Method and system for quantitatively detecting antibody drugs by using dimethyl-labeled modified peptide fragment as internal standard

Abstract

The invention belongs to the technical field of biological drug analysis, and particularly relates to a method and a system for quantitatively detecting antibody drugs by using a dimethyl-labeled modified peptide fragment as an internal standard. Sequentially carrying out dimethyl labeling on the enzymolysis peptide fragments of the antibody drugs to obtain a plurality of dimethyl-labeled modified peptide fragments which are used as internal labels; the method comprises the following steps: enriching antibody drugs in a biological sample, and carrying out enzyme digestion to obtain a plurality of unlabeled modified peptide fragments; and mixing the unlabeled modified peptide fragment with an internal standard, enriching the modified peptide fragment, and then carrying out quantitative detection on the modified peptide fragment by adopting liquid chromatography-tandem mass spectrometry. A one-to-one structural analogue internal standard is provided for the modified peptide fragment, and the matrix effect is eliminated. Meanwhile, the method is combined with a high-pH reversed-phase grading equal peptide fragment enrichment method, so that the detection sensitivity of the modified peptide fragments in the matrix is improved. The technology provides a sensitive and reliable quantitative analysis method for the modified peptide fragment, and can be used for quantitative analysis of antibody drugs in biological samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical analysis, and particularly relates to a method and system for quantitatively detecting antibody drugs using dimethyl-labeled modified peptide segments as internal standards. Background Art

[0002] Antibody-drug conjugates (ADCs) utilize chemical linkers to covalently conjugate small cytotoxins to antibodies, combining the targeting properties of antibodies with the potent cytotoxicity of small molecule toxins. The stability of ADCs is closely related to their efficacy and safety, with the choice of linker and conjugation site being key factors influencing their in vivo stability. "Site-specific stability evaluation of antibody-drug conjugate inserum using a validated liquid chromatography-mass spectrometry method" reports a universal strategy for systematically evaluating the stability of ADCs at different conjugation sites in serum, using modified peptides of conjugated cytotoxins as analytical targets. However, this method is more suitable for quantitative analysis of high-concentration serum samples. ADCs have low concentrations in blood, and the enzymatically hydrolyzed conjugated peptides exhibit significant heterogeneity and generally exhibit lower mass spectrometric response than the unconjugated peptides. Consequently, this method faces challenges in in vivo quantitative analysis due to insufficient sensitivity for low-abundance conjugated peptides and matrix interference from coelution. Quantitative analysis of conjugated peptides produced by enzymatic hydrolysis of ADC drugs can not only be used to evaluate the stability of ADC drug conjugation sites, but also be used for ADC drug pharmacokinetics research. Therefore, the development of a quantitative analysis method for conjugated peptides in low-concentration samples is of great application significance.

[0003] In addition to ADC drugs, the quantitative analysis of modified peptides (such as glycopeptides) of antibody drugs also faces major challenges. Glycosylation is one of the important post-translational modifications of antibody drugs and one of their key quality attributes. The structure of the sugar chain has a significant impact on the physicochemical properties and biological functions of antibody drugs. Abnormal sugar chain structure can affect the thermal stability and aggregation tendency of antibody drugs. In addition, differences in sugar chain composition can affect the pharmacokinetic properties of antibody drugs. Given the important impact of glycosylation on the efficacy and safety of antibodies, establishing characterization and quantitative analysis methods for glycopeptides is of great significance for the quality control and process optimization of antibody drugs. In the quantitative study of glycopeptides, due to the heterogeneity of glycosylation sites and the complexity of sugar chain structure, coupled with the low abundance of target analytes in biological samples and background interference, the accurate quantification of glycopeptides remains a challenging task.

[0004] In quantitative analysis of biological samples, stable isotope-labeled internal standards (SIL-IS) are a common technique to overcome matrix effects. However, obtaining SIL-IS for structurally complex analytes, such as conjugated peptides of ADCs or glycopeptides of antibodies, is often difficult. Existing studies have often used structural analogs of small cytotoxins as internal standards for the quantitative analysis of ADCs in matrix. However, these internal standards exhibit different chromatographic behaviors from the analytes, limiting their ability to quantify low-concentration samples. For highly heterogeneous analytes such as glycopeptides, studies have used unmodified peptides of the same sequence as internal standards for quantitative analysis of glycopeptides in biological samples. However, due to differences in chromatographic retention between unmodified and glycopeptide peptides, their ability to quantify glycopeptides is limited. Furthermore, due to the large molecular weight of antibody-based drugs, enzymatic hydrolysis produces a wide variety of peptides, and the abundance of different modified peptides varies significantly. Therefore, the simultaneous quantitative analysis of multiple modified peptides through the use of appropriate internal standards, combined with appropriate pretreatment and detection methods, remains an urgent challenge. Summary of the Invention

[0005] The purpose of the present invention is to provide a method and system for quantitative detection of antibody drugs using dimethyl-labeled modified peptides as internal standards. The antibody drug hydrolysate is dimethyl-labeled to obtain an internal standard derived from the modified peptide of the antibody drug. The antibody drug in the biological sample is then enriched and hydrolyzed, and then mixed with the internal standard for quantitative detection. This method can simultaneously perform quantitative analysis on multiple modified peptides of the antibody drug in the biological sample.

[0006] To achieve the above objectives, the present invention provides a method for quantitatively detecting antibody drugs using a dimethyl-labeled modified peptide internal standard, comprising the following steps: S1. Dissolve, denature, reduce, alkylate, and enzymatically digest the antibody drug standard in sequence, and then perform dimethyl labeling to obtain several dimethyl-labeled modified peptides; S2, enriching the antibody drugs in the biological sample to be tested, and then performing denaturation, reduction, alkylation and enzymatic cleavage to obtain several unlabeled modified peptides; S3. The dimethyl-labeled modified peptide is used as an internal standard and mixed with the unlabeled modified peptide to enrich the modified peptide, and then the unlabeled modified peptide is detected by liquid chromatography-tandem mass spectrometry (LC-HR-MS / MS).

[0007] Furthermore, in step S1, the modified peptide solution obtained after enzymatic cleavage is dimethyl labeled with a mixture of formaldehyde and sodium cyanoborohydride; and the pH value of the modified peptide solution is 5.5-6.5.

[0008] Furthermore, the formaldehyde and sodium cyanoborohydride mixture is prepared by mixing 7.5%-8.5% formaldehyde and 1-1.5 M sodium cyanoborohydride in a 1:1 volume ratio. 80 µL of the formaldehyde and sodium cyanoborohydride mixture is added to every 100 µg of peptide. The dimethyl labeling is completed at pH 6.0 at 28-32°C for 0.5-2 hours.

[0009] Furthermore, after the dimethyl labeling reaction is completed, the reaction is terminated with ammonia water, followed by acidification with formic acid solution, desalination, and finally redissolution in an aqueous solution containing trifluoroacetic acid to obtain several dimethyl-labeled modified peptides as internal standards.

[0010] Furthermore, the denaturation treatment includes treatment with solutions such as urea and sodium lauryl sulfate.

[0011] Preferably, the reagent used in the reduction treatment includes dithiothreitol.

[0012] Preferably, the reagent used in the alkylation treatment includes iodoacetamide.

[0013] Preferably, the reagents used for the enzymatic digestion treatment include trypsin, chymotrypsin, Trypsin / Lys-C mixed enzyme, etc.

[0014] Furthermore, after enzymatic digestion, the peptide is desalted and then redissolved in a sodium dihydrogen phosphate solution to obtain a modified peptide solution with a pH of 5.5-6.5. Preferably, the concentration of the sodium dihydrogen phosphate solution is 100 mM.

[0015] Furthermore, step S2 specifically includes: using protein A / G magnetic beads to capture the biological sample to be detected, washing the magnetic beads with PBS solution after capture, resuspending the washed magnetic beads in urea solution, and then reducing, alkylating and enzymatically cleaving to obtain several unlabeled modified peptides. The present invention uses immunoaffinity capture to specifically enrich the target protein, which can effectively remove background interference in the sample. The enrichment of the modified peptides in step S3 and the enrichment at the protein level in step S2 constitute a dual enrichment strategy to improve the detection sensitivity of modified peptides in biological samples.

[0016] Furthermore, in step S3, the mass ratio of the enzymatic peptide fragments after pretreatment of the biological sample to the internal standard is 100:(15-30).

[0017] Furthermore, step S3 also includes: enriching the modified peptides using different enrichment methods, and then performing quantitative detection, such as high pH reverse phase fractionation, hydrophilic interaction chromatography separation, titanium dioxide affinity enrichment, etc.

[0018] Furthermore, the antibody drugs include monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates; the liquid chromatography separation uses a UPLC column, and different chromatographic gradients are used according to the structure of the modified peptide segment. Furthermore, the method also includes: using a blank biological sample to prepare a standard curve sample containing different concentrations of the antibody drug, using the same method as step S2 for pretreatment to obtain a plurality of unlabeled modified peptide segments, after mixing with the internal standard, using liquid chromatography-tandem mass spectrometry for detection, and then using the chromatographic peak area ratio of the unlabeled modified peptide segment to the internal standard as the vertical axis and the concentration of the antibody drug in the standard curve sample as the horizontal axis to obtain a standard curve for each unlabeled modified peptide segment.

[0019] Furthermore, liquid chromatography-tandem mass spectrometry is used to obtain the chromatographic peak area ratio of each unlabeled modified peptide segment to the internal standard in the biological sample to be tested, and then substituted into the corresponding standard curve to obtain the concentration of the antibody drug in the biological sample to be tested.

[0020] The antibody drugs described in the present invention include monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates. The modified peptide segments include conjugated peptide segments derived from antibody-drug conjugates, glycopeptides derived from antibody drugs, etc.

[0021] In some embodiments, the quantitative detection method comprises the following steps: Step 1: Pre-treat the antibody drug standard to obtain a mixture containing modified peptides, desalt it, and dissolve it in 100 mM sodium dihydrogen phosphate solution at pH 6.0. Step 2: 8% formaldehyde solution and 1.2 M sodium cyanoborohydride solution were mixed at a ratio of 1:1 ( v / v ) to obtain the dimethyl labeling working solution, which is prepared immediately. Mix 100 µg of the peptide mixture with 80 µL of the dimethyl labeling working solution and react at 30°C for 1 hour in a pH 6.0 reaction system to dimethyl label the peptides. After desalting the dimethyl-labeled peptide mixture, blow dry with nitrogen gas and prepare the internal standard working solution. Step 3: Enrich the antibody drug in the biological sample through affinity capture, followed by denaturation, reduction, alkylation, and enzymatic cleavage to obtain a modified peptide mixture. Mix the biological sample enzymatic hydrolyzate with the internal standard at a ratio of 100:15 (protein mass ratio).

[0022] Step 4: Enrich the modified peptides and their internal standards, perform LC-MRM-MS / MS analysis on the enriched samples, and quantitatively analyze the modified peptides by comparing their peak area with the internal standard peptides.

[0023] The present invention also provides a quantitative detection system for antibody drugs, comprising: Internal standard preparation module: antibody drug standards are sequentially dissolved, denatured, reduced, alkylated, and enzymatically digested, followed by dimethyl labeling to obtain several dimethyl-labeled modified peptides; Biological sample pretreatment module: enriches the antibody drugs in the biological sample to be tested, and then denatures, reduces, alkylates and enzymatically digests them to obtain several unlabeled modified peptides; Quantitative detection module: The dimethyl-labeled modified peptide is used as an internal standard, mixed with the unlabeled modified peptide, and the modified peptide is enriched, and then the modified peptide is quantitatively detected by liquid chromatography-tandem mass spectrometry.

[0024] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology: 1. The present invention provides a method for quantitatively detecting antibody drugs using dimethyl-labeled modified peptides as internal standards. The method involves enzymatically cleaving and dimethyl-labeling antibody drug standards to obtain internal standards of multiple modified peptides. The biological sample containing the antibody drug is then pretreated, mixed with the internal standard, and quantitatively detected. This method can achieve simultaneous quantitative analysis of multiple modified peptides of antibody drugs.

[0025] 2. This method does not require dimethyl labeling of biological samples. Compared with the dimethyl labeling quantitative method in proteomics research, the analysis process is simpler and the cost is lower. Both methods can correct for instrument response differences and matrix effects.

[0026] 3. The modified peptide internal standard preparation method provided by this invention is versatile and suitable for the quantitative analysis of low-concentration antibody-drug conjugates and antibody-drugs in vivo. Compared with commonly used deuterated isotope internal standards, it is simpler to prepare and more cost-effective. Furthermore, this method enriches modified peptides during pretreatment, making it more conducive to the quantitative analysis of modified peptides in low-concentration samples compared to existing methods. This dual enrichment strategy can improve the detection sensitivity of modified peptides in biological samples.

[0027] 4. By optimizing the detection conditions of liquid chromatography-tandem mass spectrometry, the present invention can achieve quantitative detection of multiple modified peptides, and the quantitative error of each peptide is within ±20%, which is more accurate than the method without using an internal standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Schematic diagram of the dimethyl labeling reaction used in the present invention.

[0029] Figure 2 The responses of dimethyl-labeled coupled peptides under different labeling conditions (pH 6.0 and pH 8.0). The horizontal axis numbers represent different dimethyl-labeled coupled peptides.

[0030] Figure 3 The effect of dimethyl labeling on the mass spectrometry response of coupled peptides. 1-20 and 22-25 on the horizontal axis represent coupled peptides derived from T-DM1, and 21 is an unmodified characteristic peptide.

[0031] Figure 4 To examine the efficiency of the dimethyl labeling reaction, the figure shows the extracted ion chromatograms of a representative conjugated peptide fragment of an ADC drug, FNWYVDGVEVHNAK(DM1)TKPR (MRM transition 1040.160 / 547.22, retention time 26.4 min), and the corresponding dimethyl-labeled product (MRM transition 794.138 / 547.22, retention time 24.2 min). "DM1" in the amino acid sequence indicates that the corresponding amino acid residue is modified with DM1, and "DI" indicates that the corresponding amino acid residue is dimethylated.

[0032] Figure 5 Figure 3 is the extracted ion current chromatogram of the coupled peptides and characteristic peptides derived from T-DM1 and their corresponding internal standard peptides. Chromatographic peaks 1 to 21 are coupled peptides (1 to 20) and characteristic peptide (21) derived from T-DM1; chromatographic peaks 1' to 19' are one-to-one internal standards for the coupled peptides, 20' is one-to-one internal standard for the characteristic peptide, and chromatographic peaks 8 and 18 share the internal standard peak 8'.

[0033] Figure 6 According to the method of Example 3, samples with theoretical concentration ratios of 1:2 and 1:4 were measured. The deviation analysis results between the theoretical concentration ratios and the measured concentration ratios are shown. The acceptable range is marked with a dotted line, and the horizontal axis 1 to 21 represents the coupled peptides (1 to 20) and characteristic peptide (21) derived from T-DM1.

[0034] Figure 7The results show the comparison of the deviations between the measured concentration ratios and the theoretical concentration ratios for low-concentration plasma samples of 20 µg / mL and 50 µg / mL, respectively, when using and not using dimethyl-labeled conjugated peptides as internal standards, according to the methods of Examples 3 and 4. (A) shows the ratios of conjugated peptides in samples of different concentrations when no internal standard is used; (B) shows the ratios of conjugated peptides in samples of different concentrations when using dimethyl-labeled conjugated peptides as internal standards. The acceptable range is marked with a dotted line, and the horizontal axis 1 to 21 represents conjugated peptides (1 to 20) and characteristic peptide (21) derived from T-DM1.

[0035] Figure 8 Comparison of the results of quantitative analysis of modified peptides from plasma samples pretreated using immunoaffinity enrichment and protein precipitation. (A) RSD between replicate samples of the same concentration using 1% trichloroacetic acid in isopropanol; (B) Peak area ratio between high- and low-concentration samples using 1% trichloroacetic acid in isopropanol; (C) RSD between replicate samples using protein A / G magnetic beads; (D) Peak area ratio between low- and high-concentration samples using protein A / G magnetic beads. The horizontal axis represents the number of conjugated peptides derived from T-DM1, and the acceptable range is shown as a black dashed line.

[0036] Figure 9 The figure shows the flow chart for quantitative detection of antibody drugs in plasma using modified peptide internal standards.

[0037] Figure 10 This is the standard curve of the coupled peptide FNWYVDGVEVHNAK (DM1) TKPR in Example 3.

[0038] Figure 11 This is the standard curve of the coupled peptide ADYEK (DM1) HK in Example 3. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0041] Experimental Materials 1. Reagents: Ado-trastuzumab emtansine (T-DM1) was purchased from Roche; formaldehyde (CHO), sodium cyanoborohydride (NaBHCN), trypsin, urea, dithiothreitol, iodoacetamide, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich; a high-pH reversed-phase peptide fractionation kit was purchased from Thermo Fisher Scientific; MonoSpin™ C18 cartridges were purchased from Techtronic; Protein A / G magnetic beads were purchased from Merk-Sigma; and Oasis HLB cartridges were purchased from Waters. Phosphate-buffered saline (PBS) and triethylammonium bicarbonate (TEAB) were purchased from Thermo Fisher Scientific; chromatographic-grade acetonitrile and formic acid (FA) were purchased from Fisher Scientific.

[0042] 2. Experimental instruments: Shimadzu LC-40 AD high-performance liquid chromatography-Thermo Fisher Q Exactive HF-X mass spectrometry system; SCIEX Triple Quad™ 5500+ liquid chromatography-mass spectrometry system; low-temperature high-speed centrifuge purchased from Zhongke Zhongjia Scientific Instrument Co., Ltd.; vortex mixer purchased from Scilogex; constant temperature mixer purchased from Hangzhou Miou Instrument Co., Ltd.

[0043] 3. Solution Preparation: Dilute 50 µL of 37% formaldehyde solution with 180 µL of ultrapure water and vortex to mix to obtain an 8% formaldehyde solution. Accurately weigh 15.1 mg of solid sodium cyanoborohydride and dissolve it in 200 µL of ultrapure water to obtain a 1.2 M sodium cyanoborohydride solution. Accurately weigh 142.0 mg of solid sodium dihydrogen phosphate and dissolve it in 10 mL of water to obtain a 100 mM sodium dihydrogen phosphate solution. Weigh 240 mg of urea and dissolve it in 500 µL of ultrapure water to obtain an 8 M urea solution. Weigh 15.4 mg of powdered dithiothreitol and dissolve it in 200 µL of ultrapure water to obtain a 0.5 M dithiothreitol solution. Weigh 18.5 mg of powdered iodoacetamide and dissolve it in 200 µL of ultrapure water to obtain a 0.5 M iodoacetamide solution.

[0044] Example 1: Optimization of dimethyl labeling conditions for coupled peptides from T-DM1 Six aliquots of 100 µg of the antibody-drug conjugate T-DM1 were added to 100 µL of 8 M urea and 5 µL of 0.5 M dithiothreitol and reacted at 37°C for 60 min. Subsequently, 10 µL of 0.5 M iodoacetamide solution was added to the reaction mixture, and the reaction was incubated at 37°C in the dark for 30 min. 800 µL of 50 mM TEAB buffer was added to the reaction mixture to reduce the urea concentration in the digestion system to less than 1 M. 2 µg of trypsin was then added, and the digestion was carried out at 37°C overnight. After digestion, the samples were desalted using a Waters Oasis HLB cartridge (1 cc / 10 mg). After air drying, the samples were divided into two groups, each with three aliquots. One set of samples was re-dissolved in 200 µL of 100 mM sodium dihydrogen phosphate solution (pH 6.0), and the other set of samples was re-dissolved in 200 µL of 100 mM TEAB buffer (pH 8.0). Both sets of dissolved samples were simultaneously dimethyl labeled.

[0045] 80 μL of a 1:1 (v / v) mixture of 8% formaldehyde and 1.2 M sodium cyanoborohydride was added to the peptide solution and immediately vortexed. The mixture was then reacted at 30°C for 1 h. After the reaction, 100 μL of 2% ammonia was added to terminate the reaction, and 16 μL of 50% FA solution was added to acidify the reaction solution. Desalting was then performed, and the eluate was dried with nitrogen and redissolved in 100 μL of 30% acetonitrile ( v / v ) solution, followed by LC-MRM-MS / MS analysis. The chromatographic peaks of the dimethyl-labeled coupled peptides were integrated using data processing software, and the peak areas of the dimethyl-labeled coupled peptides in different labeling systems were calculated.

[0046] The experimental results show that the pH value of the labeling system has a significant effect on the dimethyl labeling efficiency of the coupled peptides. Figure 2 As can be seen, at pH 6.0, the response of dimethyl-labeled coupled peptides increased by 3-20 times compared to the pH 8.0 system, but the response of the unmodified peptide LLIYSASFLYSGVPSR did not differ significantly between the two pH conditions. These results indicate that the pH of the reaction system has a significant impact on the labeling efficiency of modified peptides, and the pH 6.0 labeling system is more conducive to dimethyl labeling of modified peptides.

[0047] Example 2: Effect of dimethyl labeling on mass spectrometry response of T-DM1 coupled peptides Six aliquots of 100 µg of the antibody-drug conjugate T-DM1 were added to 100 µL of 8 M urea and 5 µL of 0.5 M dithiothreitol and reacted at 37°C for 60 min. Subsequently, 10 µL of 0.5 M iodoacetamide solution was added to the reaction mixture, and the reaction was incubated at 37°C in the dark for 30 min. 800 µL of 50 mM TEAB buffer was added to the reaction mixture to reduce the urea concentration in the digestion system to less than 1 M. 2 µg of trypsin was then added, and the digestion was carried out at 37°C overnight. After digestion, the sample was desalted using a Waters Oasis HLB cartridge (1 cc / 10 mg). After air drying, the sample was divided into two groups of three. One group of samples was reconstituted in 100 µL of 30% acetonitrile in water.

[0048] Another set of samples was reconstituted in 200 µL of 100 mM sodium dihydrogen phosphate (pH 6.0) and dimethyl labeled and desalted according to the procedures in Example 1. The desalted samples were dried with nitrogen and reconstituted in 100 µL of 30% acetonitrile in water. Both sets of reconstituted samples were analyzed by LC-MRM-MS / MS. The chromatographic peaks of the coupled peptides and the dimethyl-labeled coupled peptides were integrated using data processing software, and the responses of the dimethyl-labeled coupled peptides were compared with those of the unlabeled coupled peptides.

[0049] like Figure 3 As shown, the present invention studies the effect of dimethyl labeling on the response of coupled peptides. The results show that the response of 8 coupled peptides decreased by 2 to 10 times after dimethyl labeling, the response of 7 coupled peptides increased by 2 to 20 times after labeling, and there was no significant difference in the response of 6 coupled peptides before and after labeling. This shows that there are differences in the effect of dimethyl labeling on the ionization efficiency of different coupled peptides. In general, the response of most coupled peptides is reduced or unchanged after dimethyl labeling. In the analysis of biological matrix samples, if the matrix sample is subjected to dimethyl labeling and desalting process, the pretreatment time and complicated operation steps will be increased. Therefore, the T-DM1 standard is enzymatically hydrolyzed and then dimethyl labeled to obtain an internal standard, and the analytical sample is not dimethyl labeled, which simplifies the biological sample pretreatment steps and improves the analysis throughput.

[0050] Example 3: Preparation of dimethyl-labeled conjugated peptides (internal standards) and their application in quantitative analysis of antibody-drug conjugates (1) Preparation and identification of dimethyl-labeled coupled peptides: 100 µg of antibody-drug conjugate T-DM1 was added to 100 µL of 8 M urea and 5 µL of 0.5 M dithiothreitol, and the reaction was allowed to proceed at 37°C for 60 minutes. Subsequently, 10 µL of 0.5 M iodoacetamide was added to the reaction mixture, and the reaction was incubated at 37°C in the dark for 30 minutes. 800 µL of 50 mM TEAB buffer was added to the reaction mixture to reduce the urea concentration in the enzymatic digestion system to less than 1 M. 2 µg of trypsin was then added, and the digestion was allowed to proceed overnight at 37°C. The sample was desalted using a Waters Oasis HLB cartridge (1 cc / 10 mg), dried under nitrogen purge, and reconstituted in 200 µL of 100 mM sodium dihydrogen phosphate (pH 6.0).

[0051] 80 µL of a 1:1 (v / v) mixture of 8% formaldehyde and 1.2 M sodium cyanoborohydride was added to the peptide solution and immediately vortexed. The solution was then reacted at 30°C for 1 h. After completion, the reaction was terminated with 100 µL of 2% aqueous ammonia and acidified with 16 µL of 50% FA solution. Desalting was then performed, and the eluate was dried under nitrogen. Samples used as internal standards were reconstituted in 100 µL of 0.1% aqueous TFA before use to obtain the internal standard solution.

[0052] The internal standard was redissolved in 100 μL of 30% acetonitrile aqueous solution, and the dimethyl-labeled coupled peptides were identified by LC-HR-MS / MS analysis and library search software. The chromatographic column used was a C18 column (2.1 × 100 mm, 1.8 μm, 120 Å), the mobile phase A was 0.1% FA aqueous solution, and the mobile phase B was 0.1% FA acetonitrile solution. The elution gradient was: 15% B~40% B, 40 min. The mass spectrometry data were collected using data-dependent acquisition mode, the spray voltage was set to 3800 V, and the first-level full scan mass range was m / z The ion density was 50-1800, the resolution was 70000, the secondary mass spectrometry scanning resolution was 30000, the 15 parent ions with the highest intensity were selected for secondary mass spectrometry scanning, and the normalized collision energy was 30 eV.

[0053] MaxQuant (2.4.2.0) software was used to search the database for the acquired mass spectrometry data to identify dimethyl-labeled coupled peptides. Trypsin was selected as the enzyme, and the number of missed cleavages allowed was 2. The precursor mass errors for the first search and the main search were 20 ppm and 4.5 ppm, respectively, the fragment ion mass error was 20 ppm, the false discovery rate was 0.01, and the minimum score for modified peptides was 10. The fixed modifications were iodoacetamido cysteine ​​(C2H3NO, 57.021 Da) and dimethyl modification of the peptide N-terminus (C2H 4，28.0313 Da). The variable modification is dimethyl modification of lysine residues (C2H4, 28.0313 Da).

[0054] (2) Dimethyl-labeled conjugated peptides are used as internal standards for quantitative analysis of antibody-drug conjugates: Animals were administered intravenously, and blood samples were collected at various time points for plasma separation. At each time point, 10 µL of plasma sample was captured using 100 µL of protein A / G magnetic beads for 2 h at room temperature. After capture, the beads were washed twice with 500 µL of PBS. The washed beads were resuspended in 100 µL of 8 M urea solution and 10 µL of 0.5 M dithiothreitol solution was added. The reaction was incubated at 37°C for 1 h. Subsequently, 20 µL of 0.5 M iodoacetamide solution was added to the reaction mixture and incubated in the dark for 30 min. Finally, 800 µL of 50 mM TEAB solution was added. Trypsin was added at a trypsin to protein ratio of 1:20 (w / w), and the reaction was incubated at 37°C for 2 h. The prepared internal standard solution (with a protein mass ratio of 100:15 to the internal standard) was added to the enzymatic hydrolysate, mixed well, and fractionated using a high pH reverse phase fractionation column. The fraction containing the coupled peptide was subjected to LC-MRM-MS / MS analysis.

[0055] Use blank plasma to prepare standard curve samples, perform pretreatment according to the above steps, and then perform LC-MRM-MS / MS analysis simultaneously with animal plasma samples. Use data processing software to integrate the chromatographic peaks of the conjugated peptides and internal standard peptides. Use the concentration of the antibody conjugated drug in the standard curve sample as the horizontal axis and the peak area ratio of the conjugated peptide to the internal standard peptide as the vertical axis to perform linear regression to obtain the standard curve of each conjugated peptide, as shown in Figure 2. Figure 10 and Figure 11 The concentration of the antibody-drug conjugate in animal plasma can be calculated by substituting the peak area ratio of the conjugated peptide to the internal standard peptide measured in the animal plasma sample into the standard curve.

[0056] Example 4: Quantitative Analysis of Antibody-Drug Conjugates Without Internal Standard Take 10 µL of plasma sample at each time point in the animal experiment, take 100 µL of washed protein A / G magnetic beads, capture at room temperature for 2 hours, and wash the beads twice with 500 µL PBS solution after capture. Resuspend the washed magnetic beads in 100 µL 8 M urea solution, add 10 µL 0.5 M dithiothreitol solution, and react at 37 ° C for 1 hour. Then add 20 µL 0.5 M iodoacetamide solution to the reaction solution and react in the dark for 30 minutes. Finally, add 800 µL 50 mM TEAB solution. According to the ratio of trypsin to protein of 1:20 ( w / w ) were added with trypsin and reacted at 37°C for 2 h. After enzymatic digestion, the sample was enriched using a high-pH reverse-phase fractionation column, and the enriched peptides were analyzed by LC-MRM-MS / MS.

[0057] Use blank plasma to prepare the standard curve samples, and the processing steps for the annotated curve samples are the same as above. The standard curve samples and animal plasma samples are analyzed simultaneously by LC-MRM-MS / MS. Use data processing software to integrate the chromatographic peaks of the conjugated peptide segments. Use the concentration of the antibody conjugate in the standard curve samples as the horizontal axis and the peak area of ​​the conjugated peptide segments as the vertical axis to perform linear regression to obtain the standard curve of each conjugated peptide segment. Substitute the peak area of ​​the conjugated peptide segments measured in the animal plasma samples into the standard curve for calculation to obtain the concentration of the antibody conjugate in the animal plasma.

[0058] Test Example 1 This test case is used to illustrate that under optimized labeling conditions, the coupled peptide can be completely dimethyl labeled. 100 µg of antibody-drug conjugate T-DM1 was added to 100 µL of 8 M urea and 5 µL of 0.5 M dithiothreitol, and the reaction was allowed to proceed at 37°C for 60 minutes. Subsequently, 10 µL of 0.5 M iodoacetamide was added to the reaction mixture, and the reaction was incubated at 37°C in the dark for 30 minutes. 800 µL of 50 mM TEAB buffer was added to the reaction mixture to reduce the urea concentration in the enzymatic digestion system to less than 1 M. 2 µg of trypsin was then added, and the digestion was allowed to proceed overnight at 37°C. The sample was desalted using a Waters Oasis HLB cartridge (1 cc / 10 mg), dried under nitrogen purge, and reconstituted in 200 µL of 100 mM sodium dihydrogen phosphate (pH 6.0).

[0059] 80 µL of a 1:1 (v / v) mixture of 8% formaldehyde and 1.2 M sodium cyanoborohydride was added to the peptide solution and immediately vortexed. The mixture was then reacted at 30°C for 1 h. After completion, the reaction was terminated with 100 µL of 2% ammonia solution and acidified with 16 µL of 50% FA solution. Desalting was then performed, and the eluate was dried under nitrogen and redissolved in 100 µL of 30% acetonitrile.

[0060] Sample analysis was performed using a Shimadzu LC 40 liquid chromatograph coupled to a SCIEX Triple Quad™ 5500+ mass spectrometer in MRM mode. The column was a C18 (2.1 × 100 mm, 1.8 µm, 120 Å) with mobile phase A consisting of 0.1% FA in water and mobile phase B consisting of 0.1% FA in acetonitrile. The elution gradient was as follows: 15% B (0–5 min); 40% B (i.e., linear increase in the volume of B, reaching 40% at 45 min); 90% B (45–50 min); 90% B (50–60 min); 15% B (60.1 min); and 15% B (60.1–65 min). The ion source spray voltage was set to 5500 V, the source temperature was 550°C, and the curtain gas was set to 40 psi (GAS1 and GAS2, respectively). The chromatographic peaks of the coupled peptides and dimethyl-labeled coupled peptides were extracted and integrated using data processing software to confirm whether the dimethyl-labeled samples contained unreacted coupled peptides.

[0061] Figure 4 The following are extracted ion chromatograms of the conjugated peptide FNWYVDGVEVHNAK(DM1)TKPR (MRM transitions 1040.160 / 547.22) and the dimethyl-labeled conjugated peptide (MRM transitions 794.138 / 547.22). The results show that a strong peak of the dimethyl-labeled conjugated peptide was detected in the dimethyl-labeled sample, while the original conjugated peptide was not detected. This indicates that the pH 6.0 labeling system enables complete dimethyl labeling of the conjugated peptide. Therefore, when used as an internal standard, the labeled conjugated peptide does not affect the analysis of the conjugated peptide from the antibody drug in plasma, thereby ensuring the accuracy and reliability of the quantitative analysis results.

[0062] Test Example 2 This test example is used to illustrate the calibration capability of dimethyl-labeled coupled peptides as internal standards. Plasma samples were prepared at T-DM1 concentrations of 0.1, 0.2, and 0.4 mg / mL. 10 µL of each plasma sample was pretreated according to the procedure described in Example 3, "Quantitative Analysis of Antibody-Drug Conjugates Using Dimethyl-Labeled Peptides as Internal Standards." The enriched peptides were analyzed using a Shimadzu LC 40 liquid chromatograph coupled to a SCIEX Triple Quad™ 5500+ mass spectrometer in MRM mode. The column used was a C18 (2.1 × 100 mm, 1.8 µm, 120 Å) column. The mobile phases A and B consisted of 0.1% FA in water and 0.1% FA in acetonitrile. The elution gradient was as follows: 15% B (0–5 min); 40% B (5–45 min); 90% B (45–50 min); 90% B (50–60 min); and 15% B (60.1–65 min). The ion source spray voltage was set to 5500 V, the ion source temperature was set to 550°C, and the curtain gas pressure was set to 40 psi (45 psi for both GAS1 and GAS2). The chromatographic peaks of the conjugated peptide and internal standard peptide were integrated using mass spectrometry data processing software, and the peak area ratio of the conjugated peptide to the internal standard was calculated for each plasma concentration. The matrix effect of the dimethyl-labeled conjugated peptide as an internal standard was evaluated by comparing the measured peak area ratios of the conjugated peptides with the theoretical concentration ratios for different sample concentrations.

[0063] Figure 5 Figure 3 shows the extracted ion current chromatogram of the coupled peptide and internal standard peptide from T-DM1. The retention time deviation is within 0.1~0.9 min, indicating that the dimethyl-labeled coupled peptide has good correction potential as an internal standard. Figure 6 The deviations between the theoretical and measured ratios for samples with concentration ratios of 1:2 (i.e., the ratio of plasma samples with concentrations of 0.1 mg / mL and 0.2 mg / mL) and 1:4 (i.e., the ratio of plasma samples with concentrations of 0.1 mg / mL and 0.4 mg / mL) were all within ±20%, indicating that the use of the dimethyl-labeled coupled peptide as an internal standard effectively eliminated matrix effects and corrected for instrument fluctuations.

[0064] Test Example 3 This test example is used to illustrate the effect of using dimethyl-labeled coupled peptides as internal standards on quantitative accuracy. Low-concentration plasma samples with T-DM1 concentrations of 20 µg / mL and 50 µg / mL were prepared. 10 µL of each sample with different concentrations was taken and pre-treated according to the steps in Example 4.

[0065] Then, 10 μL of each sample with different concentrations was taken and pre-treated according to the steps in Example 3, “Quantitative analysis of antibody-drug conjugates using dimethyl-labeled conjugated peptides as internal standards”.

[0066] Both peptides after different pretreatments were analyzed using a Shimadzu LC 40 liquid chromatograph coupled to a SCIEX Triple Quad™ 5500+ mass spectrometer in MRM mode. The analysis was performed using a C18 column (2.1 × 100 mm, 1.8 µm, 120 Å) with mobile phase A consisting of 0.1% FA in water and mobile phase B consisting of 0.1% FA in acetonitrile. The elution gradient was as follows: 15% B from 0 to 5 min; 40% B from 5 to 45 min; 90% B from 45 to 50 min; 90% B from 50 to 60 min; and 15% B from 60.1 to 65 min. The ion source spray voltage was set at 5500 V, the source temperature at 550°C, and the curtain gas pressure was set at 40 psi (45 psi for both GAS1 and GAS2). The chromatographic peaks of the coupled peptides and the internal standard peptide were integrated using mass spectrometry data processing software. For samples without an internal standard, the deviation between the measured and theoretical ratios of the peak areas of the coupled peptides was calculated for samples of varying concentrations. For samples with an internal standard, the deviation between the measured and theoretical ratios of the peak areas of the coupled peptides and the internal standard was calculated for samples of varying concentrations. The effect of using a dimethyl-coupled peptide as an internal standard on quantitative accuracy was evaluated by comparing the deviation between the measured and theoretical ratios under the two conditions (with and without an internal standard).

[0067] When the pretreatment was performed according to the steps of Example 3, the dimethyl-modified coupled peptide was used as a one-to-one internal standard, and the deviation between the measured ratio and the theoretical ratio of samples of different concentrations was within the range of ±25%. When the pretreatment was performed according to the steps of Example 4, without using an internal standard, the ratio deviations of 6 coupled peptides exceeded the acceptable range ( Figure 7 ). This result indicates that dimethyl-labeled coupled peptides can be used as internal standards to improve the quantitative accuracy of low-concentration samples.

[0068] Test Example 4 This test example is used to illustrate the advantages of the plasma sample pretreatment method used in the present invention in the quantitative analysis of antibody drug modified peptides. Plasma samples were prepared at 500 µg / mL and 1 mg / mL T-DM1 concentrations. Three 10 µL aliquots of each sample were diluted with 350 µL of PBST and added to Protein A / G magnetic beads. The beads were incubated for 2 h at room temperature on a vertical mixer. After capture, the beads were washed twice with 500 µL of PBS. The washed beads were resuspended in 100 µL of 8 M urea and 10 µL of 0.5 M dithiothreitol. The beads were reacted at 37°C for 1 h. Subsequently, 20 µL of 0.5 M iodoacetamide was added to the reaction mixture and incubated in the dark for 30 min. Finally, 800 µL of 50 mM TEAB solution was added. Trypsin was added at a trypsin to protein ratio of 1:20 (w / w) and incubated at 37°C for 2 h. After digestion, the samples were desalted.

[0069] Three 10 µL aliquots of each sample at different concentrations were added to 100 µL of 1% trichloroacetic acid in isopropanol, vortexed, and centrifuged to remove the supernatant. The precipitate was dissolved in 100 µL of 8 M urea solution and 10 µL of 0.5 M dithiothreitol solution was added. The reaction was incubated at 37°C for 1 h. Subsequently, 20 µL of 0.5 M iodoacetamide solution was added to the reaction mixture and incubated in the dark for 30 min. Finally, 800 µL of 50 mM TEAB solution was added. Trypsin was added at a trypsin-to-protein ratio of 1:20 (w / w), and the reaction was incubated at 37°C for 2 h. After digestion, the sample was desalted.

[0070] Desalted samples were analyzed by LC-MRM-MS / MS using a Shimadzu LC 20 liquid chromatograph coupled to a SCIEX Triple Quad™ 6500 mass spectrometer. The column used was a C18 (2.1 × 100 mm, 1.8 µm, 120 Å) column, with mobile phase A consisting of 0.1% FA in water and mobile phase B consisting of 0.1% FA in acetonitrile. The elution gradient was as follows: 15% B (0–5 min); 40% B (5–45 min); 90% B (45–50 min); 90% B (50–60 min); and 15% B (60.1–65 min). The ion source spray voltage was set to 5500 V, the source temperature to 550°C, and the curtain gas pressure was set to 40 psi (45 psi for both GAS1 and GAS2). LC-MRM-MS / MS analysis was performed, and the chromatographic peaks of the coupled peptides were integrated using data processing software. The relative standard deviation (RSD) of the peak area of ​​coupled peptides under different pretreatment conditions, as well as the deviation between the measured peak area ratio and the theoretical ratio, were calculated to evaluate the applicability of different plasma sample pretreatment methods in the quantification of antibody drug-modified peptides.

[0071] Due to the large molecular weight of antibody drugs, when protein precipitation is used for pretreatment, the antibody drugs will be co-precipitated with plasma proteins, making subsequent analysis impossible. Therefore, protein precipitation methods suitable for small molecule drugs or peptides are not suitable for bioanalysis of such drugs. This test case compares the analysis results when immunoaffinity and high abundance protein removal methods are used as pretreatment methods for antibody drugs in plasma. Figure 8 As shown, in samples pretreated with 1% trichloroacetic acid in isopropanol, the RSDs of six coupled peptides exceeded ±20% (Figure A), and the deviations of the high-to-low concentration ratios of nine coupled peptides exceeded 20% (Figure B). This indicates that the measured ratios of coupled peptides in high- and low-concentration samples deviated significantly from the theoretical ratios. In three plasma samples pretreated with immunoaffinity, the RSDs of the peak areas of coupled peptides were all within ±20% (Figure C), and the deviations of the peak area ratios of coupled peptides in high- and low-concentration samples from the theoretical ratios were within ±20% (Figure D). These results demonstrate that even after removing high-abundance proteins using 1% trichloroacetic acid in isopropanol, a strong matrix effect still exists, affecting the accuracy and reproducibility of quantification. However, the immunoaffinity pretreatment method used in this patent effectively reduces matrix interference by specifically enriching the target protein, making the quantitative analysis of antibody drugs more accurate and reliable. It should be noted that the biological sample of the present invention is not limited to plasma, but can also be saliva, breast milk, lymph fluid, synovial fluid, tissue homogenate, cell lysate, etc.

[0072] In summary, the internal standard preparation method of the present invention is simple, low-cost, and capable of accurate quantitative analysis of low-concentration samples. It also has the advantages of simple process and good versatility. It provides a one-to-one structural analog internal standard for modified peptides such as coupled peptides, eliminating matrix effects. At the same time, combined with peptide enrichment methods such as high-pH reverse-phase fractionation, it can improve the detection sensitivity of modified peptides in the matrix. This technology provides a sensitive and reliable method for the quantitative analysis of modified peptides, which can be used for the quantitative analysis of antibody drugs in biological samples.

[0073] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide as an internal standard, characterized in that: The following steps are involved: S1. Dissolve, denature, reduce, alkylate, and enzymatically digest the antibody drug standard in sequence, and then perform dimethyl labeling to obtain several dimethyl-labeled modified peptides; S2, enriching the antibody drugs in the biological sample to be tested, and then performing denaturation, reduction, alkylation and enzymatic cleavage to obtain several unlabeled modified peptides; S3. The dimethyl-labeled modified peptide is used as an internal standard, mixed with the unlabeled modified peptide, and the modified peptide is enriched, and then the unlabeled modified peptide is quantitatively detected by liquid chromatography-tandem mass spectrometry.

2. The method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide as an internal standard according to claim 1, characterized in that: In step S1, a mixture of formaldehyde and sodium cyanoborohydride is used to dimethyl label the modified peptide solution obtained after enzyme cleavage, and the pH value of the labeling reaction is 5.5-6.

5.

3. The method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide as an internal standard according to claim 2, characterized in that: The mixed solution of formaldehyde and sodium cyanoborohydride is obtained by mixing equal volumes of 7.5% to 8.5% formaldehyde and 1 to 1.5 M sodium cyanoborohydride; After the dimethylation reaction is completed, ammonia water is used to terminate the reaction, followed by adding formic acid solution for acidification, followed by desalination, and finally redissolving in an aqueous solution containing trifluoroacetic acid to obtain several dimethyl-labeled modified peptides as internal standards.

4. The method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide as an internal standard according to claim 1, characterized in that: In steps S1 and S2, the reagent used for the alkylation includes iodoacetamide; the reagent used for the reduction includes dithiothreitol; In step S1, the enzyme digestion is followed by desalting, and then the peptide is redissolved in a sodium dihydrogen phosphate solution to obtain a modified peptide solution with a pH value of 5.5-6.

5.

5. The method for quantitative detection of antibody drugs using dimethyl-labeled modified peptides as internal standards according to claim 1, characterized in that: In step S2, protein A / G magnetic beads are used to capture the biological sample to be tested. After capture, the magnetic beads are washed with PBS solution, resuspended in urea solution, and then reduced, alkylated and enzymatically digested to obtain several unlabeled modified peptides.

6. The method for quantitative detection of antibody drugs using dimethyl-labeled modified peptides as internal standards according to claim 1, characterized in that: In step S3, the mass ratio of the enzymatic peptide fragments after pretreatment of the sample to be tested to the internal standard is 100:(15-30); In step S3, the modified peptides are enriched using different enrichment methods, including high pH reverse phase fractionation, hydrophilic interaction chromatography, or titanium dioxide affinity enrichment.

7. The method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide internal standard according to claim 1, characterized in that: The antibody drugs include monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates; the liquid chromatography separation uses a UPLC column, and the chromatographic gradient is set according to the structure of the modified peptide segment.

8. The method for quantitative detection of antibody drugs using a dimethyl-labeled modified peptide as an internal standard according to any one of claims 1 to 7, characterized in that: Before analyzing the sample to be tested, a standard curve sample containing different concentrations of the antibody drug is prepared using a blank biological matrix, and pretreated according to step S2 to obtain several unlabeled modified peptides. After mixing with the internal standard, the peptides are detected using liquid chromatography-tandem mass spectrometry. Then, the chromatographic peak area ratio of the unlabeled modified peptide to the corresponding internal standard peptide is used as the ordinate, and the concentration of the antibody drug in the standard curve sample is used as the abscissa to obtain a standard curve for each unlabeled modified peptide.

9. The method for quantitative detection of antibody drugs using dimethyl-labeled modified peptides as internal standards according to claim 8, characterized in that: Liquid chromatography-tandem mass spectrometry is used to obtain the chromatographic peak area ratio of each unlabeled modified peptide segment to the corresponding internal standard peptide segment in the biological sample to be tested, and then the ratio is substituted into the corresponding standard curve to obtain the concentration of the antibody drug in the biological sample to be tested.

10. A quantitative detection system for antibody drugs, characterized in that: include: Internal standard preparation module: antibody drug standards are sequentially dissolved, denatured, reduced, alkylated, and enzymatically digested, followed by dimethyl labeling to obtain several dimethyl-labeled modified peptides; Biological sample pretreatment module: enriches the antibody drugs in the biological sample to be tested, and then denatures, reduces, alkylates and enzymatically digests them to obtain several unlabeled modified peptides; Quantitative detection module: The dimethyl-labeled modified peptide is used as an internal standard, mixed with the unlabeled modified peptide, and the modified peptide is enriched, and then the modified peptide is quantitatively detected by liquid chromatography-tandem mass spectrometry.

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