide cleaved at a given peptide bond.

[0091] As used herein, "immonium ion" refers to an ion (or ions) corresponding to an internal fragment of a peptide having a single side chain formed by a combination of -type or y-type cleavages.

[0092] As used herein, "reporter ion" refers to an ion fragmented from an isobaric labeled peptide by methods known in the art (e.g., MS 2 ).

[0093] As used herein, "isotopologue" refers to a molecule that differs from its parent molecule in that at least one atom has a different number of neutrons.

[0094] As used herein, "multipole" refers to an ion guide composed of metal rods to transport ions through a vacuum system.

[0095] As used herein, "conjugate" refers to a protein or peptide covalently linked to one or more heterologous molecules. Examples of proteins or peptides that can be covalently linked to one or more heterologous molecules include, but are not limited to, therapeutic peptides or proteins, antibodies, or fragments thereof. Examples of heterologous molecules that can be covalently linked to proteins or peptides include, but are not limited to, one or more small molecule compounds, labels, and the like.

[0096] As used herein, "peptide" refers to an amino acid-based polymer, typically composed of some combination of the twenty common naturally occurring amino acids, but may also contain or consist entirely of non-natural amino acid monomer residues. It may include a linear amino acid polymer configuration, or may include a cyclic peptide or a branched chain ketone, or any combination of all three configurations. The peptide may also have any combination of naturally occurring modifications (e.g., phosphorylation or glycosylation) or non-naturally occurring modifications (e.g., carboxamidomethylation).

[0097] "Antibodies" shall include, but are not limited to (a) immunoglobulin molecules comprising two heavy chains and two light chains and recognizing antigens; (b) polyclonal or monoclonal immunoglobulin molecules; and (c) monovalent or divalent fragments thereof. Immunoglobulin molecules may be derived from any commonly known class, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. IgG subclasses are well known to those skilled in the art, and include but are not limited to human IgG 1, IgG 2, IgG 3, and IgG 4. Antibodies may be naturally occurring and non-naturally occurring. In addition, antibodies include multispecific antibodies, such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, fully synthetic antibodies, single-chain antibodies, and fragments thereof. Antibodies may be human or non-human. Antibody fragments include but are not limited to Fab fragments, Fv fragments, and other antigen binding fragments.

[0098] As used herein, "tandem mass spectrometry tag" or "TMT" refers to a chemical label that can be used for quantitative and molecular identification based on mass spectrometry (MS). Any molecule with free thiol or primary amine or polysaccharide can be labeled. For example, the molecule can be a protein or a peptide. TMT tags usually contain at least three regions, for example, a mass reporter region, a mass normalization or balance region, and a reactive group. The reporter mass can be incremented to produce a unique mass, and the balance mass is used to compensate for the total mass of the reagent to prepare a series of reagents with the same overall mass but different reporter masses. The reactive group promotes the addition reaction of amines, thiols, oxoniums or other functional groups. It can be, for example, free thiol or reduced cysteine ​​reactive, primary amine or lysine reactive, or aminooxy reactive. Optionally, TMT can also contain one or more cleavable linker regions.

[0099] The isobaric group of TMTs enables parallel identification and multiple quantification of proteins in different samples using tandem mass spectrometry. An isobaric group of n TMTs can contain TMTs and TMTs with n-1 isotope substitutions. The chemical structure of all tags within the isobaric group of TMTs is the same, but each chemical structure contains substituted isotopes at various positions, so that the mass reporter and mass normalization regions have different molecular masses in each tag, while the TMTs have the same total molecular weight (isobaric) and structure, so that molecules labeled with different tags are indistinguishable during chromatographic or electrophoretic separation and in a single MS mode. For example, each isobaric reagent can contain a different number of heavy isotopes in the mass reporter region, which results in a unique reporter mass for sample identification and relative quantification during tandem MS / MS. After fragmentation in the MS / MS mode, sequence information is obtained from the fragmentation of the peptide back bone, and quantitative data is obtained from the fragmentation of the tag, thereby generating a mass reporter ion. Each TMT within the isobaric group produces a unique reporter mass on the MS / MS mass spectrum.

[0100] TMT labels, such as those commercially available TMT labels, can multiplex samples (such as 6-way, 10-way or more) and are reactive to cysteine ​​or amines, respectively. The position of the heavy isotope is shifted between the reporter group and the spacer, and the total mass and chemical structure of each label can remain the same (isobaric). For example, the cysteine-reactive IodoTMTsixplex reagent and the amine-reactive reagent each have six identical reporter ion masses. However, each reporter ion is unique (m / z) for the mass-to-charge ratio, or reporter mass, also referred to as the reporter ion mass. For example, the nominal reporter mass of 6TMT within the isobaric TMT6 setting range is 126-131Da (Dalton).

[0101] "IodoTMT sixplex", also known as "CysTMT", "IodoTMT", "IodoTMT6" or "IodoTMT 6 The isobaric labeling reagent group "refers to six isobaric groups of TMT, which are activated by iodoacetyl groups for covalent, irreversible labeling of sulfhydryl (—SH) groups and have Figure 1A The chemical structure shown. TMT contains a 13C and / or 15N isotope ( Figure 1A-Figure 1DThe characteristic reporter groups (shown as * in the figure). The IodoTMT sixplex isobaric labeling reagent set is commercially available from ThermoFisher Scientific (Cat. No. 90101). The IodoTMT reagent reacts specifically with reduced cysteine ​​(Cys) in peptides and proteins. The IodoTMT reagent can be differentiated by mass spectrometry (MS) in cultured cells grown or treated with different conditions, enabling quantification of the relative abundance of cysteine ​​modifications, such as S-nitrosylation, oxidation, and disulfide bonds.

[0102] "TMTsixplex", also referred to herein as "TMT6", "TMT 6 The isobaric labeling reagent set "TMT6plex" refers to six isobaric groups of TMT, which are NHS activated to covalently and irreversibly label primary amine (-NH2) groups and have Figure 1B The chemical structure shown in . The TMTsixplex isobaric labeling reagent set is commercially available from ThermoFisher Scientific (Cat. No. 90061). The reagent labels all peptides prepared from a cell or tissue sample to analyze up to six samples in a single MS analysis. According to ThermoFisher Scientific, the TMT6 reagent is optimized for use with high-resolution Thermo Scientific MS / MS platforms such as Q Exactive, Orbitrap elite TM and Orbitrap fusion TM tribrid TM Orbitraplumos TM Instrument) and Proteome discoverer TM Versions 1.0 and above fully support data analysis.

[0103] "TMT10plex" (also referred to herein as "TMT10", "TMT 10 " or "TMT10plex") refers to an isobaric labeling reagent set each having an amine-reactive NHS ester group, a spacer arm and a mass reporter and having Figure 1CIsobaric groups of ten TMTs of the chemical structure shown. Amine-reactive TMT10plex can multiplex up to 10 individual samples (10-plex). TMT10plex isobaric labeling reagent sets are commercially available from ThermoFisher Scientific (Catalog No. 90110). It is important to note that the 10-plex reagents still have six nominal masses (126-131 Da), however, four of the six reporter masses (127-130 Da) each have two unique reporter ion masses with a difference of 6.32 mDa (milliDaltons), where C 12 、N 15 The atomic pair is C 13 、N 14 Replacement. High-resolution mass spectrometry enables accurate relative quantification of these reporter ion masses and baseline resolution of their isotopologues. The reagent set enables parallel labeling of up to ten different peptide samples from cells or tissues, which can then be combined for analysis. For each sample, the unique reporter mass (i.e., TMT10 126-131Da) in the low-mass region of the high-resolution MS / MS spectrum can be used to measure relative protein expression levels during peptide fragmentation and tandem mass spectrometry. According to ThermoFisher Scientific, the TMT10 reagent is optimized for use with high-resolution Thermo Scientific MS / MS platforms such as Q Exactive, Orbitrap Elite TM and Orbitrap Fusion TM Orbitrap Elite TM Tribrid TM instrument) and by Proteome Discoverer TM 1.4 Fully supports data analysis.

[0104] "TMTpro 16plex" (also referred to herein as "TMT16", "TMT 16 ","TMTpro" or "TMTpro16plex") isobaric labeling reagent set refers to Figure 1DThe isobaric groups of 16 amine reactive NHS ester activation reagents of the chemical structure shown. Each of the 16 TMTs has a group that reacts with lysine or primary amines at the N-terminus. TMTpro 16plex is commercially available from ThermoFisher Scientific (Catalog No. A44520). According to ThermoFisher Scientific, TMTpro labeling reagents are next-generation tandem mass spectrometry tags that are optimized for use with high-resolution Thermo Scientific MS / MS platforms (such as Q Exactive and Orbitrap Fusion Tribrid instrument series, including Orbitrap EclipseTribrid and Orbitrap explororis 480 mass spectrometers), and Proteome Discoverer 2.3 fully supports data analysis.

[0105] The method of the present invention can adopt a group of 6 reporters (TMT-6plex), a group of 10 reporters (TMT-10plex), a group of 11 reporters (TMT-11plex) and up to 16 reporters (TMT-16plex) of such TMT. According to the present disclosure, other examples of TMT can also be used in the present invention. Specifically, TMT16-plex and future n-plex reagents (where n>16) will increase the number of samples that can be labeled with dual TMT labeling. TMT can be prepared using methods known in the art or obtained from commercial sources such as ThermoFisher Scientific. However, when two TMTs are used in series, they cannot have the same reporter ion mass. For example, since IodoTMT and TMT6 have the same series of reporter masses, when using an isobaric group of IodoTMT and TMT6 for dual labeling, the selected IodoTMT and TMT must not have the same reporter mass when used for dual labeling. Similarly, when more than two TMTs are employed in the same assay, such as a TMT for analyzing a sample and a mock together or a TMT for analyzing multiple samples together, each of the TMTs used in the assay must have a unique reporter mass, or none of the TMTs used in the same assay can have the same reporter mass.

[0106] The drug-antibody ratio (DAR) is an important indicator for determining ADC efficiency, safety and selectivity. Chromatographic methods, such as size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC), can be used to obtain DAR values ​​and characterize ADC. However, these methods have significant disadvantages, such as they usually show low throughput due to long separation and column regeneration time. In addition, some necessary mobile phases prevent online coupling to mass spectrometry (MS). The method based on native MS maintains non-covalent interactions, and therefore information about arrays of possible drug conjugate species can be obtained.

[0107] Another method is ion mobility spectrometry coupled to mass spectrometry (IMS-MS). IMS-MS obtains both structural and mass information in a single measurement. It has demonstrated consistency between IMS-MS and MS in DAR characterization. Therefore, IMS-MS can be used to study drug loading distribution of ADCs and characterize mAbs.

[0108] However, conventional methods for quantifying and identifying drug conjugates have the disadvantages of being haphazard, having low sensitivity, insufficient selectivity, and / or relatively long analysis times or high costs. For example, native MS, chromatography, and IMS-MS methods provide little information about site-specific drug conjugation of mAbs and whether the occupancy and position of the drug bound to the antibody alters the selectivity or efficacy of the ADC. The identification of peptides with intact drug molecules is challenging due to the size of small molecules and linkers.

[0109] The ratio can be obtained by normalizing the conjugated peptide intensity with the summed intensity of the unmodified peptide counterpart and the conjugated peptide to quantify the conjugated peptide. The Ad hoc ratio contributes to the relative estimation of the conjugate level across samples of the site of interest. A stoichiometric method can also be used, wherein the occupancy of the modification is indirectly determined by chemical removal of the modification or using a stable isotope-labeled synthetic peptide and a stable isotope-labeled cell line (see, for example, Wu et al., "Nature Methods" (Nature methods) 8,677-683 (2011); Lim et al., "Journal of proteome research" (Journal of proteome research) 16,4217-4226 (2017)). Isobaric labels specific for primary amines have been used to study the occupancy of model NIST mAbs to show the utility of stoichiometric methods based on isobaric labels (Hill et al., SciRep 8,17680 (2018)). Despite this, chemometric-based quantitative methods have not yet been integrated into drug pipelines for quantitative or ADC analysis due to, for example, complex and challenging sample preparation, detailed analytical protocols required to obtain compositional differences based on drug conjugation, etc. (Janin-Bussat et al., Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 981-982, 9-13 (2015); Le et al., Anal Chem 84, 7479-7486 (2012)).

[0110] The isobaric group of TMT can be used to label and study a group of drug polypeptide conjugates, such as ADC. Using high-resolution mass spectrometry, the baseline resolution of these reporter ion masses and isotopologues can be used for accurate relative quantification. In one aspect of the present application, TMT is used in tandem mass spectrometry (MS / MS) of samples, especially drug conjugates. 2 ) analysis is used to characterize samples based on their mass spectrometry barcodes.

[0111] A mass spectrum is a histogram obtained using a mass spectrometer. The mass spectrum of the analysis is usually plotted as a relationship between intensity (Y) and m / z (mass to charge ratio) ratio (X). The reporter ions of TMTs can be detected by their m / z ratio on the mass spectrum. The Y axis represents the signal intensity of the ion.

[0112] Signal intensity can be measured and expressed in different ways, depending on the instrument and software. For example, when using a counting detector, intensity is usually measured in counts per second (cps). When using analog detection electronics, intensity is usually measured in volts. In Fourier transform ion cyclotron resonance mass spectrometry and Orbitrap, the frequency domain signal (y-axis) is related to the power (approximately the square of the amplitude) of the signal sine wave (usually reduced to rms power). Some software calculates the report area instead of intensity. As used herein, with respect to mass spectrometry, the term "intensity" encompasses the signal intensity of ions measured by any method and expressed in any form. For example, the "intensity" of a reporter ion can include any other variation of the report intensity, report area, or the signal intensity of ions from mass spectrometry.

[0113] As used herein, a "mass spectral barcode," "MS barcode," "reporter ion mass barcode," "barcode," or "mass barcode" of a sample refers to a tandem mass spectrum (MS) containing information from a set of reporter ions. 2 ) and the measured value is unique to the sample. The unique barcode of the sample can be based on the 2 The intensity and / or presence (pattern) or absence of a set of TMT reporter ions detected.One or more barcodes can be used in a triple play analysis of a sample.

[0114] Figure 2 The use of IodoTMTsixplex and TMT6 is shown to multiplex 6 different cell lysates, where the proteins can be labeled prior to digestion or on cysteine ​​replacement peptides after digestion of each sample. Each of the six samples can be labeled with TMT, combined in an IodoTMT set and subjected to LC-MS together 2 Analyze to obtain the different barcodes of each sample in six samples.Barcode is defined by the intensity and pattern of the m / z of the reporter ion produced by TMT on tandem mass spectrometry.Specifically, each cell line marked as not distinguishable by mass produces the isobaric mass of the same peptide.However, after dissociation by collision (HCD) or via dissociation (ETD) based on electrons, a unique reporter ion is produced.It is important to note that HCD produces a series of charged fragments, in which the entire reporter is dissociated, and ETD produces the unique reporter ion mass of the second series.Similarly, TMT labeling can also be used in proteomics to compare 6,10,11 and 16 groups of reporters of different samples of any peptide, because each peptide has an N-terminal, and TMT reagents can mark N-terminal amines and / or lysines, thereby being connected to all peptides.

[0115] Therefore, the present application provides an improved method for accurately, sensitively and effectively identifying and quantifying drug conjugates. In one aspect of the present invention, a drug polypeptide conjugate (such as ADC) is labeled with a first TMT, such as a Cys-reactive IodoTMT and a second TMT, such as a TMT sequence labeled with Lys or a primary amine reaction at the N-terminus, and the labeled drug conjugate is subjected to tandem mass spectrometry. The sequential labeling scheme helps to interrogate drug conjugates. According to an embodiment of the present application, two parallel sample preparation arms can be used to label drug conjugates (conjugate samples) and unconjugated mimetics (reference or control samples). Both labels are conducive to encoding the unique reporter ion features that distinguish samples from mimetics or references. The reference sample generates a reference reporter ion channel, and the conjugate sample generates a sample reporter ion channel. Dissociate by HCD or generate unique reporter ions after dissociation based on electrons such as ETD. The number of channels in the reporter ion region, their ratios, and / or other mass spectra that perform details associated with TMT can be used to generate unique barcodes to characterize drug conjugates. For example, a barcode can include a set of different reporter ions and / or mass barcodes.

[0116] like Figure 3A As shown, the method of the present application can be used to quantify the occupancy of the drug in the conjugate, for example, by comparing the mass spectrum of the reporter ion intensity in the lower m / z region of the cysteine-containing peptide in the conjugate with the mass spectrum of the ion intensity in the mimetic control containing the polypeptide not conjugated to the drug. The method can also be used to normalize the mimetic control and the conjugate.

[0117] Another aspect of the application relates to a method for locating the conjugation site on a polypeptide, such as by using one or two TMT reporter ions to trigger additional dissociation steps to further characterize the peptide covalently attached to the drug. In one embodiment of the application, one or two TMT reporter ions are produced using a first tandem mass spectrometry analysis (such as HCD analysis). The ETD scan of the peptide is then triggered using reporter ions to obtain an ETD spectrum. The ETD spectrum is supplemented with the HCD spectrum for characterizing the conjugate, so that ETD provides sequence ions that contribute to the location of the residue and the drug conjugate. Alternatively, reporter ions are then used to trigger ECD scans to obtain ETD spectra to further analyze the peptide.

[0118] Figure 3A-3CShow wherein IodoTMT6 and TMT6 are used to mark the dual TMT workflow of mAb and its drug conjugate.Especially, IodoTMT6 is used for multiplexing 6 kinds of different samples (for example, cell lysate).In this case, protein can be marked before digestion or after digesting each sample, the replacement peptide of cysteine ​​is marked.The reporter of both IodoTMT6 and TMT6 reagent has the same nominal mass.Due to the reagent that the mass does not overlap, the peptide of isobaric labeling is produced.Sequential labeling is first carried out with the IodoTMT6 of mAb, followed by trypsin digestion and labeling of the peptide obtained by trypsin digestion with TMT6 marker.Peptides from conjugate samples and mimetics are mixed with equimolar ratios and subjected to LC-MS.The sequential labeling scheme described herein is a novel method for querying peptides, which helps to form mass spectrometry barcodes according to the embodiments of the application.

[0119] In certain embodiments, the workflow shown has two parallel sample preparation arms for drug conjugates and unconjugated mimetics. For example, both IodoTMT6 and TMT6 markers facilitate encoding of unique reporter ion features that distinguish samples from mimetics or references, such as reference samples generating reference reporter ion channels and conjugate samples generating sample reporter ion channels. Moreover, the number of channels in the reporter ion region and their ratios encode the occupancy and type of experimental measurements that can be obtained, and each experimental measurement has a different set of reporter ions or mass barcodes.

[0120] Therefore, one aspect of the present application relates to a composition comprising a peptide labeled with at least one of the first TMT and the second TMT, optionally a mixture of one or more unlabeled peptides, wherein the first TMT and the second TMT do not have the same reporter ion mass, and the mixture of peptides comprises at least one peptide conjugated to a drug and labeled with at least one of the first TMT and the second TMT. For example, the composition of the present application may include a mixture of peptides derived from a conjugate sample labeled with at least one of the first TMT and the second TMT, optionally one or more unlabeled peptides. The composition may also include a mixture of peptides derived from a conjugate sample described herein and a mixture of peptides derived from a simulation sample labeled with at least one of the third TMT and the fourth TMT, optionally one or more unlabeled peptides.

[0121] Figure 3A Drug conjugation to a single cysteine ​​is shown as seen in an alternative tryptic peptide, where IodoTMT labeling occurs at the free thiol of the mAb on the unconjugated portion prior to digestion. Sample preparation and LC-MS can remain the same regardless of the number of conjugation sites. Data-dependent MS 2 or data-dependent SPS-MS3 (Synchronous Precursor Selection and Triple Mass Spectrometry) and TMT reporter ion triggered ETD-MS can be performed in a seamless manner under the complete control of the instrument software for generating mass spectral barcodes. This novel automated 3-step method is referred to herein as "Triple Fusion", in which a specific combination of reporter ions is used to quantify, normalize, and detect drug polypeptide conjugates. In some embodiments of the present application, triple fusion is performed with dual TMT reporters to quantify drug occupancy, normalize drug conjugates in multiple samples, and detect or locate conjugation sites (e.g., by triggering additional MS2). Preferably, the drug polypeptide conjugate is an ADC.

[0122] refer to Figure 3A-3B , following the workflow depicted in the figure, all peptides carrying a single cysteine ​​conjugation site labeled with the IodoTMT labeling set produce four reporter ions (i.e., IodoTMT-126 and IodoTMT-127 and TMT-129 and TMT-130), while all non-cysteine ​​labeled peptides produce two reporter ions, TMT-129 and TMT-130, which can be used to correct for differences in sample mixing and normalization. The single reporter TMT-130 uniquely found in drug-conjugated peptides can be used to trigger additional ETD-MS 2 Scanning for site localization (see e.g. Figure 3B ). The TMT-130 reporter ion is independent of the drug conjugated to the peptide and is not dependent on the fragment of the drug. 2 Can be used for any type of drug conjugate or to screen libraries of drugs with unique reporter ions to easily identify and locate the drug.

[0123] Unlike peptides with a single cysteine, peptides with multiple cysteine ​​residues can have drugs covalently linked to one or more cysteine ​​residues. For example, the two hinge cysteines of IgG1 and IgG4 mAbs have the possibility of dual occupancy and single occupancy of any single cysteine. The four hinge cysteines on IgG2 (with IgG2-A, IgG2-B, and IgG2-A / B isotypes) even further increase the possible combination conjugation possibilities (Liu et al., MAbs 4, 17-23 (2012).

[0124] The method according to the embodiment of the present application can be used to study drug conjugates having one or more drugs conjugated to a polypeptide (such as an antibody). The one or more drugs can be within a single digested peptide. The drug molecules can be of the same type or different types. FIG. 3B to FIG. 3DShown is a study of drug conjugates containing = one, two or three drugs on a single digested peptide, where D1, D2 and D3 can be the same or unique. The number of barcodes used for quantification and normalization will not change, regardless of the number of available conjugation sites and whether they are fully or partially combined. The occupancy rate in such examples is the total drug occupancy rate of all positional isotypes and the number of normalized barcodes remains as two reporters. In addition, if the number of conjugation sites in a single peptide is greater than 2, partial conjugation produces two reporters, such as TMT-127 and TMT-130 ions, and full conjugation produces a single reporter, such as TMT-130 ions, for triggering ETD-MS2 scans for occupancy studies to locate the conjugated drug at the correct amino acid residue. As used herein, a peptide having "complete conjugation" or "complete conjugation" with a drug refers to a peptide covalently bound to a drug, and the amino acid residue can be conjugated to the drug. As used herein, a peptide with "incomplete conjugation" or "incomplete conjugation with a drug" refers to a peptide that has only some covalent bonds to a drug, but not all amino acid residues are capable of conjugating with the drug. A peptide that is fully conjugated with a drug may have one, two, three or more drugs conjugated thereto.

[0125] Although the sample preparation workflow for analyzing such conjugation reactions remains the same, the mass spectrometry parameters for TMT triggering require 1 of 2 reporters or 2 of 2 reporters to be included in the triggering setup for locating the entire multiple conjugate or a portion of the multiple conjugate, respectively. According to the present disclosure, using the methods of the present application, similar methods / protocols can be used to study drug conjugates containing more than three drugs.

[0126] Figure 4 Several exemplary experimental ADC systems analyzed by the method of the present invention are shown. For example, the NIST mAb-biotin PEO acetamide ADC has biotin PEO acetamide conjugated to mAb and a mimetic control containing only antibodies without conjugated drugs. In addition to antibodies or fragments thereof, drug conjugates with other polypeptides can also be characterized by the method of the present application. Human serum albumin (HSA) peptide is used as an example. Unlike NIST, which has only one peptide with two cysteines, the HSA peptide has multiple cysteines on the same peptide. It is tested in the method according to the embodiment of the present application, for example, in fluorescence determination and IodoTMT labeling.

[0127] exist Figure 4In the representative structure shown, two biotin PEO acetamides are conjugated to the NIST mAb, one in the light chain and one in the heavy chain of the NIST mAb. However, the NIST mAb-biotin PEO acetamide ADC can have several different isoforms in which the biotin PEO acetamides are present and attached to different cysteine ​​residues within the NIST antibody with different levels or occupancy. Similarly, although Figure 4 The representative structure of MSQC8 shown in has only one dansyl fluorophore conjugated to the mAb (MSQC4), which may have different isoforms of dansyl fluorophores conjugated to the mAb at one or more other sites.

[0128] Any ADC, including but not limited to Figure 4 Any of the ADCs shown in , and their mimetic controls can be subjected to tandem mass spectrometry analysis according to embodiments of the present application with dual TMTs, such as a TMT with Cys-reactive IodoTMT, and a TMT with reactivity to Lys or primary amines at the N-terminus. Examples of mass spectra obtained from the analysis and results from the analysis are shown in Figure 5A , Figure 5B , Figure 5C , Figure 5D , Fig. 6A , Figure 6B , Figure 7 , Figure 9A-9D As shown in etc.

[0129] In another embodiment, three different types of reporter barcodes are used to calculate drug occupancy at each cysteine ​​residue. Figure 5A HCD-MS of a dual TMT-labeled isobaric peptide showing the light chain of the NIST mAb 2Spectra. The peptide has unoccupied cysteine ​​residues labeled with IodoTMT(io) and N-terminal and terminal lysine residues labeled with TMT(tm). The HCD spectrum (left panel) consists of a series of characteristic b and y-type backbone product ions, which locate the labeling site with the corresponding TMT label. The lower mass range shows four reporter ions, which are more clearly depicted in the enlarged right panel, including the reporter ion mass of each dual TMT channel, i.e., IodoTMT-129 and TMT-128 pairs representing the simulated channel and IodoTMT-126 and TMT-130 pairs representing the conjugate sample channel. Occupancy can be derived by the IodoTMT intensity of the mimetic (A2) and conjugate (A1) or using the TMT intensity of the mimetic (B2) and conjugate (B1). The HCD spectrum provides sequence ions and spectral ratios to identify site conjugation at Cys-193 of the light chain with biotin PEO acetamide at an occupancy of approximately 50%. Similarly, for given reaction conditions for conjugate production, occupancy rates can be obtained. For example, Figure 5B The reporter ion regions for two additional sites are shown; light chain Cys-63 with approximately 65% ​​biotin PEO acetamide occupancy and heavy chain Cys-147 with approximately 100% biotin PEO acetamide occupancy, and Figure 5C and Figure 5D Displayed by HCD-MS 2 The corresponding MS of the sequenced peptides 2 Spectra (with all backbone product ions).

[0130] The dual TMT workflow uses a normalization factor between the conjugate and mock samples to correct for bias in occupancy estimates. For example, Fig. 6A and Figure 6B HCD-MS of a dual TMT-labeled isobaric peptide of the NIST mAb heavy chain is shown. 2 Spectra. The N-terminal and C-terminal lysines of the peptide were labeled with TMT. HCD spectra ( Fig. 6A ) consists of a series of characteristic b and y-type backbone product ions that locate the TMT labeling site. The lower mass range shows two reporter ions; and the reporter ions TMT-128, TMT-130 are ( Figure 6B ) represent the mimetic and conjugate sample channels, respectively. Peptides lacking cysteine ​​residues exhibit this characteristic mass number barcode of two reporter ions, where the reporter ion intensity represents the concentration of the mimetic and conjugate samples. Typically, for such fully labeled non-cysteine ​​peptides, the observed ratio is 1.

[0131] In another embodiment, the sequence ions and iminium ions corresponding to the drug fragments help determine the occupancy site of the conjugated peptide (see, e.g. Figure 7). When there are multiple potential conjugation sites on the same peptide, it is difficult to determine site positioning using conventional methods. There are usually no sequence ions that are specific to the site, and the positioning of the peptide is challenging for determining when the mass-to-charge ratio of the peptide sequence is indistinguishable. Therefore, determining the precise positioning of the conjugation site generally requires site-specific sequence ions obtained by other complementary dissociation methods known in the art. For example, ETD-MS2 is complementary to HCD-MS2. Two methods can be used to form sequence ions, which can distinguish sites or in other words locate modified residues. For example, a drug-conjugated cysteine ​​can be located in an unconjugated cysteine. The method according to an embodiment of the present application allows people to determine the occupied sites of conjugated peptides, including those with multiple conjugation sites, without using complementary dissociation methods. For example, dissociation can be performed to identify and locate the payload.

[0132] According to the present disclosure, using methods known in the art, the method of the present application can further include steps to improve mass triggering, quantitative sensitivity and accuracy. For example, HCD-MS 2 The mass-selected precursor ions of TMT-labeled peptide conjugates can be routed in an ion routing multipole (see, for example, Fig. 8A and 8B ). The resulting ion spectrum is acquired in the Orbitrap in which the TMT reporter ion is detected. The reporter ion mass ( Fig. 8A 130.14Da in ) can be used as a trigger mass selection for the same parent ion and transferred to the ion trap. Subsequently, ETD-MS 2 The ion trap is used for the mass analysis of the resulting product ions. Figure 8B Shown are the TICs and corresponding mass spectra for data-dependent scans from MS, MS 2 Triggered MS of NIST light chain cys-193 biotin PEO acetamide conjugate peptide 2 The TIC labeled 1 is a full MS scan collected in the Orbitrap. The TIC labeled 2 is an HCD-MS of mass-selected precursor ions acquired in two consecutive scans in the Orbitrap. 2 and TIC labeled 3 is a TMT-130 triggered ETD-MS acquired within the same scan cycle and mass analyzed in the Orbitrap 2 Spectra. Two MS 2 The time penalty imposed by the high-resolution Orbitrap mass analysis sequence of product ion spectra may not affect the relatively small subset of drug-conjugated peptides.

[0133] Charge-depleted ions generated by HCD-MS2 (such as the reporter ion TMT-130) can be used to trigger ETD-MS2 and generate ETD spectra. Fig. 9A and Fig. 9B Annotated HCD and ETD spectra of the Cys-193 biotin PEO acetamide conjugate peptide are shown, respectively. It is important to note that the highly selective TMT130 ion and the iminium ion from the drug are readily observed in the spectrum. In addition, the characteristic neutral loss of the reporter and the entire tag is observed in the ETD spectrum. Fig. 9B and Fig.9D The backbone fragments from both ETD and HCD are complementary and position biotin PEO acetamide on Cys-193. Although not the most abundant iminium ion, TMT130 appears to be a useful iminium ion. For example, Fig. 9C and Fig.9D The triggered mass detection of the peptide corresponding to biotin PEO acetamide conjugated at Cys-23 is shown, where TMT130 is less abundant compared to the iminium ion generated by biotin PEO acetamide. The specificity of the TMT130 mass trigger can also be controlled for interfering peptides by reducing the mass selection window of the precursor ion.

[0134] For example, co-isolation and co-fragmentation of peptides may lead to loss of specificity of the reporter ion of TMT-130, which is used to trigger ETD-MS of ADC-peptides. 2 Analysis to locate the conjugation site on the polypeptide. Higher trigger intensity thresholds or narrow separation windows (e.g., 0.4 Da) can be used to improve mass triggering (see, e.g. Fig. 10A and Fig. 10B ). Various reporter ions can influence the type of barcode used for triggering, especially when co-isolation occurs and co-fragmentation occurs due to peptides that co-elute with the same isolation window size (e.g., 2 Da). Fig. 10B ).

[0135] In another embodiment, SPS-MS 3 Can be used to further improve the specificity and accuracy of detection and quantification (see e.g. Fig.11 ). The precursor ions are dissociated in the ion trap by collision-induced dissociation (CID), and several product ion masses are separated synchronously using a trap waveform applied to the ion trap. The separated product ions are transferred to the multistage ion channel where HCD-MS is performed. 3 Fragmentation. Due to SPS, any interference due to co-fragmentation during standard HCD is mitigated, which results in reporter ions with little interference.

[0136] For example, simultaneous precursor selection is used in the presence of Figure 4 Quantification of reporter assays of the studies on MSQC8 and its unconjugated antibody analogs versus MSQC4 ADCs of the structures shown. Figure 12A-12C As shown, the application of simultaneous precursor selection allows accurate TMT reporting, which improves quantitative sensitivity and accuracy. The N=5 ions with IodoTMT-128, TMT-129 reporter ion pairs representing MSQC8 and IodoTMT-130, TMT-131 reporter ion pairs representing MSQC4 (mock) were used in SPS-MS 3 The accurate reporter ion ratio was observed after the MSQC8:MSQC4 reporter ion ratio was 1, indicating that the cysteine ​​residue was not conjugated.

[0137] Fig.13A , Fig. 13B FIG. 14 shows an experiment and results using dual TMT to quantify site-specific ADC on MSQC8 using a method according to an embodiment of the present application. Fig.13A and Fig. 13B As shown, when the labeling of ADC is not complete, for example, the conjugate is only labeled with Cys-reactive TMT, but not with ycs-reactive TMT, the detected occupancy (% ADC) is only slightly lower than that of the complete labeling. Interestingly, despite finding peptides with incomplete labeling, similar site occupancy of 55-60% was observed, which proves the robustness of the method of the present application.

[0138] Preferably, the conjugate is fully labeled with TMT. The completeness of labeling can be measured by the intensity of the reporter ion of TMT on the mass spectrometer. Figure 13C-13F The results shown in indicate that in MSQC8, the drug is conjugated to the antibody at Cys-266 and Cys-372. It was also observed that Cys-218 is the only conjugation site for dansyl-pentanediamine-SMCC on the light chain, with a site occupancy of 60% closely consistent with the average DAR of 1 observed by SLIM-IMS (Nagy et al., Anal Chem 92, 5004-5012 (2020)), and the mass analysis of MSQC8 is reduced (data not shown). The occupancy (maximum) at Cys-266 was estimated to be 60% and at Cys-372 to be 15%, while all other cysteines showed 0% conjugation (see, for example Figures 14A to 14CSuch results are consistent with the DAR0 / DAR1 ratio of 1:2 obtained from existing methods, such as, for example, light chain structure coupled to ion mobility spectrometry (SLIM-IMS) for non-destructive ion manipulation. In addition, the use of a large number of peptides to normalize the mixing differences between the two samples (i.e., MSQC8 ADC sample and MSQC4 mimetic) ensured that the occupancy estimates for each conjugation site were accurate.

[0139] The method according to the embodiment of the present application can also be used to characterize or identify the structure of a drug conjugated to a polypeptide. Fig.15 As shown, small molecule conjugates are prone to fragmentation, resulting in complex spectra. The fragment masses from the drug are specific to each drug and can be used for the identification of the drug. Fig.15 A range of ammonium ions is shown for the SigmMAb dansyl-pentanediamine-SMCC conjugate, which is significantly different from the iminium ions of biotin PEO acetamide. Fig. 8A and Figure 8B By using a specific reporter ion m / z to trigger ETD, the method of the present invention allows the use of ETD-MS 2 Unambiguous site localization is performed regardless of the structure of the small molecule drug that tends to generate fragments. In other words, the TMT reporter m / z is specific, while the reporter from the drug depends on the structure of the molecule to change m / z.

[0140] According to other embodiments of the present application, the dual TMT mass spectrometry analysis according to the present invention can also be used to analyze the reaction time course of the conjugation reaction (see, for example Figure 16A-16B , Fig.17A and Fig. 17B ).exist Fig.16A and Fig. 16B In the dual TMT protocol described in , a single TMT is used on synthetic peptide standards (such as HSA peptide) with varying numbers of cysteine ​​residues for multiplexed reaction time course experiments combining TMT with fluorescence.

[0141] In other embodiments of the present application, the dual TMT mass spectrometry analysis according to the present invention can be used for multiplexed quantification of drug polypeptide conjugates to increase throughput and reduce costs. Fig.18A A multiplexed format is shown, where additional drug conjugate samples are added to the original workflow. Multiplexing allows multiple reaction conditions to be monitored in a single analysis, which is superior to separate workflows for each sample. Careful selection of TMT reagents with non-overlapping reporter ion masses allows multiplexing of even higher numbers of samples. Multiplexing also changes the occupancy of the reporter ion feature, where for n number of samples, the number of reporters in the barcode is (2n+2). Fig.18AIn the example of , the addition of the second sample produces a total of 6 reporters. Again, these reporters come from equal amounts of IodoTMT and TMT reagents. The number of reporter ions in the barcode used for normalization is half the number of barcodes used for occupancy, and is (2n+2) / 2. This is always the case in the dual TMT strategy, where only amine-reactive TMT reporter ions are used for normalization of samples. It is important to note that the number of reporter ions in the mass-triggered barcodes used for peptide drug conjugates is independent of the number of samples. In contrast, the number of reporters used for mass triggering depends on the number of possible conjugation sites: m. Typically, when m=1, a single reporter is observed, and when m>1, and the drug is not conjugated at all sites, two reporter ions are observed.

[0142] In certain embodiments of the present application, multiplexed triple fusion analysis can be used to analyze multiple samples containing conjugates with more than one conjugation site. For example, a synthetic peptide with 1-3 cysteine ​​residues conjugated to the fluorescent molecule DCAM-3 is analyzed using the method of the present application. Fig. 16B The method of using the present application using iodoTMT is shown 6 The time points of the reaction were monitored in combination with IAA to block unreacted cysteines. 6 Not with TMT 10 used together as a dual labeling method, since the upper limit of multiplexing under the assay conditions is 4. Currently, TMT 16 The availability of (16-way) and future n-way reagents (where n>16) will greatly facilitate the number of samples for the dual TMT approach. Fluorescence-based quantification was used as a standard and compared with TMT-based occupancy estimates. Fig. 16C The triple fusion workflow of the present application for monitoring the reaction of multiple drugs D1-D5 in a multiplexed manner is shown. The scheme represents labeling with IAA or IodoTMT to achieve dual labeling. The occupancy of each drug can be estimated using the report intensity to show the rank order of reactivity D5>D3>D1 and the failed reaction of D2, D4 where the occupancy is zero. Fig.16D Mass triggering via a single TMT reporter ion specific to each drug molecule, namely TMT127 for D1, TMT129 for D2, and TMT130 for D3 is shown.

[0143] Fig.17A and Fig. 17BResults from multiplexed experiments are shown, wherein the reaction time points are simulated by mixing the percentage ratios of peptide-DACM-3 conjugation: 0, 20, 40, 60 and 80. Each peptide mixture is prepared separately so that the unconjugated peptide counterpart is first reduced and all free thiols are blocked with IAA. The unconjugated counterparts of the conjugated peptide mixture and each peptide sequence are used for two parallel experiments. First, fluorescence measurements are performed to ensure that DACM is conjugated and mixed accurately. The fluorescence intensity of all peptides with 1-3 cystine residues shows a linear reaction from no conjugation (simulations) to 80% conjugation. Next, the conjugate ratio is 0, 20, 40, 60, 80 of five peptide-DACM and mimics, each sample is labeled with IodoTMT 126, 127, 128, 129, 130 and 131 respectively and equimolar amounts are mixed for reporter ion quantification. HCD-MS2 reporter ions reflect the unique barcodes of the reactants as the reaction proceeds and reaches a stable reaction. The reporter intensity of the peptide mixture relative to its mimetic decreases with the expected increase in conjugation. The correlation of the observed conjugation level to the expected or theoretical conjugation level is obtained based on the TMT reporter ion. It is important to note that the TMT reporter ion intensity is inversely proportional to the conjugation level. In particular, the dynamic compression of the TMT ratio for low intensity reporter ions mainly affects high drug conjugates.

[0144] For example, the algorithm reported by Savisky et al. can be used to correct for ratio compression (Savisky et al., 2013, Journal of proteome research 12, 3586-3598), the entire contents of which are incorporated herein by reference. The SPS-MS3 routines available on current instruments can also improve ratio compression (McAlister et al., Anal Chem 86, 7150-7158 (2014). However, without any correction, the methods of the present application can provide accurate estimates of low-level conjugation, where the TMT reaction is a linear function of conjugation. Detection of low stoichiometries of conjugates is beneficial because most off-target conjugations are undesirable and can be monitored more accurately. Multiplexed TMT ratios and / or inverse TMT ratios (site occupancy) are compared to fluorescence-based yield estimates. The linear dynamic range of fluorescence-based measurements is higher than the dynamic range of TMT reporter occupancy due to ratio compression. However, TMT reporters with high intensity are also channels with low levels of conjugation, which can be measured with fewer ratio compression effects and missing values ​​(Lim et al., "he detection of low-stoichiometr" (Journal of proteomeresearch) 16, 4217-4226 (2017)).

[0145] In another embodiment, the method of the present application includes the use of a robotic system. For example, the triple fusion workflow of the present application can be implemented on an AsayMap Bravo liquid handling robotic system, where double labeling with TMT reagents and sample multiplexing can increase the accuracy and precision of TMT-based quantification. Fig.18A An overall scheme is shown where automation can be beneficial, particularly for robust monitoring of reactions at the point of synthesis. Simultaneous analysis of multiple drug conjugation reactions can be useful when rapid optimization of reaction conditions is required. Additionally, sequential conjugation reactions also require estimation of drug production at intermediate steps in addition to the final product to optimize overall yield. The speed and reproducibility of any automated platform offering multiplexing is needed to reduce human error during sample processing.

[0146] In one embodiment, the sample multiplexing scheme of the present application can analyze up to four ADC samples in duplicate at two different concentrations using dual TMT labeling of the samples (eg, MSQC8). Fig.18B Shown from TMT 10 and IodoTMT 6 The dual TMT reporter selection of reagents makes all masses unique. TMT 10 (10-plex) reagents have 6 of the 10 isotopes, and the isotopologues of these isotopes are labeled with TMT 10 xN or TMT 10 xC, the difference is 6.32mDa (milliDaltons), where C 12 、N 15 The atomic pair is C 13 、N 14 Replace. TMT 10 The xC isotopologue mass is the same mass as the IodoTMT reagent and cannot be used in parallel. Once the samples are multiplexed with the correct combination of reporter ions, high resolution mass spectrometry allows baseline separation of the reporter ion mass and its isotopologue. Four different ADC samples and either the mimic or unconjugated samples were bi-TMT labeled as shown in the scheme, such that the trypsin containing cysteine ​​peptide was an isobaric mixture of 5 TMT reporters and 5 iodoTMT reporters. After MS2, the barcode consisted of 10 reporter ions.

[0147] Four MSQC8 drug conjugate mimics were prepared at two concentrations; the original sample, and another by diluting to half the concentration with MSQC4 and MSQC8. Each of these samples was a duplicate sample in the double TMT labeling. Figure 19 shows the reporter ion of the Cys-218 peptide after HCD-MS2. The reporter of the mimic (MSQC4) was labeled with TMT 10 -126 and IodoTMT6 -130 pairs of double labeling, and four samples were labeled with TMT 10 xN、IodTMT 6 x reagent labeling, where x ranges from 127 to 130. Replicate 1 of MSQC8 was labeled with dual reagent x = 127, and replicate 2 of MSQC8 was labeled with dual reagent x = 130. Similarly, for two replicate samples of an equimolar mixture of MSQC8 and MSQC4 labeled reagents, where x = 128 and 129. The site occupancy at Cys-218 for the ith sample is given in Eq. 1. Fig. 20A The use of non-cysteine ​​peptide sequences is shown, now with five TMTs on MS2-HCD (one mock and four samples) 10 The barcodes of the reporter ions are used to correct for sample concentrations in multiplexed experiments. The normalization factor for the ith sample is given by Equation 2. The corrected occupancy of each ADC sample can be obtained by Equation 3. Fig. 20B Normalized occupancies of four samples obtained in a single acquisition are shown. Mainly these multiplexed experiments can also be used to identify drug conjugates using trigger mass.

[0148] In one embodiment of the present application, when the reaction is carried out via an intermediate step or the reference material is not available (see, for example, Fig. 20A and Fig. 20B ), the relative occupancy between any two reaction steps can be determined. Fig.16A and Fig. 16B As shown, the occupancy of a peptide conjugated to a fluorophore drug mimetic DACM-3 can be evaluated by a two-step antibody conjugation reaction. An example of a two-step antibody conjugation reaction is a transglutaminase reaction followed by click-addition of a cytotoxic payload. See, for example, Huggins et al., Molecules. September 2019; 24(18):3287, the contents of which are incorporated herein by reference in their entirety. Any ADC prepared by a two-step antibody conjugation reaction can be analyzed by the method of the present application.

[0149] The present invention will now be described in more detail with reference to the following specific non-limiting examples. It will be appreciated by those skilled in the art that the technology disclosed in the following examples represents the technology found by the inventor, and the technology works well in the practice of the present invention, and therefore can be considered to constitute the preferred mode of its practice. However, it will be understood by those skilled in the art based on the present disclosure that many changes can be made to the disclosed specific embodiments and similar or similar results can still be obtained without departing from the scope of the present invention.

[0150] Example

[0151] Example 1: Dual TMT labeling and conjugation of unconjugated mAb

[0152] Four different ratios (1:1, 1:9, 9:1 and 1:3) of reduced and drug-conjugated NIST monoclonal antibody ((mAb), Sigma Aldrich, catalog number 8671) (sample #1) were prepared into non-reduced and IodoTMT TM Labeled NISTmAb (sample #2). NIST mAb was conjugated using iodoacetamide (IAA) (Sigma Aldrich, catalog number 16125) or biotin PEO iodoacetamide (Sigma Aldrich, catalog number B2059). For sample #1, 100 μg of NIST (50 μl 2 mg / ml NIST) was prepared by combining 10 μl of stock solution (10 mg / ml) with 40 μl of a solution containing 8 M guanidine hydrochloride (GuHCl) (Sigma Aldrich, catalog number G3272) and 4 mM ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich, catalog number 03620). The NIST aliquots were then reduced by adding 10 μl 1 M DTT (Sigma Aldrich, catalog number 1019777001) and incubated at 37°C for 1 hour. Alternatively, TCEP can also be used as a reducing agent instead of DTT. While DTT is generally active under neutral to alkaline pH conditions, TCEP has a wider pH range and is a stronger reducing agent.

[0153] NIST was conjugated with 1M IAA. 1M IAA was prepared by adding 300μl trimethylammonium carbonate (TEAB) (ThermoFisher, part of the TMT labeling kit) to an Eppendorf tube, vortexed and sonicated for 20 minutes to equilibrate the TMT reagent to room temperature before use. 24μl 1M IAA was then added to the reduced NIST sample, which was incubated in the dark at room temperature for 1 hour. During the incubation, 15μl 1M DTT was added to the sample to quench the conjugation reaction. The sample was desalted by combining it with a buffer exchange solution (8M GuHCl+4M EDTA) and passed through a Zebra TM A spin desalting column (ThermoFisher, catalog number 89882) was placed.

[0154] To conjugate the reduced NIST sample with biotin PEO iodoacetamide, the sample buffer was exchanged to PBS (phosphate buffered saline) without sulfhydryl groups at a pH of about 7.5. A 20 mM biotin PEO iodoacetamide stock solution was prepared immediately before use (190 μl PBS was added to 2 mg biotin PEO iodoacetamide in an Eppendorf tube). The reduced NIST sample in PBS was combined with 5 μl 20 mM biotin PEO iodoacetamide and mixed. The reaction was incubated on ice or at room temperature for 2 hours. After the incubation period, the sample was desalted. The sample was placed in a buffer exchange solution (8 M GuHCl + 4 M EDTA) and was filtered through a Zebra ELISA kit with a molecular weight cutoff of 7 kDa. TM Spin the desalting column for a run.

[0155] Sample #2 (IodoTMT) was prepared by generating 3 aliquots of NIST (50 μl of 2 mg / ml NIST combined with 10 μl of stock solution (10 mg / ml)). TM Labeled non-reduced NIST sample) and added to 40μl solution (8M GuHCl+4mM EDTA). Each aliquot was combined with 50μl of a solution containing 8M GuHCl and 4mM EDTA to compensate for the volume. To reduce the samples for subsequent IodoTMT labeling, 10μl DTT was added to each aliquot and the samples were incubated at 37°C for 1 hour. The following four ratios of sample #1 and sample #2 were prepared: 1) 1:9 ratio (10μl reduced NIST: 90μl non-reduced NIST), 2) 1:3 ratio (25μl reduced NIST: 75μl non-reduced NIST), 3) 1:1 ratio (50μl reduced NIST: 50μl non-reduced NIST) and 4) 9:1 ratio (90μl reduced NIST: 10μl non-reduced NIST). Each mixed sample contained 100μg NIST, and 2μl of IodoTMT TM Labeled A1 (add 10 μl methanol to 200 μg IodoTMT TM The samples were then incubated in the dark at 37°C for 1 hour. To quench the labeling reaction, 4 μl of 0.5 M DTT was added to each sample and incubated for another 15 minutes at 37°C in the dark. The sample buffer was exchanged with TEAB and IodoTMT was digested with 4 μl of trypsin (1 mg / ml) at 37°C. TMThe labeled proteins were added for 4 hours to overnight. Trypsin was quenched with 2 μl 98% formic acid, and 2 μl TMT-labeled B1 (800 μg dissolved in 40 μl anhydrous acetonitrile) was added to each of the 4 samples. The samples were incubated at 37°C for 1 hour in the dark, and the reaction was quenched by adding 8 μl 5% hydroxylamine at room temperature for 15 minutes.

[0156] In addition to Sample #1 and Sample #2, a reference or mock sample, called Sample #3, was prepared and analyzed using IodoTMT T Tag labeling. Sample #3 was prepared similarly to the protocol used for sample #2, except that four samples were generated instead of three. Sample #3 was trypsinized individually and labeled with TMT-labeled B2. Sample #1 and sample #2 were each mixed with 132 μl of sample #3 at a 1:1 volume ratio, which resulted in TMT-labeled proteins with unique isobaric reporter ion masses forming four multiplexed samples for mass spectrometry. Up to four reaction mixtures can be combined with the IodoTMT TM and amine-reactive TMT10plex TM Dual labeling multiplexing of five unique reporter masses using ELISA reagent (ThermoFisher, catalog number 90110). The dual labeled four conjugated samples and a single mock / reference control sample were combined in a volume ratio of 1:1:1:1:1, which resulted in a single multiplexed sample for liquid chromatography-mass spectrometry.

[0157] Fig.13A , Fig. 13B FIG. 14 shows an experiment and results of quantifying site-specific ADC on MSQC8 using dual TMT using a method according to an embodiment of the present application. Fig.13A As shown, when labeling of ADC is not complete, for example, the conjugate is labeled with only Cys-reactive TMTs, such as IodoTMT128 and IodoTMT130, but not Lys-reactive TMTs, such as TMT129 and TMT131, for each of MSQC-4 and MSQC-8, respectively, the site occupancy detected in single labeling (e.g., 55%-60% ADC) is slightly less than the site occupancy with dual labeling. Non-conjugated peptides with dual labeling, such as IodoTMT 128 and TMT129 for MSQC-4, and IodoTMT130 and TMT 131 for MSQC-8, provide a 60% more accurate assessment (measurement assessment from two reporters instead of one reporter). The completion of labeling is measured by the intensity of the reporter ion of TMT on the mass spectrometer. It is known that MSQC8 has several conjugation sites. Fig. 13BThe results shown in indicate that in MSQC8, the drug is conjugated to the antibody at Cys-266 and Cys-372. Cys-218 is the only conjugation site for dansyl-pentanediamine-SMCC on the light chain, where the site occupancy of 60% is closely consistent with the average DAR observed by SLIM-IMS of 1 (e.g., Nagy, G. et al. Anal-Chem-92, 5004-5012 (2020), which found drugs bound to both chains of the antibody). In addition, the mass analysis of MSQC8 occupancy of 60% Cys-266 (maximum) and 15% Cys-372 was reduced, while all other cysteines showed 0% conjugation (e.g., see Figure 14). This result is consistent with the DAR0 / DAR1 ratio of 1:2 obtained from existing methods, such as light chain structure for non-destructive ion manipulation combined with ion mobility spectrometry (SLIM-IMS). It is important to note that the use of a large number of peptides to normalize for mixing differences between the two samples (ie, MSQC8 ADC sample and MSQC4 mimetic) ensures that the occupancy estimates for each conjugation site are accurate.

[0158] Example 2: Dual TMT labeling of conjugated mAbs

[0159] The conjugated mAb was labeled using a dual TMT labeling protocol. MSQC8 (Sigma Aldrich mAb antibody-drug conjugate mimetic) and MSQC4 (Sigma Aldrich mAb standard) were used as conjugated samples and reference or mock samples, respectively. The samples were labeled using different IodoTMT following the sample protocol as described for labeling NIST mAb. TM Label samples and trypsinize individually to generate peptides. Follow the procedure for TMT or IodoTMT. TM The resulting peptide mixtures from MSQC8 and MSQC4 were labeled with different amine-reactive TMT tags, respectively, following the same protocol for labeling NIST mAbs. After TMT double labeling, the samples were mixed in a 1:1 volume ratio, which produced a multiplexed single sample for mass spectrometry.

[0160] Example 3: Automation of TMT labeling in AssayMap Bravo

[0161] The double TMT labeling scheme is implemented in the AssayMAP Bravo (Agilent) robot system, which automates protein sample preparation before analysis with LC-MS. The double TMT labeling of unconjugated NIST mAb conjugation is performed as previously described. The single plate scheme is digested in solution according to the manufacturer's instructions, which is incorporated herein by reference in its entirety (see World Wide Web: agilent.com / cs / library / applications / application-protease-digestion-in-solution-assaymap-5994-1682en-agilent.pdf). The automated sample preparation is programmed to distribute a minimum volume of 5 μl, and the sample is desalted with reversed-phase protein using RP-W box applications as known in the art. Different sample ratios (e.g., 1:1, 1:9, 9:1, and 1:3) are performed using the reformatting tool in the AssayMAP Bravo robot system.

[0162] Example 4: Multiplex TMT labeling of conjugated peptides

[0163] Synthetic mAbs with 1-3 cysteine ​​residues were conjugated with N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM-3; ThermoFisher, catalog number D10251) via an adapted manufacturer's protocol for protein labeling. A stock solution of 16.76 mM DACM-3 was prepared. 1 mg of each peptide was dissolved in 1 mL of a solution containing 100 mM PBS, 0.1 M NaCl, 10 mM EDTA (pH 8.0) and 50 μl 16.76 mM DACM-3. The peptides were sealed with a light protection cap and incubated at ambient temperature for about 5 minutes. Mass spectrometry was used to confirm that the peptides were fully conjugated. The relative fluorescence (Relative Fluorescence Units or RFU) of the samples was measured using a fluorescence plate reader (Spectramax M5, Molecular Devices) and a standard curve of N-acetyl-L-cysteine ​​(cysteine ​​peptide standards from NIST monoclonal antibodies were synthesized by Biomatik Corporation to 99.99% purity) (excitation = 385 nm, emission = 465 nm, 455 nm cutoff). The relative concentration of free or conjugated thiols for each peptide was calculated based on regression analysis of the internal N-acetyl-L-cysteine ​​curve. It was determined that N-acetyl-L-cysteine ​​reacted substantially 100% with DACM-3.

[0164] After the DACM-3 of peptide is conjugated, peptide is labeled with TMT. Each DACM-3 conjugated peptide is mixed with unlabeled peptide counterpart to produce five kinds of mixtures of various stoichiometric ratios (0, 0.2, 0.4, 0.6 and 0.8). Unlabeled peptide is used as control. According to the disclosure, the sample is evaluated with fluorescence determination known in the art to ensure that the DACM-3 labeling works properly before TMT labeling. As previously described for the TMT labeling of tryptic peptides, amine reactive 6-plexTMT labeling is carried out to each sample. Finally, five kinds of mixtures and controls of labeling are combined with a volume ratio of 1: 1, and methods known in the art are used to analyze using methods known in the art.

[0165] Example 5: LC-MS 2 (LC-MS / MS) Analysis

[0166] Using LC-MS 2 The samples analyzed were analyzed on an Agilent Infinity 12900 UHPLC with an AdvanceBio Peptide Mapping column (Agilent, catalog number 864600-911) at 65°C. A 50-minute liquid chromatography (LC) gradient program using LC-MS grade water with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B was performed according to the following scheme: 0 min, 2% B; 35 min, 30% B; 40 min, 80% B; 45 min, 85% B; 45.5 min, 2% B; and then re-equilibrated at 2% B for 5 minutes. The flow rate was set to 0.2 mL / min and the injection volume was 2 μl. The mass spectrometer was used with a data dependent (dd) MS known in the art. 2 HCD (MS / MS-HCD) and electron transfer dissociation (ETD) methods were operated in positive ionization mode. The following interface conditions were used: emitter voltage, +2600 V; vaporizer temperature, 325° C.; ion transfer tube, 325° C.; sheath gas, 55 (arb (arb)): auxiliary gas, 10 (arb); and purge gas, 1 (arb).

[0167] The following internal mass spectrometer settings were used for MS scans: RF (radio frequency) lens, 60%; AGC (automatic gain control) target, 1e6; maximum injection time, 50 ms; and 1 μscan in analytical mode at 70K resolution on an Orbitrap (OT) mass analyzer. The method was performed on any MS as known in the art. 2A series of filters were included in sequence before the HCD event. A monoisotopic peak selection filter was included and set to peptides for all methods, and an intensity filter of 1e5 was used. Some methods used an optional charge state filter to select precursor charge states 2-6. In addition, some methods used an optional dynamic exclusion (DE) filter to more effectively identify peptides in the sample with a 12s or 3s exclusion window, and exhibited the following common parameters: exclusion n=1 times; + / -3ppm; isotope exclusion; and a single charge state for each precursor. One method involves Apex detection, which uses the following parameters: expected peak width, 6s; expected apex window, 30%.

[0168] Five times ddMS 2 OT-HCD (data-dependent MS / MS-Orbitrap detection-higher energy collisional dissociation) scans were performed with the following settings: quadrupole separation, 1.6 m / z separation window; detector type, Orbitrap, automatic m / z normal scan range, 70K resolution, 100 m / z first mass; AGC target, 2e5, ions injected over all available parallel time, maximum injection time 50 ms; 1 μscan, profile. 2 After OT-HCD, targeted mass triggering (TMT) is implemented, which is a scan that is triggered only when the system detects product ions from a user-defined list. Targeted ion masses include specific TMT reporter ions to detect payloads in the reporter mass list (e.g., 126 to 131 Da), and only ions within the top 10 most intense mass-to-charge ratios are used for all mass triggers. The following conditions are used for ddMS 2 OT-ETD (Data-dependent MS / MS-Orbitrap detection-electron transfer dissociation): MS n Level, 2; quadrupole separation, 1.6 m / z separation window; ETD reaction time 50 ms; detector type, Orbitrap, automatic m / z normal scan range, 30K resolution; AGC target, 5e4, ions injected over all available parallel time, maximum injection time 22 ms; 1 μscan, profile. 2 OT-HCD and ddMS 2 The number of dependent scans between OT-ETD was set to 1.

[0169] Data from various mass spectrometry analyses were analyzed using Xcalibur TMData acquisition and interpretation software (ThermoFisher, catalog number OPTON-3096 7, MaxQuant and Perseus source proteomics data analysis software (MaxPlanck Institute), and R 3.6 statistical programming software (R Foundation for Statistical Computing, Vienna, Austria) were used for analysis. Site occupancy was estimated using the IodoTMT reporter ion peak area by equation 1, where A2 is the area of ​​the unconjugated sample and A1 is the area of ​​the conjugated sample. The normalization factor was calculated using the TMT reporter ion by equation 2, where B1 is the peak area of ​​the conjugated sample and B2 is the peak area of ​​the unconjugated sample:

[0170]

[0171]

[0172] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but the present invention is intended to cover modifications consistent with the spirit and scope of the present invention, as defined in the appended claims.

Claims

1. A method for analyzing a conjugate comprising a drug covalently linked to a polypeptide, the method comprising: (i) contacting a sample comprising the conjugate with a first tandem mass tag (TMT), thereby labeling the polypeptide of the conjugate with the first TMT; (ii) digesting the polypeptide of the conjugate labeled with the first TMT to produce a first mixture comprising one or more unlabeled peptides and one or more peptides labeled with the first TMT; (iii) contacting the first mixture with a second tandem mass tag (TMT) to obtain a second mixture comprising one or more peptides labeled with at least one of the first TMT and the second TMT, and optionally one or more unlabeled peptides; (iv) contacting a control sample comprising a polypeptide not covalently linked to the drug with a third TMT, thereby labeling the polypeptide not covalently linked to the third TMT; (v) digesting the polypeptide not covalently linked to the drug and labeled with the third TMT to produce a third mixture comprising one or more unlabeled peptides and one or more peptides labeled with the third TMT; (vi) contacting the third mixture with a fourth tandem mass tag (TMT) to obtain a fourth mixture comprising one or more peptides labeled with at least one of the third TMT and the fourth TMT, and optionally one or more unlabeled peptides; (vii) combining the second mixture with the fourth mixture and subjecting the combination to liquid chromatography (LC) to produce an eluent of the LC; as well as (viii) subjecting the eluate to tandem mass spectrometry to obtain a mass spectrum of a peptide comprising at least one reporter ion of the first TMT, the second TMT, the third TMT, and the fourth TMT; as well as (ix) detecting a mass-to-charge ratio associated with the at least one reporter ion, thereby analyzing the conjugate in the sample, wherein none of the first TMT, the second TMT, the third TMT and the fourth TMT have the same reporter ion mass, the first TMT and the third TMT are selected from a first isobaric group of TMTs, the second TMT and the fourth TMT are selected from a second isobaric group of TMTs, and the first isobaric group of TMTs reacts with an unconjugated amino acid residue capable of forming a covalent bond with the drug, and the second isobaric group of TMTs reacts with a lysine or a free amine at the N-terminus of the peptide.

2. A method for determining the occupancy ratio of a conjugation site in the conjugate, the method comprising: 1) obtaining a mass spectrum of a peptide labeled with both the first TMT and the second TMT and a peptide labeled with both the third TMT and the fourth TMT using the method according to claim 1, wherein the mass spectrum includes a reporter ion of the first TMT, a reporter ion of the third TMT, a reporter ion of the second TMT, and a reporter ion of the fourth TMT; 2) detecting a mass-to-charge ratio (m / z) associated with the reporter ion in the mass spectrum; as well as 3) determining the occupancy ratio of the conjugation site in the conjugate based on the intensity of the reporter ion of the first TMT and the intensity of the reporter ion of the third TMT in the mass spectrum, or the intensity of the reporter ion of the second TMT and the intensity of the reporter ion of the fourth TMT, preferably the occupancy ratio is determined by the following equation: (the intensity of the reporter ion of the third TMT-the intensity of the reporter ion of the first TMT) / the intensity of the reporter ion of the third TMT, or (Intensity of the reporter ion of the fourth TMT−Intensity of the reporter ion of the second TMT) / Intensity of the reporter ion of the fourth TMT.

3. The method according to claim 2, wherein the occupancy ratio of the conjugation site in the conjugate is determined at various time points, wherein additional TMT is used to label the polypeptide at different time points.

4. A method for normalizing the sample comprising the conjugate with the control sample, the method comprising: 1) obtaining a mass spectrum of a peptide labeled only with the second TMT and a peptide labeled only with the fourth TMT using the method according to claim 1, wherein the mass spectrum includes a reporter ion of the second TMT and a reporter ion of the fourth TMT, but does not include a reporter ion of the first TMT or a reporter ion of the third TMT; 2) detecting a mass-to-charge ratio (m / z) associated with the reporter ion; and 3) normalizing the sample with the control sample by the ratio of the intensity of the reporter ion of the second TMT to the intensity of the reporter ion of the fourth TMT.

5. A method for locating a drug conjugation site in the conjugate, the method comprising: 1) obtaining a mass spectrum of a peptide labeled only with the second TMT using the method according to claim 1, wherein the mass spectrum includes only reporter ions of the second TMT but does not include reporter ions of the first TMT, the third TMT or the fourth TMT; as well as 2) Triggering a second tandem mass spectrometry analysis on the peptides labeled only with the reporter ions of the second TMT to locate the drug conjugation site.

6. The method of claim 5, wherein the peptide is fully conjugated to the drug.

7. A method for locating a drug conjugation site in the conjugate, the method comprising: 1) obtaining a mass spectrum of a peptide labeled only with the first TMT and the second TMT using the method according to claim 1, wherein the mass spectrum includes only reporter ions of the first TMT and the second TMT, but does not include reporter ions of the third TMT or the fourth TMT; as well as 2) triggering a second tandem mass spectrometry analysis on the peptides labeled only with the first TMT and the second TMT, thereby locating the drug conjugation site.

8. The method of claim 7, wherein the peptide is incompletely conjugated to the drug.

9. The method according to any one of claims 1 to 8, wherein the tandem mass spectrometry is high energy collision induced dissociation tandem mass spectrometry (HCD-MS2).

10. The method according to any one of claims 5 to 8, wherein the second tandem mass spectrometry is electron transfer dissociation tandem mass spectrometry (ETD-MS2) or electron capture dissociation tandem mass spectrometry (ECD-MS2).

11. The method according to any one of claims 5 to 8, wherein a higher trigger intensity threshold and / or a narrow separation window are used to improve the triggering of the second tandem mass spectrometer.

12. The method according to any one of claims 1 to 8, wherein simultaneous precursor selection using three-in-one technology is applied to the tandem mass spectrometry to improve the specificity and accuracy of the detection and quantification.

13. The method according to any one of claims 1 to 8, wherein each of the first TMT and the third TMT comprises a mass reporter, a mass normalizer, and a cysteine-reactive group covalently linked to each other, and each of the second TMT and the fourth TMT comprises a mass reporter, a mass normalizer, and an amine-reactive group covalently linked to each other.

14. The method of claim 13, wherein each of the first TMT and the third TMT is selected from the isobaric set of IodoTMT sixplex.

15. The method of claim 13, wherein each of the second TMT and the fourth TMT is selected from an isobaric group of TMT6plex, TMT10plex, or TMT pro16plex.

16. The method of any one of claims 1 to 8, wherein the conjugate is an antibody drug conjugate (ADC).

17. The method according to any one of claims 1 to 8, wherein the analysis of samples comprising one or more conjugates is multiplexed.

18. The method according to any one of claims 1 to 8, wherein the conjugate or the ADC comprises one or more drugs conjugated to a cysteine ​​residue of the polypeptide or the antibody.

19. The method according to any one of claims 1 to 8, wherein the conjugate or the ADC comprises one or more drugs conjugated to a lysine residue of the polypeptide or the antibody.

20. The method of claim 19, wherein each of the first TMT, the second TMT, the third TMT, and the fourth TMT comprises a mass reporter, a mass normalizer, and an amine-reactive group covalently linked to each other. 21 . The method of claim 20 , wherein each of the first TMT, the second TMT, the third TMT, and the fourth TMT is selected from TMT6plex, TMT10plex, or TMTpro16plex.

22. A method according to any one of claims 1 to 8, wherein the drug conjugation site in the conjugate is characterized by a mass spectral barcode comprising (2n+2) reporter ions, and the conjugate is normalized by the mass spectral barcode, wherein the mass spectral barcode comprises n+1 reporter ions, and n is the number of samples analyzed by the method.

23. A system for performing any of the methods according to claims 1 to 8.

Images