Methods for identifying scrambled disulfides in biomolecules

Abstract

A method for identifying one or more non-native disulfide bonds in a biomolecule (e.g., an antibody) is disclosed. In one example, the method includes performing digestion of a biomolecule under non-reducing conditions to provide a sample containing a plurality of biomolecule fragments, contacting the sample with a separation column, applying a first mobile phase gradient containing trifluoroacetic acid (TFA) and a small molecule additive to the separation column, applying a second mobile phase gradient containing TFA and a small molecule additive in acetonitrile (ACN) to the separation column, performing a partial reduction procedure on the eluted sample, applying the partially reduced eluted sample components to a mass spectrometer, and performing mass spectrometry on the partially reduced eluted sample components to identify one or more non-native disulfide bonds in the biomolecule.

Description

[Technical Field]

[0001] Sequence Listing Reference This application incorporates by reference the Sequence Listing filed in computer readable format as file 10870WO01-Sequence, created on December 17, 2021, and containing 12,466 bytes.

[0002] FIELD OF THE INVENTION Embodiments herein relate to mass spectrometry, and more particularly to methods for improving the ability to use mass spectrometry to identify low abundance scrambled disulfides in biomolecules. [Background technology]

[0003] Disulfide bonds are present in a large number of proteins (nearly one-third) in the eukaryotic proteome. Disulfide bond formation involves a reaction between the sulfhydryl (SH) side chains of two cysteine ​​residues. Native disulfide bond formation acts to stabilize proteins, and disulfide bonds are important for effective protein functionality.

[0004] Therapeutic monoclonal antibodies can bind to specific epitopes on cell surface receptors or other biological targets, and the efficacy and stability of such therapeutic antibodies depend on the proper formation of native disulfide bonds. Alternatively, the formation of non-native (e.g., scrambled) disulfide bonds in proteins, including but not limited to therapeutic monoclonal antibodies, can lead to destabilization, improper folding, aggregate formation, and the inability of a particular protein to function effectively. Therefore, elucidating the presence of scrambled disulfides in proteins, such as therapeutic antibodies, is important. Current challenges in determining the presence of disulfide scrambling in proteins include low abundance and, therefore, the difficulty of detecting disulfide scrambling when relying on methodologies such as mass spectrometry. Discussed herein are methods for improving the ability to use mass spectrometry to identify low-abundance scrambled disulfides in biomolecules, such as therapeutic monoclonal antibodies. Summary of the Invention

[0005] In one aspect, the present invention provides a method for identifying one or more non-native disulfide bonds in a biomolecule. The method includes performing digestion of a biomolecule under non-reducing conditions to obtain a sample containing multiple fragments of the biomolecule; contacting the sample with a separation column under conditions that allow the sample components to bind to a column substrate; applying a first mobile phase gradient to the separation column, the first mobile phase gradient comprising trifluoroacetic acid (TFA) and a small molecule additive at a concentration of about 1-2 mM; applying a second mobile phase gradient to the separation column, the second mobile phase gradient comprising TFA in acetonitrile (ACN) and a small molecule additive at a concentration of about 1-2 mM; performing a partial reduction procedure by treating the eluted sample components with tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 10-100 μM; applying the partially reduced, eluted sample components to a mass spectrometer; and performing mass spectrometry on the partially reduced, eluted sample components to identify one or more non-native disulfide bonds in the biomolecule.

[0006] In some embodiments, the small molecule additive in the first mobile phase is glycine.

[0007] In some embodiments, the small molecule additive in the first mobile phase is glycine, and the glycine concentration is about 1 mM.

[0008] In some embodiments, the small molecule additive in the first mobile phase is glycine, and the glycine concentration is about 2 mM.

[0009] In some embodiments, the small molecule additive in the second mobile phase is glycine, and the glycine concentration is about 1 mM.

[0010] In some embodiments, the small molecule additive in the second mobile phase is glycine, and the glycine concentration is about 2 mM.

[0011] In some embodiments, the small molecule additive in one or more of the first and second mobile phases is selected from alanine, serine, valine, N-acetylglycine, methionine, β-alanine, aspartic acid, or N-methylglycine.

[0012] In some embodiments, the TFA concentration in the first mobile phase is about 0.05% to 0.1% TFA in HO.

[0013] In some embodiments, the TFA concentration in the second mobile phase comprises about 0.05% TFA in 80% ACN and 20% H2O, or about 0.1% TFA in 80% ACN and 20% H2O.

[0014] In some embodiments, the biomolecule is a monoclonal antibody of an IgG1, IgG2, IgG3, IgG4, or mixed isotype.

[0015] In some embodiments, the biomolecule is recombinantly produced.

[0016] In some embodiments, the partial reduction procedure is carried out for a duration of 500 ms to 3 s.

[0017] In some embodiments, performing digestion of the biomolecule includes performing a modification and alkylation step to obtain a modified alkylated biomolecule, performing a pre-digestion step on the modified alkylated biomolecule to obtain a pre-digested modified alkylated biomolecule, and performing a digestion step on the pre-digested modified alkylated biomolecule after the pre-digestion step to obtain a sample that is contacted with a separation column.

[0018] In some embodiments, the denaturation and alkylation step comprises denaturing the biomolecule in 7-9 M urea in the presence of an alkylating agent at a pH of about 5.5-5.9. Optionally, the denaturation and alkylation step is performed at a temperature of 45-55°C. Optionally, the alkylating agent is N-ethyl maleimide (NEM) at a concentration of 5-15 mM. Optionally, the alkylating agent is iodoacetamide (IAM) at a concentration of about 0.5-5 mM. Optionally, the method comprises performing the denaturation and alkylation step for 20-40 minutes.

[0019] In some embodiments, performing a pre-digestion step comprises incubating the modified alkylated biomolecule in the presence of recombinant Lys-C protease at a pH of 5 to 5.6. Optionally, the pre-digestion step is performed at a temperature of 35 to 40°C. Optionally, the pre-digestion step is performed for a duration of 30 to 90 minutes. Optionally, the ratio of recombinant Lys-C protease to modified alkylated biomolecule is 1:5 to 1:20, respectively.

[0020] In some embodiments, performing the digestion step comprises incubating the pre-digested modified alkylated biomolecule in the presence of recombinant Lys-C protease and trypsin protease at a pH of 5 to 5.6. Optionally, the ratio of recombinant Lys-C protease to pre-digested modified alkylated biomolecule during the digestion step is about 1:5 to 1:20, respectively. Optionally, the ratio of trypsin protease to pre-digested modified alkylated biomolecule is about 1:2 to about 1:10, respectively. Optionally, the digestion step is performed at a temperature of 35 to 40°C. Optionally, the digestion step is performed for 2 to 4 hours.

[0021] In some embodiments, the partially reduced eluted sample components include one or more disulfide peptides and corresponding reduction partner peptides. Optionally, each of the one or more disulfide peptides and corresponding reduction partner peptides simultaneously enters a mass spectrometer. Optionally, the mass spectrometer is a tandem mass spectrometer, and performing mass analysis includes acquiring an MS1 ​​spectrum and an MS2 spectrum. Optionally, a parallel reaction monitoring (PRM) inclusion list is constructed using the corresponding reduction partner peptides. Optionally, a disulfide identification confidence score is assigned to the one or more disulfide peptides based on a confidence scoring system.

[0022] In some embodiments, the confidence scoring system includes steps of displaying whether the MS1 mass of the disulfide peptide has been identified by mass spectrometry, displaying whether the MS1 mass of a first reduction partner peptide corresponding to the disulfide peptide has been identified by mass spectrometry, displaying whether the MS1 mass of a second reduction partner peptide corresponding to the disulfide peptide has been identified by mass spectrometry, displaying whether the MS2 mass of the first reduction partner peptide has been identified with a score greater than a predetermined threshold, and / or displaying whether the MS2 mass of the second reduction partner peptide has been identified with a score greater than a predetermined threshold; for each of the displaying steps of the confidence scoring system, assigning a single point where the corresponding peptide has been identified and not assigning a point where the corresponding peptide has not been identified; summing the single points; and assigning a disulfide identification confidence score based on the summation, where a larger sum indicates a higher confidence.

[0023] In some embodiments, performing mass spectrometry includes determining a disulfide scrambling percentage for each of one or more non-native disulfide bonds in the biomolecule. In an example, the disulfide scrambling percentage is the ratio of the average peak area of ​​the peptide containing the non-native disulfide bond to the sum of the average peak area of ​​the peptide containing the non-native disulfide bond plus the average peak area of ​​another two peptides containing a native disulfide bond corresponding to the cysteine ​​residue involved in the non-native disulfide bond.

[0024] In some embodiments, the concentration of TCEP is between 20 μM and 80 μM.

[0025] In some embodiments, the concentration of TCEP is about 40 mM.

[0026] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range where those values ​​represent the upper and lower limits of the range, and such ranges and all values ​​within such ranges are encompassed within the scope of the present disclosure. Each of the values ​​described above or discussed herein may be expressed with a 1%, 5%, 10%, or 20% variance. For example, a 10 mM concentration may be expressed as 10 mM ± 0.1 mM (1% variance), 10 mM ± 0.5 mM (5% variance), 10 mM ± 1 mM (10% variance), or 10 mM ± 2 mM (20% variance). Other embodiments will be apparent from a review of the detailed description below. [Brief explanation of the drawings]

[0027] Embodiments will be readily understood from the following detailed description taken in conjunction with the accompanying drawings and appended claims, and are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings. [Figure 1A]Illustrative examples of native and non-native disulfide bonds in biomolecules (e.g., therapeutic monoclonal antibodies) are shown. [Figure 1B] An example of how disulfide scrambling occurs in biomolecules is shown. [Figure 2] 1 shows the total ion current (TIC) and extracted ion chromatograph (EIC) for the disulfide formed from GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2) analyzed by liquid chromatography mass spectrometry (LC-MS), and tandem mass spectrometry demonstrating the ability to detect peptide fragments corresponding to GPSVFPLAPCSR (SEQ ID NO: 1) but not TYTCNVDHKPSNTK (SEQ ID NO: 2). [Figure 3] An exemplary LC-MS analysis of trypsin-digested monoclonal antibody (mAb) treated with various concentrations of tris(2-carboxyethyl)phosphine (TCEP) is shown. After separation by high-performance liquid chromatography (HPLC) at a flow rate of 50 μl / min (0.02% TFA and 0.08% FA), the mAb was digested with trypsin under non-reducing conditions to preserve disulfide bonds. After separation, the column eluate was treated with various concentrations of TCEP (0 mM, 0.4 mM, 0.8 mM, 2 mM, and 4 mM) and NH4OH (final concentration 0.12%) and then analyzed by mass spectrometry. TCEP and NH4OH were added to the eluate using a mixing T-tube at a flow rate of 2 μL / min. Data are presented as counts to mass-charge ratio (m / z). The disulfides and corresponding reduced peptides (R1 and R2) at various TCEP concentrations are shown. [Figure 4]1 is a graph showing MS signal of the peptide fragment VVSVLTVLHQDWLNGK (SEQ ID NO: 3) corresponding to mAb1 as a function of TCEP, NHOH, and glycine concentrations. Five conditions are shown, including control (no TCEP, no NHOH, no glycine), Sample 1 (2 mM TCEP, 0.12% NHOH), Sample 2 (2 mM TCEP, 0.12% NHOH, 2 mM glycine), Sample 3 (2 mM TCEP, 2 mM glycine, no NHOH), and Sample 4 (2 mM glycine only). Compared to the control, 2 mM TCEP in the presence of 0.12% NHOH causes a decrease in MS signal (Sample 1). Inclusion of 2 mM glycine in a sample containing 2 mM TCEP and 0.12% NHOH results in a slight improvement over the Sample 1 condition (Sample 2). 2 mM TCEP and 2 mM glycine in the absence of NH4OH (Sample 3) similarly provides only a slight improvement over the conditions of Samples 1 and 2. The sample containing only 2 mM glycine (Sample 4) shows approximately a 10-fold signal enhancement compared to the sample containing TCEP and glycine. For each of the samples shown in Figure 4, the conditions included a loading amount of 0.1 μg of mAb1 with 0.05% TFA in the mobile phase. [Figure 5A] Figure 5A shows the MS signals of two peptide fragments corresponding to mAb1, VVSVLTVLHQDWLNGK (SEQ ID NO: 3) (Figure 5A) and DTLMISR (SEQ ID NO: 4) (Figure 5B), as a function of TCEP concentration. Sample conditions included a loading amount of mAb1 of 0.1 μg, 0.05% TFA, and 2 mM glycine, and were treated with various concentrations of TCEP (0 μM, 20 μM, 40 μM, 80 μM, 200 μM, 400 μM, 800 μM, and 2000 μM). As shown, the MS signals decrease with increasing concentrations of TCEP. [Figure 5B]Figure 5A shows the MS signals of two peptide fragments corresponding to mAb1, VVSVLTVLHQDWLNGK (SEQ ID NO: 3) (Figure 5A) and DTLMISR (SEQ ID NO: 4) (Figure 5B), as a function of TCEP concentration. Sample conditions included a loading amount of mAb1 of 0.1 μg, 0.05% TFA, and 2 mM glycine, and were treated with various concentrations of TCEP (0 μM, 20 μM, 40 μM, 80 μM, 200 μM, 400 μM, 800 μM, and 2000 μM). As shown, the MS signals decrease with increasing concentrations of TCEP. [Figure 6A] Figure 6 shows MS signals of peptide fragments generated from trypsin digestion of mAb1 in the presence of various concentrations of TCEP. Figure 6A shows the MS signals for the disulfides corresponding to peptides NQVSLTCLVK (SEQ ID NO: 5) and WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 6), and Figure 6B shows the MS signals for the corresponding individual reduced peptides. Sample conditions included a loading amount of 1 μg of mAb1, 0.05% TFA, and 2 mM glycine, treated with the indicated TCEP concentrations. [Figure 6B] Figure 6 shows MS signals of peptide fragments generated from trypsin digestion of mAb1 in the presence of various concentrations of TCEP. Figure 6A shows the MS signals for the disulfides corresponding to peptides NQVSLTCLVK (SEQ ID NO: 5) and WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 6), and Figure 6B shows the MS signals for the corresponding individual reduced peptides. Sample conditions included a loading amount of 1 μg of mAb1, 0.05% TFA, and 2 mM glycine, treated with the indicated TCEP concentrations. [Figure 7A]Figure 7A shows the relative abundance of peptide fragments generated from trypsin digestion of mAb2 without reduction with TCEP, and Figure 7B shows the relative abundance of peptide fragments generated from trypsin digestion of mAb2 after partial reduction with 40 μM TCEP. For each of Figures 7A-7B, the reduced peptides correspond to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain disulfide peptides in their non-reduced form. The HPLC eluate was mixed with 40 μM TCEP to induce partial reduction of the disulfides, along with the addition of 2 mM glycine, resulting in a 10- to 20-fold enhancement of the MS signal compared to samples lacking glycine. The post-elution partial reduction methodology results in the disulfide peptides entering the mass spectrometer simultaneously with their reduced partner peptides. [Figure 7B] Figure 7A shows the relative abundance of peptide fragments generated from trypsin digestion of mAb2 without reduction with TCEP, and Figure 7B shows the relative abundance of peptide fragments generated from trypsin digestion of mAb2 after partial reduction with 40 μM TCEP. For each of Figures 7A-7B, the reduced peptides correspond to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain disulfide peptides in their non-reduced form. The HPLC eluate was mixed with 40 μM TCEP to induce partial reduction of the disulfides, along with the addition of 2 mM glycine, resulting in a 10- to 20-fold enhancement of the MS signal compared to samples lacking glycine. The post-elution partial reduction methodology results in the disulfide peptides entering the mass spectrometer simultaneously with their reduced partner peptides. [Figure 8A]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8B]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8C]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8D]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8E]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8F]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 8G]Figures 8A-8E show the relative abundance of peptide fragments generated from tryptic digestion of mAb2, showing the MS signals of the disulfide peptide and reduced partner peptide as a function of small molecule additives (e.g., glycine) and / or ion-pairing agents (e.g., TFA or FA) in the mobile phase. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. The sample conditions for each of Figures 8A-8G were as follows: Containing 5 μg of digested mAb2 and 40 μM TCEP, Figure 8A contains 0.05% TFA without glycine, Figure 8B contains 2 mM glycine and 0.05% TFA, Figure 8C contains 0.1% FA without glycine, Figure 8D contains 0.1% FA without glycine, Figure 8E contains 2 mM glycine and 0.1% FA, Figure 8F contains 0.1% FA without glycine, and Figure 8G contains 2 mM glycine and 0.1% FA. [Figure 9A] Figures 9A-9B show the MS signals of peptide fragments generated from trypsin digestion of mAb2. Figures 9A-9B show the reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide peptide in their non-reduced form. The conditions shown in each of Figures 9A-9B include: 40 μM TCEP, 2 mM glycine, and 0.05% TFA; 2000 μM TCEP, 0.12% NHOH, and 0.05% TFA; and 2000 μM TCEP, 0.12% NHOH, and 0.1% FA. Figure 9B shows the same data as Figure 9A, but zoomed the y-axis to show the improvement in MS signal with 40 μM TCEP and 2 mM glycine. The loading amount of mAb2 for each of Figures 9A-9B was 5 μg. [Figure 9B]Figures 9A-9B show the MS signals of peptide fragments generated from trypsin digestion of mAb2. Figures 9A-9B show the reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide peptide in their non-reduced form. The conditions shown in each of Figures 9A-9B include: 40 μM TCEP, 2 mM glycine, and 0.05% TFA; 2000 μM TCEP, 0.12% NHOH, and 0.05% TFA; and 2000 μM TCEP, 0.12% NHOH, and 0.1% FA. Figure 9B shows the same data as Figure 9A, but zoomed the y-axis to show the improvement in MS signal with 40 μM TCEP and 2 mM glycine. The loading amount of mAb2 for each of Figures 9A-9B was 5 μg. [Figure 10A] Figures 10A-10B show the MS signals of peptide fragments generated from tryptic digestion of mAb2. Data corresponding to the reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) (labeled R1) and TYTCNVDHKPSNTK (SEQ ID NO: 2) (labeled R2) are shown, which contain disulfide peptides in their non-reduced forms as indicated. Figure 10A shows the relative abundance of the disulfides and the corresponding reduced partner peptides. Figure 10B shows the m / z for the second stage (MS2) of mass spectrometry analysis, in which ions from the first stage (MS1) were selectively fragmented to generate the MS2 spectrum. For Figures 10A-10B, sample conditions included a 5 μg loading of mAb2, 40 μM TCEP, 2 mM glycine, and 0.05% TFA. [Figure 10B]Figures 10A-10B show the MS signals of peptide fragments generated from tryptic digestion of mAb2. Data corresponding to the reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) (labeled R1) and TYTCNVDHKPSNTK (SEQ ID NO: 2) (labeled R2) are shown, which contain disulfide peptides in their non-reduced forms as indicated. Figure 10A shows the relative abundance of the disulfides and the corresponding reduced partner peptides. Figure 10B shows the m / z for the second stage (MS2) of mass spectrometry analysis, in which ions from the first stage (MS1) were selectively fragmented to generate the MS2 spectrum. For Figures 10A-10B, sample conditions included a 5 μg loading of mAb2, 40 μM TCEP, 2 mM glycine, and 0.05% TFA. [Figure 10C] An exemplary mAb containing 16 unique cysteine ​​residues is shown. [Figure 11] Figure 11 shows the MS / MS spectra of the disulfide and the corresponding reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7). Peptide fragments were generated from trypsin digestion of mAb2. The data shown in Figure 11 indicate that the MS / MS spectrum of the disulfide contains a significant degree of complexity compared to the corresponding reduced partner peptide, and that performing post-column partial reduction with 40 μM TCEP allows for simpler characterization of any detectable scrambled disulfides. [Figure 12]Cysteine-containing peptides generated from trypsin digestion of mAb2 are shown, with the cysteine ​​residue number and an indication of whether the cysteine ​​is located on the heavy (H) or light (L) chain of mAb2 shown for reference. Tryptic peptides include: LSCAGSGFTFR (SEQ ID NO: 8), AEDTAVYYCAK (SEQ ID NO: 9), GPSVFPLAPCSR (SEQ ID NO: 1), STSESTAALGCLVK (SEQ ID NO: 7), TYTCNVDHKPSNTK (SEQ ID NO: 2), TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK (SEQ ID NO: 10), CK, NQVSLTCLVK (SEQ ID NO: 37), WQEGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 11), DIVMTQSPLSLPVTPGEPASISCR (SEQ ID NO: 12), VEAEDVGFYYCMQALQTPYTFGQGTK (SEQ ID NO: 13), SGTASVVCLLNNFYPR (SEQ ID NO: 14), VYACEVTHQGLSSPVTK (SEQ ID NO: 15), and SFNRGEC (SEQ ID NO: 16). [Figure 13] All possible scrambled disulfide bonds from mAb2 are shown, with the exception of the hinge region peptide YGPPCPPCPAPEFLGGPSVFLFPPKPK (SEQ ID NO: 46). The hinge region peptide is excluded because such peptides contain more than one cysteine ​​(e.g., two) and therefore form more than one disulfide bond; therefore, a scrambled version of this peptide would be expected to be very large and complex. Tandem mass spectrometry (MS / MS) using the targeted MS2 approach disclosed herein was used to identify the scrambled disulfide bonds. Five micrograms of trypsin-digested mAb2 was separated and partially reduced using 40 μM TCEP. 2 mM glycine was used to enhance the MS signal. Each disulfide bond is coded according to the confidence level of the determination (not detected, low confidence, medium confidence, high confidence, very high confidence). 71.6% of all possible scrambled disulfide bonds were identified with high or very high confidence, 17% of all possible scrambled disulfide bonds were identified with medium confidence, and 11.3% of all possible scrambled disulfide bonds were identified with low confidence. [Figure 14A] Figure 14 shows various trypsin digestion protocols used to generate peptide fragments, including disulfide peptides and the corresponding reduced partner peptides, for mAb2. Three different protocols are shown, including the standard operating procedure (SOP) for mAb2 (Figure 14A), the SOP for mAb3 (Figure 14B), and a low pH digestion kit (Figure 14C). Note that the SOP for mAb2 was used to generate the data shown in Figure 13. [Figure 14B] Figure 14 shows various trypsin digestion protocols used to generate peptide fragments, including disulfide peptides and the corresponding reduced partner peptides, for mAb2. Three different protocols are shown, including the standard operating procedure (SOP) for mAb2 (Figure 14A), the SOP for mAb3 (Figure 14B), and a low pH digestion kit (Figure 14C). Note that the SOP for mAb2 was used to generate the data shown in Figure 13. [Figure 14C] Figure 14 shows various trypsin digestion protocols used to generate peptide fragments, including disulfide peptides and the corresponding reduced partner peptides, for mAb2. Three different protocols are shown, including the standard operating procedure (SOP) for mAb2 (Figure 14A), the SOP for mAb3 (Figure 14B), and a low pH digestion kit (Figure 14C). Note that the SOP for mAb2 was used to generate the data shown in Figure 13. [Figure 15A] The alkylating agents iodoacetamide (IAM) and N-ethylmaleimide (NEM) are shown used with the procedure shown in Figure 14 to label free cysteine ​​residues prior to trypsin digestion. [Figure 15B] The alkylating agents iodoacetamide (IAM) and N-ethylmaleimide (NEM) are shown used with the procedure shown in Figure 14 to label free cysteine ​​residues prior to trypsin digestion. [Figure 16]14A-14C show overlays of UV chromatograms corresponding to trypsin-digested mAb2 using each of the exemplary digestion procedures shown, specifically, mAb2 SOP, mAb3 SOP, and the low pH digestion kit. The sample run to obtain the chromatograms contained 5 μg of trypsin-digested mAb2 in the presence of 2 mM glycine (or trypsin with low pH-resistant recombinant LysC in the case of the low pH digestion protocol). [Figure 17A] Selected time windows corresponding to the overlay of the UV chromatograms shown in Figure 16 are shown. Figure 17A shows the time window from about 14 minutes to 30 minutes, and Figure 17B shows the time window from about 34 minutes to 49 minutes. [Figure 17B] Selected time windows corresponding to the overlay of the UV chromatograms shown in Figure 16 are shown. Figure 17A shows the time window from about 14 minutes to 30 minutes, and Figure 17B shows the time window from about 34 minutes to 49 minutes. [Figure 18] Graphs showing MS signals for various native disulfides corresponding to mAb2 obtained by each of the three different digestion procedures exemplarily shown in Figures 14A-14C are shown. Native disulfides include C152H-C208H, C139H-C219L, C139H-C219L (deletion, indicating miscleavage), C22H-C96H, C372H-C430H, C266H-C326H, C139L-C199L, C23L-C93L, C231H-C231H, and C234H3-C234H. For each disulfide, the conditions, from left to right, include mAb2 SOP, mAb3 SOP, and the low-pH digestion kit procedure. Data shown as MS signals include all isotopic peaks of all major charge states. The sample run to generate the data in Figure 18 contains 5 μg of trypsin-digested mAb2 in the presence of 2 mM glycine (or trypsin with low pH-tolerant recombinant LysC in the case of the low pH digestion protocol). [Figure 19A]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb2 digest are shown. For each domain, the peptides shown were generated using the mAb2 SOP, mAb3 SOP, or low-pH digestion kit and are exemplarily shown in Figures 14A-14C. Figure 19A shows the VH / VL domain, Figure 19B shows the CH1 / CL domain, Figure 19C shows the CH2 domain, and Figure 19D shows the CH3 domain. Figure 19A (top) contains peptide DYAMTWVR (SEQ ID NO: 17), and Figure 19A (bottom) contains peptide SGQSPQLLIYLGSNR (SEQ ID NO: 18). Figure 19B (top) contains peptide DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK (SEQ ID NO: 19), and Figure 19B (bottom) contains peptide ADYEK (SEQ ID NO: 20). Figure 19C (top) contains peptide DTLMISR (SEQ ID NO: 39) and (bottom) contains peptide VVSVLTVLHQDWLNGK (SEQ ID NO: 38). Figure 19D (top) contains peptide GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 21) and (bottom) contains peptide TTPPVLDSDGSFFLYSR (SEQ ID NO: 22). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 19B]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb2 digest are shown. For each domain, the peptides shown were generated using the mAb2 SOP, mAb3 SOP, or low-pH digestion kit and are exemplarily shown in Figures 14A-14C. Figure 19A shows the VH / VL domain, Figure 19B shows the CH1 / CL domain, Figure 19C shows the CH2 domain, and Figure 19D shows the CH3 domain. Figure 19A (top) contains peptide DYAMTWVR (SEQ ID NO: 17), and Figure 19A (bottom) contains peptide SGQSPQLLIYLGSNR (SEQ ID NO: 18). Figure 19B (top) contains peptide DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK (SEQ ID NO: 19), and Figure 19B (bottom) contains peptide ADYEK (SEQ ID NO: 20). Figure 19C (top) contains peptide DTLMISR (SEQ ID NO: 39) and (bottom) contains peptide VVSVLTVLHQDWLNGK (SEQ ID NO: 38). Figure 19D (top) contains peptide GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 21) and (bottom) contains peptide TTPPVLDSDGSFFLYSR (SEQ ID NO: 22). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 19C]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb2 digest are shown. For each domain, the peptides shown were generated using the mAb2 SOP, mAb3 SOP, or low-pH digestion kit and are exemplarily shown in Figures 14A-14C. Figure 19A shows the VH / VL domain, Figure 19B shows the CH1 / CL domain, Figure 19C shows the CH2 domain, and Figure 19D shows the CH3 domain. Figure 19A (top) contains peptide DYAMTWVR (SEQ ID NO: 17), and Figure 19A (bottom) contains peptide SGQSPQLLIYLGSNR (SEQ ID NO: 18). Figure 19B (top) contains peptide DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK (SEQ ID NO: 19), and Figure 19B (bottom) contains peptide ADYEK (SEQ ID NO: 20). Figure 19C (top) contains peptide DTLMISR (SEQ ID NO: 39) and (bottom) contains peptide VVSVLTVLHQDWLNGK (SEQ ID NO: 38). Figure 19D (top) contains peptide GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 21) and (bottom) contains peptide TTPPVLDSDGSFFLYSR (SEQ ID NO: 22). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 19D]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb2 digest are shown. For each domain, the peptides shown were generated using the mAb2 SOP, mAb3 SOP, or low-pH digestion kit and are exemplarily shown in Figures 14A-14C. Figure 19A shows the VH / VL domain, Figure 19B shows the CH1 / CL domain, Figure 19C shows the CH2 domain, and Figure 19D shows the CH3 domain. Figure 19A (top) contains peptide DYAMTWVR (SEQ ID NO: 17), and Figure 19A (bottom) contains peptide SGQSPQLLIYLGSNR (SEQ ID NO: 18). Figure 19B (top) contains peptide DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK (SEQ ID NO: 19), and Figure 19B (bottom) contains peptide ADYEK (SEQ ID NO: 20). Figure 19C (top) contains peptide DTLMISR (SEQ ID NO: 39) and (bottom) contains peptide VVSVLTVLHQDWLNGK (SEQ ID NO: 38). Figure 19D (top) contains peptide GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 21) and (bottom) contains peptide TTPPVLDSDGSFFLYSR (SEQ ID NO: 22). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 20A] A table showing the disulfide scrambling percentage for mAb2 digests as a function of various digestion protocols is shown. Specifically, the mAb2 SOP, mAb3 SOP, and low pH digestion kit were individually tested on mAb2 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambled percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. For each digestion protocol shown in Figures 20A-C, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 20B]A table showing the disulfide scrambling percentage for mAb2 digests as a function of various digestion protocols is shown. Specifically, the mAb2 SOP, mAb3 SOP, and low pH digestion kit were individually tested on mAb2 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambled percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. For each digestion protocol shown in Figures 20A-C, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 20C] A table showing the disulfide scrambling percentage for mAb2 digests as a function of various digestion protocols is shown. Specifically, the mAb2 SOP, mAb3 SOP, and low pH digestion kit were individually tested on mAb2 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambled percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. For each digestion protocol shown in Figures 20A-C, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 20D] 20A-20C show the formula for determining disulfide scrambling percentage, used to determine the scrambling percentage in the experiments summarized in FIG. 20A-20C. [Figure 20E] A representative example of interference corresponding to the C208H-C93L disulfide peptide included in the table in Figure 20A above is shown. [Figure 21A]The relative abundances of high-abundance scrambled disulfides, as determined by quantification, are shown in Figures 20A-20C. In particular, Figure 21A shows C152H-C139H, which correspond to the disulfides of the corresponding reduction partner peptides STSESTAALGCLVK (SEQ ID NO: 7) and GPSVFPLAPCSR (SEQ ID NO: 1). Figure 21B shows C23L-C22H, which correspond to the disulfides of the corresponding reduction partner peptides DIVMTQSPLSLPVTPGEPASISCR (SEQ ID NO: 12) and LSCAGSGFTFR (SEQ ID NO: 8). For each of Figures 21A-21B, the relative abundance is shown as a function of the digestion protocol (mAb2 protocol, mAb3 protocol, or low pH digestion protocol). As shown in both Figures 21A-B, when digestion was performed using the mAb2 and mAb3 protocols, the chromatographs readily distinguish the high-abundance disulfides, whereas the chromatographs of scrambled disulfides are indistinguishable from the baseline noise under the low pH digestion conditions. For each condition, samples contained 5 μg of trypsin-digested mAb2 (or trypsin with low-pH-tolerant recombinant LysC in the low pH digestion protocol) and 2 mM glycine. [Figure 21B]The relative abundances of high-abundance scrambled disulfides, as determined by quantification, are shown in Figures 20A-20C. In particular, Figure 21A shows C152H-C139H, which correspond to the disulfides of the corresponding reduction partner peptides STSESTAALGCLVK (SEQ ID NO: 7) and GPSVFPLAPCSR (SEQ ID NO: 1). Figure 21B shows C23L-C22H, which correspond to the disulfides of the corresponding reduction partner peptides DIVMTQSPLSLPVTPGEPASISCR (SEQ ID NO: 12) and LSCAGSGFTFR (SEQ ID NO: 8). For each of Figures 21A-21B, the relative abundance is shown as a function of the digestion protocol (mAb2 protocol, mAb3 protocol, or low pH digestion protocol). As shown in both Figures 21A-B, when digestion was performed using the mAb2 and mAb3 protocols, the chromatographs readily distinguish the high-abundance disulfides, whereas the chromatographs of scrambled disulfides are indistinguishable from the baseline noise under the low pH digestion conditions. For each condition, samples contained 5 μg of trypsin-digested mAb2 (or trypsin with low-pH-tolerant recombinant LysC in the low pH digestion protocol) and 2 mM glycine. [Figure 22] Figure 14 shows UV overlays of the high-abundance scrambled disulfide (C152H-C139H) digested via each of the three different methods described above in Figure 14: mAb2 SOP, mAb3 SOP, and the low-pH digestion kit procedure. The UV chromatogram overlays show that the C152H-C139H disulfide corresponding to the reduced partner peptides STSESTAALGCLVK (SEQ ID NO: 7) and GPSVFPLAPCSR (SEQ ID NO: 1) is generated by the mAb2 and mAb3 digestion protocols, but not by the low-pH digestion protocol. Sample conditions for all three digestion procedures included 5 μg of trypsin-digested mAb2 (or trypsin with low-pH-resistant recombinant LysC in the case of the low-pH digestion protocol) and 2 mM glycine. [Figure 23A]Figure 23 shows various trypsin digestion protocols used to generate fragments containing disulfide peptides and the corresponding reduced partner peptides for mAb 5. Three different protocols are shown, including the mAb4 protocol (Figure 23A), the mAb5 protocol (Figure 23B), and the low pH digestion kit (Figure 23C). [Figure 23B] Figure 23 shows various trypsin digestion protocols used to generate fragments containing disulfide peptides and the corresponding reduced partner peptides for mAb 5. Three different protocols are shown, including the mAb4 protocol (Figure 23A), the mAb5 protocol (Figure 23B), and the low pH digestion kit (Figure 23C). [Figure 23C] Figure 23 shows various trypsin digestion protocols used to generate fragments containing disulfide peptides and the corresponding reduced partner peptides for mAb 5. Three different protocols are shown, including the mAb4 protocol (Figure 23A), the mAb5 protocol (Figure 23B), and the low pH digestion kit (Figure 23C). [Figure 24] 23A-23C show overlays of UV chromatograms corresponding to trypsin-digested mAb5 using each of the exemplary digestion procedures shown, specifically, the mAb4 protocol, the mAb5 protocol, and the low pH digestion kit. The sample run to obtain the chromatograms contained 5 μg of trypsin-digested mAb5 in the presence of 2 mM glycine (or trypsin with low pH-resistant recombinant LysC in the case of the low pH digestion protocol). [Figure 25A] Selected time windows corresponding to the overlay of the UV chromatograms shown in Figure 24 are shown. Figure 25A shows the time window from about 15 minutes to 33 minutes, and Figure 25B shows the time window from about 35 minutes to 67 minutes. [Figure 25B] Selected time windows corresponding to the overlay of the UV chromatograms shown in Figure 24 are shown. Figure 25A shows the time window from about 15 minutes to 33 minutes, and Figure 25B shows the time window from about 35 minutes to 67 minutes. [Figure 26]Graphs depicting MS signals for various native disulfides corresponding to mAb5 obtained by each of the three different digestion procedures exemplarily shown in Figures 23A-23C are shown. Native disulfides include C22H-C96H, C145H-C201H, C221H-C213L, C221H-C213L (deletion, indicating miscleavage), C227H-C227H, C230H-C230H, C262H-C322H, C368H-C426H, C23L-C88L, and C133L-C193L. For each disulfide, the conditions, from left to right, include the mAb4 protocol, the mAb5 protocol, and the low-pH digestion kit procedure. Data shown as MS signals include all isotopic peaks of all major charge states. The sample run to generate the data in Figure 26 contains 5 μg of trypsin-digested mAb5 in the presence of 2 mM glycine (or trypsin with low pH-resistant recombinant LysC in the case of the low pH digestion protocol). [Figure 27A] The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb5 digest are shown. For each domain, the peptides shown were generated using the mAb4 protocol, the mAb5 protocol, or the low-pH digestion kit, as exemplarily shown in Figures 23A-23C. Figure 27A shows the VH / VL domain, Figure 27B shows the CH1 / CL domain, Figure 27C shows the CH2 domain, and Figure 27D shows the CH3 domain. Figure 27A (top) contains peptide EVQLVESGGGLVQPGGSLR (SEQ ID NO: 23), and Figure 27A (bottom) contains peptide DIQMTQSPSSLSASVGDR (SEQ ID NO: 24). Figure 27B (top) contains peptide GPSVFPLAPSSK (SEQ ID NO: 25), and Figure 27B (bottom) contains peptide ADYEK (SEQ ID NO: 40). Figure 27C (top) contains peptide FNWYVDGVEVHNAK (SEQ ID NO: 26) and (bottom) contains peptide ALPAPIEK (SEQ ID NO: 27). Figure 27D (top) contains peptide DELTK (SEQ ID NO: 28) and (bottom) contains peptide TTPPVLDSDGSFFLYSK (SEQ ID NO: 29). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 27B] The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb5 digest are shown. For each domain, the peptides shown were generated using the mAb4 protocol, the mAb5 protocol, or the low-pH digestion kit, as exemplarily shown in Figures 23A-23C. Figure 27A shows the VH / VL domain, Figure 27B shows the CH1 / CL domain, Figure 27C shows the CH2 domain, and Figure 27D shows the CH3 domain. Figure 27A (top) contains peptide EVQLVESGGGLVQPGGSLR (SEQ ID NO: 23), and Figure 27A (bottom) contains peptide DIQMTQSPSSLSASVGDR (SEQ ID NO: 24). Figure 27B (top) contains peptide GPSVFPLAPSSK (SEQ ID NO: 25), and Figure 27B (bottom) contains peptide ADYEK (SEQ ID NO: 40). Figure 27C (top) contains peptide FNWYVDGVEVHNAK (SEQ ID NO: 26) and (bottom) contains peptide ALPAPIEK (SEQ ID NO: 27). Figure 27D (top) contains peptide DELTK (SEQ ID NO: 28) and (bottom) contains peptide TTPPVLDSDGSFFLYSK (SEQ ID NO: 29). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 27C]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb5 digest are shown. For each domain, the peptides shown were generated using the mAb4 protocol, the mAb5 protocol, or the low-pH digestion kit, as exemplarily shown in Figures 23A-23C. Figure 27A shows the VH / VL domain, Figure 27B shows the CH1 / CL domain, Figure 27C shows the CH2 domain, and Figure 27D shows the CH3 domain. Figure 27A (top) contains peptide EVQLVESGGGLVQPGGSLR (SEQ ID NO: 23), and Figure 27A (bottom) contains peptide DIQMTQSPSSLSASVGDR (SEQ ID NO: 24). Figure 27B (top) contains peptide GPSVFPLAPSSK (SEQ ID NO: 25), and Figure 27B (bottom) contains peptide ADYEK (SEQ ID NO: 40). Figure 27C (top) contains peptide FNWYVDGVEVHNAK (SEQ ID NO: 26) and (bottom) contains peptide ALPAPIEK (SEQ ID NO: 27). Figure 27D (top) contains peptide DELTK (SEQ ID NO: 28) and (bottom) contains peptide TTPPVLDSDGSFFLYSK (SEQ ID NO: 29). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 27D]The peak areas of peptides from various mAb domains (e.g., VH / VL, CH1, CL, CH2, CH3) of the mAb5 digest are shown. For each domain, the peptides shown were generated using the mAb4 protocol, the mAb5 protocol, or the low-pH digestion kit, as exemplarily shown in Figures 23A-23C. Figure 27A shows the VH / VL domain, Figure 27B shows the CH1 / CL domain, Figure 27C shows the CH2 domain, and Figure 27D shows the CH3 domain. Figure 27A (top) contains peptide EVQLVESGGGLVQPGGSLR (SEQ ID NO: 23), and Figure 27A (bottom) contains peptide DIQMTQSPSSLSASVGDR (SEQ ID NO: 24). Figure 27B (top) contains peptide GPSVFPLAPSSK (SEQ ID NO: 25), and Figure 27B (bottom) contains peptide ADYEK (SEQ ID NO: 40). Figure 27C (top) contains peptide FNWYVDGVEVHNAK (SEQ ID NO: 26) and (bottom) contains peptide ALPAPIEK (SEQ ID NO: 27). Figure 27D (top) contains peptide DELTK (SEQ ID NO: 28) and (bottom) contains peptide TTPPVLDSDGSFFLYSK (SEQ ID NO: 29). For each peptide and corresponding digestion protocol shown, sample conditions included 5 μg of digested mAb2 and 2 mM glycine. [Figure 28]Figure 1 shows cysteine-containing peptides generated from trypsin digestion of mAb5. For reference, the cysteine ​​residue number and an indication of whether the cysteine ​​is located on the heavy (H) or light (L) chain of mAb5 are shown. Tryptic peptides include: LSCAASGFTSSSYAMNWVR (SEQ ID NO: 30), AEDTAVYYCAK (SEQ ID NO: 41), STSGGTAALGCLVK (SEQ ID NO: 31), DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK (SEQ ID NO: 32), SCDK (SEQ ID NO: 33), TPEVTCVVVDVSHEDPEVK (SEQ ID NO: 34), NQVSLTCLVK (SEQ ID NO: 42), WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 43), VTITCR (SEQ ID NO: 35), FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLTFGQGTR (SEQ ID NO: 36), SGTASVVCLLNNFYPR (SEQ ID NO: 44), VYACEVTHQGLSSPVTK (SEQ ID NO: 45), and GEC / SFNRGEC. With regard to cysteine ​​C213L (GEC / SFNRGEC), GEC is a predicted / expected tryptic peptide, while SFNRGEC contains a very common and abundant tryptic cleavage. When dealing with specific disulfide bonds, the presence of one form confirms the presence of the other, since both the GEC and SFNRGEC peptides contain the same cysteine ​​residue. Thus, the C213L residue is represented herein as GEC / SFNRGEC, where SFNRGEC corresponds to SEQ ID NO: 16. [Figure 29]All possible scrambled disulfide bonds from mAb5 are shown, except for the hinge region peptide THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 47). Tandem mass spectrometry (MS / MS) using the targeted MS2 approach discussed herein was used to identify the scrambled disulfide bonds. Five micrograms of trypsin-digested mAb5 was separated and partially reduced using 40 μM TCEP. 2 mM glycine was used to enhance the MS signal. The digestion procedure included the mAb4 protocol (see FIG. 23A). Each disulfide bond is coded according to the confidence level of the determination (not detected, low confidence, medium confidence, high confidence, very high confidence). 63.3% of all possible scrambled disulfide bonds were identified with high or very high confidence, 20% of all possible scrambled disulfide bonds were identified with medium confidence, and 16.7% of all possible scrambled disulfide bonds were identified with low confidence. [Figure 30A] A table showing the disulfide scrambling percentage for mAb5 digests as a function of various digestion protocols is shown. Specifically, the mAb4 protocol, the mAb5 protocol, and the low pH digestion kit were individually tested for mAb5 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambling percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. The formula for determining disulfide scrambling percentages used to determine the scrambling percentages in the experiments summarized in Figures 30A-30C is the formula shown in Figure 20D. For each digestion protocol shown in Figures 30A-30C, the sample conditions included 5 μg digested mAb5 and 2 mM glycine. [Figure 30B]A table showing the disulfide scrambling percentage for mAb5 digests as a function of various digestion protocols is shown. Specifically, the mAb4 protocol, the mAb5 protocol, and the low pH digestion kit were individually tested for mAb5 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambling percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. The formula for determining disulfide scrambling percentages used to determine the scrambling percentages in the experiments summarized in Figures 30A-30C is the formula shown in Figure 20D. For each digestion protocol shown in Figures 30A-30C, the sample conditions included 5 μg digested mAb5 and 2 mM glycine. [Figure 30C] A table showing the disulfide scrambling percentage for mAb5 digests as a function of various digestion protocols is shown. Specifically, the mAb4 protocol, the mAb5 protocol, and the low pH digestion kit were individually tested for mAb5 to determine whether the rate of disulfide scrambling depended on the digestion protocol. All possible disulfides and the corresponding scrambling percentages, depending on the digestion protocol used, are shown. The data shown include all isotopic peaks of all major charge states. The formula for determining disulfide scrambling percentages used to determine the scrambling percentages in the experiments summarized in Figures 30A-30C is the formula shown in Figure 20D. For each digestion protocol shown in Figures 30A-30C, the sample conditions included 5 μg digested mAb5 and 2 mM glycine. [Figure 31]A series of m / z determinations corresponding to the dimeric disulfide of STSGGTAALGCLVK (SEQ ID NO: 31) are shown. The top chromatograph shows the m / z for the disulfide alone, the middle chromatograph shows the m / z for the fully reduced peptide corresponding to the disulfide peptide alone, and the bottom chromatograph shows the m / z for the partially reduced peptide corresponding to the disulfide peptide. Together, the data show the altered isotopic peak pattern in the partially reduced sample. The isotopic peaks are sharper in the top chromatograph due to data collected at higher MS1 resolution (without TCEP, hence "disulfide only"). MS1 resolution was reduced when performing post-column TCEP reduction using targeted MS2 methodology to maximize the number of MS2 scans collected to increase signal near the apex of each reduced peptide peak. DETAILED DESCRIPTION OF THE INVENTION

[0028] Before the present invention is described, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Any embodiments or features of embodiments may be combined with each other, and such combinations are expressly encompassed within the scope of the present invention. In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification and which show exemplary embodiments that may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope.

[0029] Various operations may be described as multiple separate operations in a manner that may be helpful in understanding the embodiments, however, the order of description should not be construed to imply that these operations are order dependent.

[0030] The description may use perspective-based descriptions such as top / bottom, back / front, and top / bottom, etc. Such descriptions are used merely for ease of discussion and are not intended to limit the application of the disclosed embodiments.

[0031] The terms "coupled" and "connected," along with their derivatives, may be used. It should be understood that these terms are not intended as synonyms for each other. Rather, in particular embodiments, "connected" may be used to indicate that two or more elements are in direct physical or electrical contact with each other. "Coupled" may mean that two or more elements are in direct physical or electrical contact. However, "coupled" may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other.

[0032] For purposes of illustration, a phrase in the form "A / B" or "A and / or B" means (A), (B), or (A and B). For purposes of illustration, a phrase in the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C). For purposes of illustration, a phrase in the form "(A)B" means (B) or (AB), i.e., A is an optional element.

[0033] The description may use the terms "embodiment" or "embodiments," each of which may refer to one or more of the same or different embodiments. Furthermore, when used in reference to embodiments, the terms "comprising," "including," "having," etc. are synonymous and generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0034] With respect to the use of any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specific recited numerical value, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0036] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0037] Abbreviations used herein ACN: acetonitrile AU: absorbance units CH:Steady weight CL: Stationary light Cys: cysteine DDA: Data-Dependent Acquisition EIC: Extracted ion chromatograph E / S: Enzyme / Substrate FA: Formic acid HILIC: Hydrophilic Interaction Liquid Chromatography HPLC: High-Performance Liquid Chromatography IAM: Iodoacetamide IgG: immunoglobulin G LC: Liquid chromatography LC-MS: Liquid chromatography-mass spectrometry mAb: monoclonal antibody MPA: Mobile phase A MPB: Mobile phase B MS: Mass spectrometry MS / MS: Tandem mass spectrometry MS1: the first mass spectrometer of the tandem mass spectrometer MS2: second mass spectrometer in a tandem mass spectrometer MW: molecular weight NEM: N-ethylmaleimide PRM: Parallel Reaction Monitoring RPLC: reversed-phase liquid chromatography RPLC-MS / MS: reversed-phase liquid chromatography tandem mass spectrometry TCEP: Tris(2-carboxyethyl)phosphine TFA: Trifluoroacetic acid TIC: Total ion current UV: Ultraviolet light VH: Variable weight VL: Variable Light

[0038] definition The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") composed of four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR," or "V"). H ) and heavy chain constant region (C H 1 domain, C H 2 domain, and C H In various embodiments, the heavy chain may be of the IgG isotype. In some cases, the heavy chain is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain is of the isotype IgG1 or IgG4, and optionally includes a chimeric hinge region of the isotype IgG1 / IgG2 or IgG4 / IgG2. Each light chain comprises a light chain variable region ("LCVR, light chain variable region or "V"). L ) and light chain constant region (C L ) V H Area and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V Lconsists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. For a review of antibody structure, see Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, 27(1) Dev. Comp. Immunol. 55-77 (2003); and M. Potter, Structural correlates of immunoglobulin diversity, 2(1) Surv. Immunol. Res. 27-42 (1983).

[0039] The term antibody also encompasses "bispecific antibodies," which include heterotetrameric immunoglobulins capable of binding to two or more different epitopes. One half of a bispecific antibody, containing a single heavy chain and a single light chain and six CDRs, binds to one antigen or epitope, while the other half of the antibody binds to a different antigen or epitope. In some cases, bispecific antibodies can bind to the same antigen but to different or non-overlapping epitopes. In some cases, both halves of a bispecific antibody have the same light chain, while retaining the dual specificity. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527 (December 30, 2010).

[0040] The term "antigen-binding portion" of an antibody (or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), (iii) a Fd fragment (composed of the VH and CH1 domains), (iv) a Fv fragment (composed of the VL and VH domains of a single arm of an antibody), (v) a dAb fragment (composed of the VH domain; Ward et al. (1989) Nature 241:544-546), (vi) an isolated CDR, and (vii) a scFv (composed of the two domains (VL and VH) of an Fv fragment linked by a synthetic linker to form a single protein chain, where the pair of VL and VH domains forms a monovalent molecule). Other forms of single chain antibodies, such as diabodies, are also encompassed by the term "antibody" (see, e.g., Holliger et al. (1993) 90 PNAS USA 6444-6448; and Poljak et al. (1994) 2 Structure 1121-1123).

[0041] Furthermore, antibodies and antigen-binding fragments thereof can be obtained using standard recombinant DNA techniques commonly known in the art (see Sambrook et al., 1989). Methods for generating human antibodies in transgenic mice are also known in the art. For example, using VELOCIMMUNE® technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies against a desired antigen having human variable regions and mouse constant regions are first isolated. VELOCIMMUNE® technology involves the generation of transgenic mice whose genomes contain human heavy chain variable regions and human light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing the human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions, and the DNA is then expressed in a cell capable of expressing a fully human antibody.

[0042] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the invention may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. This term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. This term is not intended to include antibodies isolated from or produced in a human subject.

[0043] As used herein, the term "disulfide" refers to a covalent bond derived from two thiol groups. In proteins, such as monoclonal antibodies, these bonds are formed between the thiol groups of two cysteine ​​amino acids. Disulfide bonds stabilize the globular structure of proteins and contribute to maintaining proteins in their respective conformations, thus playing an important role in protein folding and stability. As discussed herein, a disulfide or disulfide peptide encompasses two peptides covalently linked via cysteine ​​residues on each corresponding peptide, and each of the two peptides in reduced form is referred to as a "reduced partner peptide." Such disulfide peptides can be generated by protease digestion (e.g., trypsin protease digestion and / or recombinant Lys-C protease digestion) under non-reducing conditions in which the disulfide bond remains intact. Such disulfide peptides can then be reduced to their corresponding reduced partner peptides using a reducing agent (e.g., DTT, TCEP, etc.). As used herein, the term "scrambled disulfide" or "disulfide scrambling" encompasses disulfide bonds that are non-native to a particular biomolecule, such as a monoclonal antibody.

[0044] The term "hydrophilic interaction chromatography" or HILIC is intended to include a process using a hydrophilic stationary phase and a hydrophobic organic mobile phase, in which hydrophilic compounds are retained longer than hydrophobic compounds. In certain embodiments, the method utilizes a water-miscible solvent mobile phase.

[0045] As used herein, the term "sample" includes a mixture of molecules containing at least an analyte molecule (e.g., a disulfide peptide and / or corresponding reduced partner peptide, such as obtained from a monoclonal antibody), which is manipulated according to the methods of the invention, including, for example, separation, analysis, extraction, or enrichment.

[0046] The terms "analysis" or "analyzing," as used herein, are used interchangeably and refer to any of a variety of methods for separating, detecting, isolating, purifying, solubilizing, and / or characterizing a molecule of interest (e.g., a biomolecule, including, but not limited to, a monoclonal antibody, a disulfide peptide, and / or the corresponding reduced partner peptide). Examples include, but are not limited to, solid-phase extraction, solid-phase microextraction, electrophoresis, mass spectrometry (e.g., ESI-MS, tandem mass spectrometry (MS / MS), or MALDI-MS), liquid chromatography, e.g., high performance, e.g., reversed-phase, normal-phase, or size-exclusion, ion-pair liquid chromatography, liquid-liquid extraction, e.g., accelerated fluid extraction, supercritical fluid extraction, microwave-assisted extraction, membrane extraction, Soxhlet extraction, precipitation, clarification, electrochemical detection, staining, elemental analysis, Edmund degradation, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection, and combinations thereof.

[0047] "Electrospray ionization mass spectrometry" or "ESI-MS" is a technique used in mass spectrometry to generate ions using electrospray, in which high voltage is applied to a liquid to create an aerosol. For example, electrospray generates ions from proteins / peptides in solution, allowing fragile molecules to be ionized intact and preserving noncovalent interactions. Electrospray ionization is the ion source of choice for combining liquid chromatography with mass spectrometry (LC-MS). Analysis can be performed online by directly feeding the liquid eluting from the LC column into the electrospray, or offline by collecting fractions that are subsequently analyzed in a classical nanoelectrospray mass analyzer. LC-MS can be used for protein characterization, including biomarker quantification, sequence variant analysis, and the identification and quantification of disulfide peptides and their corresponding reduced-partner peptides.

[0048] "Tandem mass spectrometry" or "MS / MS" or "MS2 Tandem mass spectrometry is a technique used to analyze biomolecules such as proteins and peptides. In tandem mass spectrometry, a first mass analyzer (MS1) selects ions of one specific mass-to-charge ratio (m / z) (or range of mass-to-charge ratios) from ions supplied via an ion source (e.g., a sample ionized via ESI, MALDI, etc.). The ions are fragmented, and a second mass analyzer (MS2) records the mass spectrum of the fragment ions. Tandem mass spectrometry involves three distinct steps: selection, fragmentation, and detection. The separation of these steps can be achieved spatially or temporally. Typical tandem mass spectrometry instruments in space include QqQ (triple quadrupole), QTOF (quadrupole time-of-flight), and hybrid ion trap / FTMS (Fourier transform mass spectrometry). Temporal tandem MS / MS instruments include ion trap and FT-ICR MS (Fourier transform ion cyclotron resonance mass spectrometry). The fragmentation step is generally achieved by colliding selected ions with a neutral gas in a process called collisional activation (CA) or collision-induced dissociation (CID).

[0049] "Partially reduced" or "partial reduction," as discussed herein, encompasses treating a sample (e.g., a biomolecule or fragment of a biomolecule) with a reducing agent at a concentration and / or for a time such that some of the disulfide bonds present in the sample are reduced to their corresponding reduced counterparts, but not all of the disulfides present in the sample are reduced.

[0050] "Parallel reaction monitoring" or "PRM," as discussed herein, refers to a targeted proteomics technique used to quantify multiple proteins / peptides in the same experiment. In PRM, all fragment ions, rather than only selected fragment ions, are measured after fragmentation of a selected precursor. PRM is typically performed on an Orbitrap or Time-of-Flight (ToF) analyzer. PRM can reduce assay development time because target transitions (product ions) do not need to be preselected. PRM eliminates most interferences, improving the precision and attomole-level limits of detection and quantitation.

[0051] "Data-dependent acquisition" or "DDA," as discussed herein, refers to an acquisition mode in tandem mass spectrometry, in which the mass spectrometer selects the most intense peptide ions in the first stage of tandem mass spectrometry, which are then fragmented and analyzed in the second stage of mass spectrometry.

[0052] General Description Therapeutic antibodies are increasingly being used in the treatment of diseases such as cancer, infectious diseases, and other conditions. Therapeutic antibodies function, for example, by binding to antigens (e.g., present on target cells) to attract disease-fighting molecules and / or induce cell death via an immune system response, and / or block processes (e.g., viral entry into cells or receptor-ligand interactions) that otherwise result in infection and / or disease. To be effective, therapeutic antibodies must be stable and properly folded. Improper folding and / or reduced stability can contribute to the ineffectiveness of such molecules. One reason for improper folding and / or reduced stability includes the formation of non-natural (e.g., scrambled) disulfide bonds at some point in the therapeutic antibody development process. Therefore, there is a need for a method that easily enables the determination of scrambled disulfide bonds in therapeutic biomolecules, including, but not limited to, therapeutic antibodies.

[0053] Disclosed herein is a new methodology for mass spectrometry-based characterization of scrambled disulfide bonds in biomolecules. The disclosed methodology improves the ability to reliably detect and quantify scrambled disulfide bonds in biomolecules, including, but not limited to, monoclonal antibodies. As discussed herein, it has been surprisingly found that the improvement in mass spectral signal by including a small molecule additive (e.g., glycine) in a liquid chromatography mobile phase solution is specifically dependent on the concentration of the reducing agent used to partially reduce sample components eluted from the liquid chromatography separation column. It has also been surprisingly found that the improvement in mass spectral signal and the ability to use mass spectrometry to detect reduced partner peptides corresponding to disulfide peptide fragments of biomolecules are highly dependent on the choice of ion-pairing agent included in the mobile phase during liquid chromatography separation. Furthermore, it has surprisingly been discovered that biomolecule digestion conditions before the digested sample components are separated and analyzed by mass spectrometry can reliably induce artificial disulfide scrambling under certain conditions, and that this artificial disulfide scrambling can be avoided by performing the digestion procedure at an acidic pH. The above findings, as disclosed herein, enable an improved ability to reliably detect and quantitate scrambled disulfide bonds in biomolecules using tandem mass spectrometry (MS / MS). Thus, the disclosed methodology has a wide range of applications in enabling the ability to screen therapeutic biomolecules (e.g., monoclonal antibodies) for disulfide scrambling, which in turn can improve the efficacy of therapeutics derived from the use of such molecules.

[0054] The separation of protease-digested biomolecules by liquid chromatography methods discussed herein can include the use of one or more buffer gradients. One or more of the buffers can, in some instances, include an ion-pairing agent, including, but not limited to, formate, acetate, TFA, and salt. In a preferred embodiment, the ion-pairing agent includes TFA.

[0055] When two buffers are used, the concentration of the first buffer can decrease while the concentration or percentage of the second buffer increases over the course of a chromatographic run. For example, the percentage of the first buffer can decrease from about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% to about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% over the course of a chromatographic run. As another example, the percentage of the second buffer may increase over the course of the run from about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% to about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40%. Optionally, the concentrations or percentages of the first and second buffers may be returned to their starting values ​​at the end of the chromatographic run. As an example, the percentage of the first buffer may be varied in five steps: from 85% to 63%, to 59%, to 10%, to 85%. The percentage of the second buffer in the same steps changes from 15% to 37%, to 41%, to 90%, to 15%. The percentage can change gradually as a linear gradient or nonlinearly (e.g., stepwise). For example, the gradient can be multiphasic (e.g., biphasic, triphasic, etc.). In some embodiments, the methods described herein use a decreasing acetonitrile buffer gradient corresponding to an increase in the polarity of the mobile phase without the use of an ion-pairing agent.

[0056] In some embodiments, applying a mobile gradient to the separation column comprises applying a first mobile gradient buffer to the separation column, wherein the first mobile phase buffer comprises TFA and a small molecule additive (e.g., an amino acid), and applying a second mobile gradient buffer to the separation column, wherein the second mobile phase buffer comprises TFA and a small molecule additive (e.g., an amino acid) in ACN.

[0057] In various embodiments, the small molecule additive is selected from glycine, alanine, serine, valine, N-acetylglycine, methionine, β-alanine, aspartic acid, or N-methylglycine. In some embodiments, the amino acid is selected from glycine, alanine, serine, or valine. In some embodiments, the amino acid is alanine. In some embodiments, the amino acid is serine. In some embodiments, the amino acid is valine. In some embodiments, the amino acid in the first mobile phase buffer is glycine. In some embodiments, the amino acid in the second mobile phase buffer is glycine. In some embodiments, the amino acid in the first and second mobile phase buffers is glycine. In some embodiments, the small molecule additive (e.g., amino acid) in the first and / or second mobile phase buffer is a small molecule (e.g., a modified amino acid) or one of the other amino acids identified above or herein.

[0058] The concentration of the small molecule additive (e.g., amino acid) in the mobile phase buffer is about 0.5 mM to about 5 mM, for example, about 0.5 mM to about 3 mM, about 1 mM, and about 2 mM, including 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, and the like. The concentration of the small molecule additive (e.g., an amino acid) may be less than 5 mM. In some embodiments, the small molecule additive is glycine at a concentration of less than 5 mM. In some embodiments, the amino acid in the first mobile phase buffer is glycine, and the concentration is about 1 to about 2 mM glycine. In some embodiments, the amino acid in the second mobile phase buffer is glycine, and the concentration is about 1 to about 2 mM glycine. In some embodiments, the concentration of glycine in the first mobile phase buffer is about 1 mM. In some embodiments, the concentration of glycine in the first mobile phase buffer is about 2 mM. In some embodiments, the amino acid in the second mobile phase buffer is glycine, and the concentration is about 1 to about 2 mM glycine. In some embodiments, the concentration of glycine in the second mobile phase buffer is about 1 mM. In some embodiments, the concentration of glycine in the second mobile phase buffer is about 2 mM. In some embodiments, the amino acid in both the first and second mobile phase buffers is glycine, and the concentration is about 1 to about 2 mM glycine.

[0059] In some embodiments, the TFA concentration in the first mobile phase is about 0.03% to 0.15% TFA in HO, e.g., about 0.03% to 0.1%. In some embodiments, the TFA concentration is about 0.05% to about 0.1% TFA in HO. For example, the TFA concentration is about 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% in HO. In some embodiments, the TFA concentration in the second mobile phase comprises about 0.05% TFA in 80% ACN and 20% HO, or about 0.1% TFA in 80% ACN and 20% HO. In some embodiments, the concentration of ACN in the second mobile phase is about 60% to 100%, e.g., 80% to 100%, including 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0060] In some embodiments, the sample comprises peptides. In some embodiments, the sample comprises peptides linked via cysteine ​​residues through disulfide bond linkages. For example, the sample may comprise disulfide peptides obtained by proteolytic digestion of a monoclonal antibody or other biomolecule under non-reducing conditions. In some embodiments, the monoclonal antibody is an IgG1, IgG2, IgG3, IgG4, or mixed isotype monoclonal antibody.

[0061] In some embodiments, the method includes preparing the sample before contacting the sample with the separation column under conditions that allow sample components to bind to the substrate. In some embodiments, sample preparation includes contacting the sample with a denaturing / alkylation solution under conditions that allow denaturation and alkylation of the sample. In an example, the denaturant is urea. A concentration of urea sufficient to cause sample denaturation (e.g., protein denaturation) can be 7 to 9 M urea, e.g., 8 M urea. In an example, the denaturing / alkylation solution includes an alkylating agent. In one example, the alkylating agent can include iodoacetamide (IAM). Additionally or alternatively, the alkylating agent can include N-ethylmaleimide (NEM). In an example, the alkylating agent in the denaturing / alkylation solution is at a concentration of about 0.5 mM to about 10 mM, e.g., about 1 mM to about 8 mM. In some examples, the alkylating agent in the denaturing / alkylation solution is NEM, and the concentration is about 6 mM to about 10 mM, e.g., 8 mM. In some examples, the alkylating agent in the denaturing / alkylating solution is IAM, and the concentration is about 0.5 mM to about 5 mM, for example, about 1 mM, or about 2 mM, or about 3 mM, or about 4 mM, or about 5 mM, for example, 2.4 mM. In some examples, the denaturing / alkylating solution is contacted with the sample for a period of about 10 minutes to about 60 minutes, for example, 20 minutes, 30 minutes, 40 minutes, or 50 minutes. In examples, the denaturing / alkylating solution is at a temperature of about 40°C to about 60°C, for example, about 50°C. In examples, the pH of the denaturing / alkylating solution is acidic. For example, the pH may be about 5 to about 6.5, for example, about 5.5 to about 6.0, for example, about 5.7.

[0062] In some examples, preparing the sample before contacting the sample with the separation column under conditions that allow sample components to bind to the substrate includes contacting the sample with a pre-digestion solution after contacting the sample with a denaturing / alkylating solution. In examples, the pre-digestion solution contains a protease, for example, a serine protease. In examples, the protease is endoproteinase LysC. In examples, the protease is a recombinant protease, for example, recombinant endoproteinase LysC. In examples, the pre-digestion solution contains the protease at an enzyme / substrate ratio of about 1:2 to about 1:20, for example, about 1:5 to about 1:15, for example, about 1:10, respectively. In examples, the pre-digestion solution is contacted with the sample for about 30 minutes to 2 hours, for example, 1 hour. In examples, the pre-digestion solution is at a temperature of about 35 to 40°C, for example, about 37°C. In an example, the pre-digestion solution is of an acidic pH, such as a pH of about 5 to about 6, such as about 5.2 to 5.5, such as about 5.3.

[0063] In some examples, preparing the sample prior to contacting the sample with the separation column under conditions that allow sample components to bind to the substrate includes contacting the sample with a denaturing / alkylating solution, contacting the sample with a pre-digestion solution, and then contacting the sample with a digestion solution. In examples, the digestion solution includes a first protease, e.g., a serine protease. In examples, the first protease is endoproteinase LysC. In examples, the first protease is a recombinant protease, e.g., recombinant endoproteinase LysC. In examples, the digestion solution includes the first protease at an enzyme / substrate ratio of about 1:2 to about 1:20, e.g., about 1:5 to about 1:15, e.g., about 1:10, respectively. In examples, the digestion solution includes a second protease, e.g., a serine protease. In examples, the second protease is trypsin. In an example, the digestion solution comprises the second protease at an enzyme / substrate ratio of about 1:2 to about 1:10, e.g., about 1:5, respectively. In an example, the digestion solution is contacted with the sample for about 1 hour to about 4 hours, e.g., 3 hours. In an example, the digestion solution is at a temperature of about 35-40°C, e.g., about 37°C. In an example, the digestion solution is at an acidic pH, e.g., about 5 to about 6, e.g., about 5.2-5.5, e.g., about 5.3.

[0064] As discussed herein, a partial reduction procedure is performed on sample components eluted from a liquid chromatography column after their separation. It can be understood that prior to the partial reduction procedure, the sample components include unreduced, digested biomolecules (e.g., monoclonal antibodies). The partial reduction procedure involves treating the eluted sample components with a reducing agent. In an example, the reducing agent is TCEP. In an example, the concentration of the reducing agent used to partially reduce the eluted sample components is about 20 μM to about 100 μM, e.g., about 30 μM to about 60 μM, e.g., about 40 μM. As discussed in Example 1 below and at least in Figures 6A-6B, it was surprisingly found that the MS signal of the reduced partner peptide is highly dependent on a specific range of TCEP concentrations (about 20 μM to about 100 μM), and that reduced MS signals are associated with TCEP concentrations outside the specific range (e.g., both higher and lower concentrations). This finding was particularly surprising because it was expected that higher concentrations of TCEP (e.g., 400 μM to 2 mM or higher) would result in increased abundance of the reduced partner peptide corresponding to the disulfide peptide, thereby resulting in increased MS signal for the corresponding reduced partner peptide at higher TCEP concentrations. Therefore, the finding that lower TCEP concentrations (e.g., 20 to 100 μM, e.g., about 40 μM) improved, rather than diminished, the ability to detect the corresponding reduced partner peptide was unexpected. In examples, the partial reduction procedure involves further treating the eluted sample components with NH4OH. In examples, the final percentage of NH4OH is about 0.05% to about 0.2%, e.g., about 0.12%. In examples, the partial reduction procedure is performed on the eluted sample components for a duration of about 0.5 seconds to about 5 seconds, e.g., about 1 to 3 seconds, e.g., about 2 seconds. In examples, the efficiency corresponding to the partial reduction procedure is about 1 to 3%.

[0065] In some embodiments, the separation column is a liquid chromatography (LC) separation column. Liquid chromatography, including HPLC, can be used to separate structures such as peptides, including disulfide peptides. Various forms of liquid chromatography can be used to separate these structures, including anion exchange chromatography, reversed-phase HPLC, size-exclusion chromatography, high-performance anion exchange chromatography, and normal phase (NP) chromatography (including NP-HPLC) (see, e.g., Alpert et al., J. Chromatogr. A 676:191-202 (1994)). Hydrophilic interaction chromatography (HILIC) is a variant of NP-HPLC that can be performed using a partially aqueous mobile phase, allowing for normal-phase separation of peptides, disulfide peptides, carbohydrates, nucleic acids, and many proteins. The elution order in HILIC is from least polar to most polar, which is the opposite of that in reversed-phase HPLC. HPLC can be performed on HPLC systems from, for example, Waters (eg, a Waters 2695 Alliance HPLC system), Agilent, Perkin Elmer, Gilson, and the like.

[0066] NP-HPLC, preferably HILIC, can be used in some examples in the methods described herein. NP-HPLC separates analytes based on polar interactions between the analytes and a stationary phase (e.g., a substrate). Polar analytes associate with and are retained by the polar stationary phase. The adsorption strength increases with increasing analyte polarity, and the interaction of polar analytes with the polar stationary phase (relative to the mobile phase) increases elution time. The use of more polar solvents in the mobile phase decreases the retention time of the analytes, while more hydrophobic solvents tend to increase retention time.

[0067] Various types of substrates can be used with NP-HPLC, for example, for column chromatography, including silica, amino, amide, cellulose, cyclodextrin, and polystyrene substrates. For example, examples of useful substrates that can be used in column chromatography include polysulfoethylaspartamide (e.g., manufactured by PolyLC), sulfobetaine substrates such as ZIC®-HILIC (e.g., manufactured by SeQuant), POROS® HS (e.g., manufactured by Applied Biosystems), POROS® S (e.g., manufactured by Applied Biosystems), polyhydroethylaspartamide (e.g., manufactured by PolyLC), Zorbax 300 SCX (e.g., manufactured by Agilent), PolyGLYCOPLEX® (e.g., manufactured by PolyLC), Amide-80 (e.g., manufactured by Tosohaas), TSK GEL® Amide-80 (e.g., manufactured by Tosohaas), polyhydroxyethyl A (e.g., manufactured by PolyLC), Glyco-Sep-N (e.g., manufactured by Oxford GlycoSciences), and Atlantis HILIC (e.g., manufactured by Waters). In some embodiments, the disclosed methods include columns utilizing one or more of the following functional groups: carbamoyl groups, sulfopropyl groups, sulfoethyl groups (e.g., poly(2-sulfoethylaspartamide)), hydroxyethyl groups (e.g., poly(2-hydroxyethylaspartamide)), and aromatic sulfonic acid groups.

[0068] In some embodiments, reversed-phase HPLC can be used with the methods described herein. Reverse-phase HPLC separates analytes based on non-polar interactions between the analytes and a stationary phase (e.g., a substrate). Non-polar analytes associate with and are retained by the non-polar stationary phase. The adsorption strength increases with the non-polarity of the analyte, and the interaction of non-polar analytes with the non-polar stationary phase (relative to the mobile phase) increases elution time. The use of more non-polar solvents in the mobile phase decreases the retention time of the analytes, while more polar solvents tend to increase retention time.

[0069] The column temperature can be maintained constant throughout the chromatographic run, for example, using a commercially available column heater. In some embodiments, the column is maintained at a temperature of about 18°C ​​to about 70°C, e.g., about 30°C to about 60°C, about 40°C to about 50°C, e.g., about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C. In some embodiments, the column temperature is about 40°C.

[0070] The mobile phase flow rate can be about 0 to about 100 mL / min. For analytical purposes, flow rates typically range from 0 to 10 mL / min, while for preparative HPLC, flow rates greater than 100 mL / min can be used. For example, the flow rate can be about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5 mL / min (or higher for preparative HPLC). Substituting a column with the same packing, the same length, but a smaller diameter requires a reduction in flow rate to maintain the same retention times and peak resolution as seen with the wider diameter column. In some embodiments, a flow rate equivalent to about 1 mL / min is used for a 4.6 x 100 mm, 5 μm column.

[0071] In some embodiments, the run time can be about 15 to about 240 minutes, e.g., about 20 to about 70 minutes, about 30 to about 60 minutes, about 40 to about 90 minutes, about 50 to about 100 minutes, about 60 to about 120 minutes, or about 50 to about 80 minutes.

[0072] In examples, following the partial reduction procedure, the partially reduced sample is analyzed by mass spectrometry. In examples, the partially reduced sample includes a disulfide peptide and a corresponding reduction partner peptide. In examples, the disulfide peptide and the corresponding reduction partner peptide enter the mass spectrometer at approximately the same time.

[0073] In an example, analyzing a sample by mass spectrometry involves acquiring MS1 and MS2 spectra via reliance on a tandem mass spectrometer configuration. In an example, acquiring MS2 spectra involves a targeted MS2 approach. Such a targeted MS2 approach can include targeting only protease-digested (e.g., trypsin-digested) reduced partner peptides corresponding to disulfide peptides containing cysteine ​​residues. In an example, targeting only corresponding reduced partner peptides containing cysteines allows for dramatically simpler characterization of any scrambled disulfides compared to approaches in which MS / MS is used in attempts to target disulfide peptides, due to the exponentially greater number of possible fragmentations of disulfide peptides compared to their corresponding reduced partner peptides. Using a monoclonal antibody as an example, such a monoclonal antibody can contain a specific number of cysteine ​​residues (e.g., 16). By using the partial reduction procedure discussed herein, only 15 reduced tryptic peptides containing cysteine ​​(15 tryptic peptides minus the antibody hinge region) need to be targeted in the MS2 step. Thus, in an example, analyzing a sample by mass spectrometry can involve building a PRM inclusion list using only cysteine-containing protease-generated fragments and scanning across the entire gradient. In an example, MS1 resolution can be reduced as the number of MS2 scans increases.

[0074] In an example, analyzing a sample by mass spectrometry includes assigning a disulfide identification confidence score for each possible disulfide peptide probability in a particular biomolecule. Assigning a disulfide identification confidence score, in an example, includes assigning a point for each affirmatively answered query among several queries associated with the generated MS / MS data. In an example, the queries may include, but are not limited to, whether the MS1 mass of the disulfide peptide has been identified, whether the MS1 mass of a first corresponding reduced peptide has been identified, whether the MS1 mass of a second corresponding reduced peptide has been identified, whether a bispecific MS2 identification of the first corresponding reduced peptide has been identified with a score greater than a predetermined threshold, and whether a bispecific MS2 identification of the second corresponding reduced peptide has been identified with a score greater than another predetermined threshold. In an example, the predetermined thresholds are the same, although different predetermined thresholds are within the scope of the present disclosure. In an example, analyzing a sample by mass spectrometry includes assigning a scrambling percentage for each possible disulfide bond in a particular biomolecule (e.g., a monoclonal antibody). [Example]

[0075] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

[0076] Example 1: Optimized post-column partial reduction of disulfides and MS signal enhancement Antibodies (e.g., monoclonal antibodies) and other biomolecules have native disulfide bonds that contribute to their stability and effective functionality. However, under certain conditions (e.g., alkaline conditions), non-native (also referred to herein as scrambled) disulfide bonds can arise, thereby reducing stability and functional effectiveness. Figure 1A shows a mAb with multiple native disulfide bonds (left) and the same mAb with several scrambled disulfide bonds (right). Figure 1B shows a simplified scheme illustrating how disulfide bonds can rearrange (e.g., scramble) under basic conditions. The present disclosure relates to detecting scrambled disulfide bonds in biomolecules, with an emphasis on mAbs.

[0077] Therapeutic mAbs (or other therapeutic biomolecules) with scrambled disulfides may exhibit reduced functionality; therefore, the ability to detect scrambled disulfides is important. Figure 2 shows that unambiguous identification of low-abundance scrambled disulfides in mAbs can be difficult. Figure 2 shows the disulfide (C133H-C202H, relative abundance 0.2%) formed from the corresponding reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2) analyzed by liquid chromatography-mass spectrometry (LC-MS) (upper panel) and tandem mass spectrometry (MS / MS) analysis (lower panel). Although the EIC shows the m / z ratio corresponding to the disulfide, MS / MS analysis is too complex to identify the fragment corresponding to TYTCNVDHKPSNTK (SEQ ID NO: 2).

[0078] Complications associated with disulfide pair fragmentation in MS / MS analysis can be reduced by at least partially reducing the disulfide pair prior to mass spectral analysis. Referring to Figure 3, a series of plots showing the counts versus mass-to-charge (m / z) of disulfides and the corresponding reduced partner peptides as a function of increasing TCEP concentrations (0 mM, 0.4 mM, 0.8 mM, 2 mM, and 4 mM) is shown. Briefly, the mAb was trypsin-digested under nonreducing conditions to preserve the disulfide bond, and the nonreduced digests were separated by HPLC. After separation, TCEP at the indicated concentrations was allowed to react with the eluted disulfides for 1–3 s (e.g., partial reduction) before entering the mass spectrometer. Furthermore, to increase the effectiveness of TCEP in disulfide reduction, NH4OH (final concentration 0.12%) was added along with TCEP. Using this methodology, the disulfides co-elute with the reduced peptide, ensuring that all components have exactly the same retention time. For the experimental procedure, the digested mAb was passed through the column at 50 μL / min (0.02% TFA, 0.08% FA), and TCEP and NH OH were added to the separated sample via a syringe and a mixing T-tube. The experiment shows the highest signal for the reduced partner peptide at 2 mM TCEP.

[0079] Because low-abundance disulfides can be difficult to detect, identify, and quantitate if the MS signal is too low, experiments were performed to determine whether adding glycine to a mobile phase eluent containing TCEP could improve the MS signal of scrambled disulfides. Specifically, experiments were performed to determine whether glycine could improve the MS signal in samples that also contained 2 mM TCEP. The experimental procedure involved examining the MS signal of the peptide fragment VVSVLTVLHQDWLNGK (SEQ ID NO: 3) from mAb1 under various conditions, as shown in Figure 4. The results show that 2 mM TCEP suppresses the MS signal enhancement otherwise observed with the addition of glycine (2 mM). Proceeding from left to right, Sample 1, containing TCEP and NH4OH, showed a decrease in signal compared to a control lacking TCEP, NH4OH, and glycine. Addition of 2 mM glycine to a sample containing TCEP and NH4OH (Sample 2) showed only a slight improvement, and removal of NH4OH while maintaining the addition of TCEP and glycine (Sample 3) showed only a slight improvement in MS signal over Sample 3. Only Sample 4 (2 mM glycine in the absence of TCEP and NH4OH) showed substantial MS signal enhancement, indicating that 2 mM TCEP suppresses any MS signal enhancing effect of 2 mM glycine (compare Sample 3 with Sample 4).

[0080] This inhibitory effect of TCEP on glycine-induced MS signal enhancement was further investigated in a dose-response study. Figure 5A shows the MS signal of the peptide fragment VVSVLTVLHQDWLNGK (SEQ ID NO: 3) from mAb1 over a range of TCEP concentrations from 0 μM to 2000 μM, and Figure 5B shows the MS signal of another peptide DTLMISR (SEQ ID NO: 4) over the same range of TCEP concentrations. Both Figures 5A and 5B show that the MS signal decreases with increasing concentrations of TCEP. For each of Figures 5A-5B, the sample conditions included mAb1 at a concentration of 0.1 μg, 0.05% TFA, and 2 mM glycine, as well as the indicated concentrations of TCEP.

[0081] The results in Figures 5A-5B indicated that higher MS signals were associated with lower TCEP concentrations. Further experiments were performed to determine whether lower concentrations of TCEP could at least partially circumvent the suppression effect on MS signals while retaining the ability to detect the reduced partner peptide corresponding to the disulfide-linked peptide pair. The MS signals for the disulfide corresponding to peptides NQVSLTCLVK (SEQ ID NO: 5) and WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 6) are shown in Figure 6A, and the MS signals for the corresponding reduced partner peptides are shown in Figure 6B. For each of Figures 6A-6B, various TCEP concentrations ranging from 0 μM to 2000 μM were tested, similar to those discussed above for Figures 5A-5B. The sample conditions for Figures 6A-6B included a loading amount of 1 μg of trypsin-digested mAb1, 0.05% TFA, and 2 mM glycine, along with the indicated TCEP concentrations. The data show that increasing concentrations of TCEP suppress the MS signal corresponding to the disulfide (Figure 6A), and that there is a range (approximately 20 μM to approximately 100 μM) where TCEP effectively reduces disulfide-linked peptide pairs without undesirably suppressing the MS signal. The increase in the MS signal of the corresponding reduced partner peptide (Figure 6B) is desirable for MS / MS identification. The best MS signal was found at approximately 40 μM TCEP.

[0082] Therefore, further experiments were performed using 40 μM TCEP to partially reduce the disulfide to the corresponding reduction partner peptide. Referring to Figures 7A-7B, the relative abundances of the disulfide consisting of GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7) and the corresponding reduction partner peptide are shown without (Figure 7A) and with (Figure 7B) partial reduction of the disulfide via treatment with 40 μM TCEP. The analyzed peptide fragments were generated from trypsin digestion of mAb2 prior to HPLC. When partial reduction of the disulfide was not performed, no signal was observed for the corresponding reduction partner peptide (Figure 7A). However, when the disulfide peptide was partially reduced with 40 μM TCEP, a good signal for the corresponding reduction partner peptide was observed (Figure 7B).

[0083] Referring to Figures 8A-8B, glycine was observed to improve the MS signal of the disulfide peptide and the corresponding reduced partner peptide by more than 10-fold (e.g., 10- to 20-fold) in the presence of 40 μM TCEP and TFA (0.05%). Figures 8C-8G show data demonstrating whether FA with or without glycine can be used to improve detection of the reduced partner peptide. For each of Figures 8A-8E, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and TYTCNVDHKPSNTK (SEQ ID NO: 2), which contain the disulfide peptide in their non-reduced form. For each of Figures 8F-8G, the reduced partner peptide corresponds to GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7), which contain the disulfide in their non-reduced form. Each of Figures 8A-8G shows the relative abundance of peptide fragments generated from tryptic digestion of mAb2 treated with 40 μM post-column separation TCEP. As shown, glycine (e.g., 2 mM) is required to detect the corresponding reduced partner peptide whether the ion-pairing agent is TFA (see Figures 8A-8B) or FA (see Figures 8C-8E and 8F-8G).

[0084] Referring to Figures 9A-9B, MS signals of peptide fragments generated from trypsin digestion of mAb2 are shown. The disulfides correspond to those of the reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7). The same data is shown in both Figures 9A-9B, with Figure 9B showing a y-axis zoom of Figure 9A. The data show that the largest MS signals for both the disulfides and the corresponding reduced partner peptides were observed when partial reduction was performed with 40 μM TCEP and the mobile phase contained 2 mM glycine and 0.05% TFA, compared to samples treated with 2 mM TCEP, 0.12% NH4OH, and 0.05% TFA, or 2 mM TCEP, 0.12% NH4OH, and 0.1% FA. The data obtained under conditions where the mobile phase contained 2 mM TCEP, 0.12% NH4OH, and 0.1% FA demonstrates that it is possible to detect the reduced partner peptide using FA without glycine. However, this appears to require conditions with high concentrations of other components (2 mM TCEP and 0.12% NH4OH), which, as discussed above, are desirable to avoid in order to obtain improved MS signals. At milder concentrations (e.g., 40 μM TCEP, 0% NH4OH), detection of reduced peptides requires TFA in the mobile phase.

[0085] Example 2: Optimization of MS / MS detection of scrambled disulfides The partial reduction strategy discussed above, using TCEP (e.g., 20-100 μM), is estimated to result in a typical reduction efficiency of 1-3%, in addition to the low abundance of scrambled disulfides to begin with. Therefore, it was recognized that even with a reasonable MS1 signal, the MS2 scan may not be present in data-dependent acquisition (DDA) and / or may be too weak if the MS2 scan occurs at the end of the peak.

[0086] To illustrate these issues, 5 μg of trypsin-digested mAb2 was subjected to tandem mass spectrometry after partial reduction with 40 μM TCEP. The mobile phase contained 2 mM glycine and 0.05% TFA. Figure 10A shows the relative abundance (MS1) of the disulfide peptide and the corresponding reduced partner peptides GPSVFPLAPCSR (SEQ ID NO: 1) (labeled R1) and TYTCNVDHKPSNTK (SEQ ID NO: 2) (labeled R2). Figure 10B shows the relative abundance as a function of m / z for the second stage of MS / MS analysis (MS2). As shown, even with adequate MS1 signal, the MS2 signal can be weak or absent.

[0087] Therefore, we developed a targeted MS2 approach that addresses all reduced cysteine-containing peptides to ensure that MS2 scans are present and near the apex of each peak. Specifically, referring to Figure 10C, an exemplary diagram of a mAb containing 16 unique cysteine ​​residues, as shown, is shown, corresponding to 15 tryptic reduced peptides, including the hinge. Targeting all disulfide combinations separately, without relying on the partial reduction strategy described above, would yield 120 unique disulfide peptides, 8 of which are natural. Therefore, by performing partial reduction, it is only necessary to target approximately 15 reduced tryptic peptides containing cysteines. Therefore, the methodology may involve building a parallel reaction monitoring (PRM) inclusion list using approximately 15 peptides and scanning across the entire gradient. The number of reduced peptides (e.g., approximately 15) is described as an estimate due to the exclusion of hinge peptides, but there may potentially be other miscleaved peptides that could be included depending on user preference. Furthermore, MS1 resolution may be degraded with increasing numbers of MS2 scans.

[0088] Figure 11 shows the MS / MS spectra of the disulfide and the corresponding reduced partner peptides, GPSVFPLAPCSR (SEQ ID NO: 1) and STSESTAALGCLVK (SEQ ID NO: 7). Peptide fragments were generated from trypsin digestion of mAb2. The data shown in Figure 11 indicate that the MS / MS spectra of disulfides contain a significant degree of complexity compared to the corresponding reduced partner peptides, and that performing post-column partial reduction with 40 μM TCEP allows for simpler characterization of any detectable scrambled disulfides. In other words, reduced peptides have much simpler MS / MS spectra than disulfide peptides, due to the increased complexity of disulfide MS / MS spectra resulting from exponentially more possible fragmentations compared to single peptides.

[0089] To facilitate MS / MS detection of scrambled disulfides, the following confidence scoring system was developed. The confidence scoring system consists of five yes / no queries. The first query determines whether the MS1 mass of a specific disulfide has been identified. The second query determines whether the MS1 mass of a first reduction partner peptide has been identified. The third query determines whether the MS1 mass of a second reduction partner peptide has been identified. Based on the present disclosure, it can be understood that the first reduction partner peptide and the second reduction partner peptide, when linked via a cysteine ​​residue, comprise the disulfide mentioned in the first query. The fourth query determines whether the byonic MS2 ID of the first reduction partner peptide has a score greater than a predetermined threshold (e.g., >200). The fifth query determines whether the byonic MS2 ID of the second reduction partner peptide has a score greater than another predetermined threshold (e.g., >200). For each query, a "yes" response equates to 1 point, and each "no" response equates to none or 0 points. Thus, disulfide ID confidence scores are as follows: 0 = not detected, 1 = low confidence, 2 = medium confidence, 3 = high confidence, and 4 or 5 = very high confidence. To improve precision in confidence determination, an option may be to run additional samples without partial reduction (e.g., in the absence of TCEP) to determine whether the reduced peptide peak disappears at the same retention time. Some issues that may complicate such confidence determination schemes are that short (e.g., 2-3 residue peptides) may not be detectable, and dimeric disulfide peptides have the same m / z as their corresponding reduced partner peptides (see below with respect to Figure 31).

[0090] Example 3: Quantitation of mAb2 disulfide scrambling The targeted MS2 approach designed above for identifying scrambled disulfides and assigning confidence values ​​to them was applied to mAb2. Specifically, mAb2 was subjected to trypsin digestion under non-reducing conditions, the peptide fragments were separated by HPLC, and the eluted components were partially reduced with 40 μM TCEP before MS / MS analysis (e.g., targeted MS2). 2 mM glycine was used to enhance the MS signal. Figure 12 shows tryptic peptides of mAb2 containing cysteine ​​residues and the corresponding cysteine ​​labels, including the residue number and designation of whether the residue is on the light (L) or heavy (H) chain.

[0091] The results are shown in Figure 13, which shows all possible scrambled disulfide bonds from mAb2 coded by confidence level. 71.6% of the scrambled disulfide bonds were identified with high or very high confidence, 17% with medium confidence, and 11.3% with low confidence. Scrambled peptides corresponding to hinges were excluded from the analysis. The data showed that the majority of all possible scrambled disulfides were identifiable to some degree. The results raised the question of whether disulfide scrambling could be artificially induced by the protocol used for trypsin digestion. Therefore, this issue was investigated, as discussed below.

[0092] Figures 14A-14C show different digestion protocols corresponding to the mAb2 protocol (used to generate the data shown in Figure 14A and Figure 13), the mAb3 protocol (Figure 14B), and the low-pH digestion kit (Promega, Madison, WI). As shown in Figures 14A-14C, each protocol involves similar steps (e.g., buffer exchange, denaturation, alkylation, and digestion), but there are some differences as indicated. Each protocol includes an alkylation step, which involves alkylation with iodoacetamide (IAM) in the mAb2 and mAb3 protocols and N-ethylmaleimide (NEM) in the low-pH digestion kit. The structures of IAM and NEM and how they label cysteine ​​residues are shown for reference in Figures 15A and 15B, respectively. Another major difference between the protocols centers on the pH at which the denaturation, alkylation, and digestion steps are performed. Furthermore, the low pH digestion kit procedure includes an additional pre-digestion step with recombinant Lys-C protease prior to the digestion step further comprising trypsin. Importantly, the denaturation / alkylation step for the low pH digestion kit procedure is performed at pH 5.7, the pre-digestion step is performed at pH 5.3, and the digestion step is performed at pH 5.3. This is in contrast to similar steps corresponding to the mAb2 and mAb3 protocols, which are performed at a higher pH (e.g., 7.5).

[0093] Referring to Figure 16, UV chromatographs are shown demonstrating that the low pH kit procedure yields results comparable to those of the mAb2 and mAb3 procedures, despite the low pH associated with the low pH kit procedure. Figure 16 shows the UV chromatograph from about 9 minutes to about 72 minutes. Figure 17A shows a portion of the UV chromatograph to highlight the time window from about 14 minutes to about 30 minutes, and Figure 17B shows another portion of the UV chromatograph from Figure 16 to highlight the time window from about 34 minutes to about 49 minutes. Taken together, Figures 16-17B demonstrate that comparable trypsin digestion can be achieved using the lower pH associated with the low pH kit procedure compared to higher pH trypsin digestion procedures (e.g., the mAb2 and mAb3 procedures). For the UV chromatographs shown in Figures 16-17B, each sample corresponds to 5 μg of mAb2 digested via the mAb2 procedure, the mAb3 procedure, and the low pH digestion kit procedure.

[0094] The same samples were used to assess whether there were any discernible differences in MS signal depending on the digestion procedure used (e.g., mAb2 procedure, mAb3 procedure, or low pH digestion kit procedure). Referring to Figure 18, a graph is shown depicting the MS signal for several different native disulfide peptides corresponding to trypsin-digested mAb2 (or trypsin with low pH-resistant recombinant LysC in the case of the low pH digestion protocol). As can be seen in Figure 18, comparable signal intensities were observed for each native disulfide, regardless of whether the mAb2 procedure, mAb3 procedure, or low pH digestion kit procedure was used to generate the disulfide peptide fragments.

[0095] Figures 19A-19D show that low-pH digestion has similar digestion efficiency across the entire protein compared to basic digestion procedures (e.g., mAb2 and mAb3 procedures). The bars in Figures 19A-19D represent MS1 peak integrations of non-cysteine-containing peptides within all of the various regions of the mAb (e.g., VH, VL, CH1, CH2, CH3, CL). The data in Figures 19A-19D, combined with the data shown in Figure 18, confirm that the reported scrambled disulfide levels (see Figures 20A-20C) are accurate for the low-pH digestion conditions. Because the data suggested comparable digestion and MS signal levels in samples digested under acidic conditions (e.g., the low-pH digestion kit procedure), we assessed whether the amount of scrambled disulfide initially identified (see Figure 13) was artifactually caused by the choice of digestion procedure. Figures 20A-20C each show a table depicting the disulfide peptides detected via the procedures discussed above, including the targeted MS2 approach. Figures 20A-20C show all possible scrambled disulfide linkages from mAb2, shown as heat maps corresponding to the quantified abundance of scrambled disulfides for each of three different digestion procedures (see Figures 14A-14C), corresponding to the mAb2 procedure, the mAb3 procedure, and the low pH digestion kit procedure. For each possible scrambled disulfide, the scrambled percentage was calculated as a function of the specific digestion method (e.g., the mAb2 procedure, the mAb3 procedure, or the low pH kit digestion procedure). The formula for calculating the scrambled percentage is shown in Figure 20D. Briefly, the scrambled percentage was determined by dividing the peak area of ​​the scrambled disulfide by the sum of the average peak areas of both native disulfides and the peak area of ​​the scrambled disulfide. As can be seen in Figures 20A-20C, all scrambled disulfides were quantified at less than 0.01% using the low pH digestion procedure, compared to higher percentages identified using the high pH digestion procedure (e.g., mAb2 and mAb3 procedures). There were interferences with some of the disulfides, and Figure 20E shows representative examples of such interferences.

[0096] Figure 20A, like Figure 20C (C23L-C22H), includes ellipses highlighting the identified, particularly high-abundance scrambled disulfides (C152H-C139H). These were further investigated as discussed with respect to Figures 21A-21B. Specifically, Figures 21A-21B show the relative abundance of high-abundance scrambled disulfides when the trypsin digestion procedure was performed at a higher pH (e.g., the mAb2 and mAb3 procedures) or a lower pH (the low-pH digestion kit procedure). Figure 21A shows C152H-C139H corresponding to the reduction partner peptides STSESTAALGCLVK (SEQ ID NO: 7) and GPSVFPLAPCSR (SEQ ID NO: 1), while Figure 21B shows C23L-C22H corresponding to the disulfide of the corresponding reduction partner peptides DIVMTQSPLSLPVTPGEPASISCR (SEQ ID NO: 12) and LSCAGSGFTFR (SEQ ID NO: 8). For each of Figures 21A-B, relative abundance is shown as a function of digestion protocol (mAb2 protocol, mAb3 protocol, or low pH digestion protocol). As shown in both Figures 21A-B, EICs readily distinguish high-abundance disulfides from baseline noise when digestions were performed using the mAb2 and mAb3 protocols, whereas EICs of scrambled disulfides were indistinguishable from baseline noise under the low pH digestion conditions. For each condition, samples contained 5 μg of trypsin-digested mAb2 (or trypsin with low-pH-resistant recombinant LysC in the low pH digestion protocol) and 2 mM glycine. This observation is consistent with the data shown in Figure 22, which shows UV chromatographs of the C152H-C139H scrambled disulfide (corresponding to the reduced partner peptides STSESTAALGCLVK (SEQ ID NO:7) and GPSVFPLAPCSR (SEQ ID NO:1)), which were shown to contain high abundance scrambled disulfides when samples were generated using either the mAb2 or mAb3 digestion protocol. Specifically, Figure 22 shows the absence of corresponding peaks for samples prepared via the low pH digestion kit procedure, compared to the observed peaks seen when samples were prepared via the mAb2 and mAb3 digestion procedures.

[0097] Example 4: Quantitation of mAb5 disulfide scrambling A series of experiments similar to those discussed above with respect to Example 3 was performed on another monoclonal antibody, designated herein as mAb5. Figures 23A-23C show the different digestion protocols that were investigated, specifically the mAb4 procedure, the mAb5 procedure, and the same low pH digestion kit procedure discussed above and shown in Figure 14C. Details of each procedure are shown in Figures 23A-23C, with the primary difference being the lower pH of the denaturation / alkylation step (e.g., pH 5.7) and digestion step (e.g., pH 5.3) associated with the low pH digestion kit procedure compared to the mAb4 and mAb5 digestion procedures.

[0098] Figure 24 shows an overlay of UV chromatograms corresponding to trypsin-digested mAb5 (or trypsin with low-pH-resistant recombinant LysC in the case of the low-pH digestion protocol) using each of the digestion procedures (e.g., mAb4 procedure, mAb5 procedure, and low-pH digestion kit procedure) illustratively shown in Figures 23A-23C. Similar to what was shown above for the mAb2 antibody (see Figures 16-17B), Figure 24 shows that comparable digestion was achieved compared to digestion using the mAb4 and mAb5 digestion procedures, despite the low pH associated with the low-pH digestion kit procedure. The sample run to obtain the chromatogram shown in Figure 24 contained 5 μg of trypsin-digested mAb5 (or trypsin with low-pH-resistant recombinant LysC in the case of the low-pH digestion protocol) in the presence of 2 mM glycine. Figures 25A-25B depict a portion of the entire chromatograph shown in Figure 24 for better visual resolution to highlight the fact that the digestion of mAb5 was similar regardless of whether higher pH digestion conditions (e.g., mAb4 and mAb5 digestion procedures) or lower pH digestion conditions (e.g., low pH digestion kit procedure) were used.

[0099] The same samples discussed with respect to Figures 24-25B were used to assess whether there were any discernible differences in MS signal depending on the digestion procedure used (e.g., mAb4 procedure, mAb5 procedure, or low pH digestion kit procedure). Referring to Figure 26, a graph is shown depicting the MS signal (e.g., peak area) for several different native disulfide peptides corresponding to trypsin-digested mAb5 (or, in the case of the low pH digestion protocol, trypsin with low pH-resistant recombinant LysC). As can be seen in Figure 26, comparable signal intensities were observed for each native disulfide, regardless of whether the mAb4 procedure, mAb5 procedure, or low pH digestion kit procedure was used to generate the disulfide peptide fragments.

[0100] Figures 27A-D show that low pH digestion has similar digestion efficiency across the entire protein compared to basic digestion procedures (e.g., mAb4 and mAb5 procedures). The bars in Figures 27A-D represent MS1 peak integrations of non-cysteine-containing peptides within all of the various regions of the mAb (e.g., VH, VL, CH1, CH2, CH3, CL). The data in Figures 27A-D, combined with the data shown in Figure 26, confirm that the reported scrambled disulfide levels (see Figures 30A-C) are accurate for the low pH digestion conditions.

[0101] The targeted MS2 approach discussed above for identifying and assigning confidence values ​​to scrambled disulfides was applied to mAb5. Specifically, mAb5 was subjected to trypsin digestion under non-reducing conditions via the mAb4 digestion procedure, peptide fragments were separated by HPLC, and the eluted components were partially reduced with 40 μM TCEP prior to MS / MS analysis (e.g., targeted MS2). 2 mM glycine was used to enhance the MS signal. Figure 28 shows tryptic peptides of mAb5 containing cysteine ​​residues and the corresponding cysteine ​​labels, including the residue number and designation of whether the residue is on the light (L) or heavy (H) chain.

[0102] The results are shown in Figure 29, which shows all possible scrambled disulfide bonds from mAb2 coded by confidence level. 63.3% of the scrambled disulfide bonds were identified with high or very high confidence, 20% with medium confidence, and 16.7% with low confidence. Scrambled peptides corresponding to hinges were excluded from the analysis. The data showed that the majority of all possible scrambled disulfides were identifiable to some degree. Again, the results raised the question of whether disulfide scrambling could be artificially caused by the protocol used for trypsin digestion.

[0103] Therefore, this issue was investigated in a manner similar to that discussed above for the mAb2 antibody. Specifically, experiments were performed for the mAb5 antibody to determine whether the amount of scrambled disulfides identified (see Figure 29) was artificially caused by the choice of digestion procedure (e.g., a higher pH digestion procedure). Figures 30A-30C each show tables depicting disulfides detected via the procedures discussed above, including the targeted MS2 approach combined with a confidence scoring system that assigns a confidence level to each identified disulfide peptide fragment. Shown in Figures 30A-30C are all possible scrambled disulfide bonds from mAb5, coded by confidence level in the same manner as discussed for Figure 29, for each of three different digestion procedures corresponding to the mAb4 procedure, the mAb5 procedure, and the low pH digestion kit procedure (see Figures 23A-23C). For each possible scrambled disulfide, the scrambling percentage was calculated as a function of the particular digestion method (e.g., mAb4 procedure, mAb5 procedure, or low pH kit digestion procedure). The formula for calculating the scrambling percentage is shown in Figure 20D and discussed above. As can be seen in Figures 30A-30C, all scrambled disulfides quantitated at less than 0.01% when using the low pH digestion procedure, compared to the higher percentages identified when using the high pH digestion procedures (e.g., mAb2 and mAb3 procedures). For some disulfides, there were interferences similar to those discussed above and shown illustratively in Figure 20E.

[0104] Taken together, the methodology discussed herein can be effectively used to identify low abundance scrambled disulfides in biomolecules (e.g., monoclonal antibodies).

[0105] These examples demonstrate that post-column TCEP partial reduction is a simple and effective method for identifying scrambled disulfide peptides by generating simpler MS / MS spectra (compared to those of intact disulfide peptides), with all components sharing precisely the same retention time. Furthermore, experiments show that digestion conditions under more basic conditions (e.g., pH 7.5) can induce artificial disulfide scrambling. Thus, the abundance of artificially scrambled disulfide peptides may depend on the digestion protocol, and free thiols may contribute to disulfide scrambling but are not solely responsible for it. Performing non-reducing digestion under more acidic conditions (e.g., pH 5.7) can prevent artificial disulfide scrambling. Finally, the mAbs discussed herein (e.g., mAb2 and mAb5) were shown to contain negligible levels of actual scrambled disulfides.

[0106] While specific embodiments have been illustrated and described herein, those skilled in the art will recognize that a wide variety of alternative and / or equivalent embodiments or implementations calculated to achieve the same purpose may be substituted for the illustrated and described embodiments without departing from the scope of the invention. Those skilled in the art will readily appreciate that the embodiments may be implemented in a wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. It is manifestly intended, therefore, that the embodiments be limited only by the claims and equivalents thereof.

Claims

1. 1. A method for identifying one or more non-native disulfide bonds in a biomolecule, comprising: performing digestion of the biomolecule under non-reducing conditions to obtain a sample comprising a plurality of fragments of the biomolecule; contacting the sample with a separation column under conditions that allow sample components to bind to a column matrix; applying a first mobile phase gradient to the separation column, the first mobile phase gradient comprising trifluoroacetic acid (TFA) and a small molecule additive at a concentration of about 1-2 mM; applying a second mobile phase gradient to the separation column, the second mobile phase gradient comprising TFA in acetonitrile (ACN) and the small molecule additive at a concentration of about 1-2 mM; performing a partial reduction procedure by treating the eluted sample components with tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 10-100 μM; applying the partially reduced eluted sample components to a mass spectrometer; and performing mass spectrometry on the partially reduced eluted sample components to identify the one or more non-native disulfide bonds in the biomolecule.

2. 2. The method of claim 1, wherein the small molecule additive in the first mobile phase is selected from glycine, alanine, serine, valine, N-acetylglycine, methionine, β-alanine, aspartic acid, or N-methylglycine.

3. The method of claim 2 , wherein the small molecule additive is glycine.

4. 4. The method of claim 1, wherein the small molecule additive in the second mobile phase is selected from glycine, alanine, serine, valine, N-acetylglycine, methionine, β-alanine, aspartic acid, or N-methylglycine.

5. The method of claim 4 , wherein the small molecule additive is glycine.

6. The TFA concentration in the first mobile phase is 2 about 0.05% to 0.1% TFA in O; The method according to any one of claims 1 to 5.

7. The TFA concentration in the second mobile phase is 80% ACN and 20% H 2 About 0.05% TFA in O, or 80% ACN and 20% H 2 7. The method of any one of claims 1 to 6, comprising about 0.1% TFA in O.

8. 8. The method according to any one of claims 1 to 7, wherein the partial reduction procedure is carried out for a duration of between 500 ms and 3 s.

9. performing the digestion of the biomolecule, performing a modification and alkylation step to obtain a modified alkylated biomolecule; performing a pre-digestion step on the modified alkylated biomolecule to obtain a pre-digested modified alkylated biomolecule; 9. The method of claim 1, further comprising, after the pre-digestion step, performing a digestion step on the pre-digested modified alkylated biomolecules to obtain the sample to be contacted with the separation column.

10. 10. The method of claim 9, wherein the denaturing and alkylating step comprises denaturing the biomolecule in 7-9 M urea in the presence of an alkylating agent at a pH of about 5.5-5.

9.

11. 11. The method of claim 9 or 10, further comprising carrying out the modification and alkylation steps at 45-55°C.

12. The method described in claim 10 or 11, wherein the alkylating agent is N-ethylmaleimide (NEM) at a concentration of 5 to 15 mM.

13. The method of claim 10 or 11, wherein the alkylating agent is iodoacetamide (IAM) at a concentration of about 0.5 to 5 mM.

14. 14. The method of any one of claims 9 to 13, further comprising carrying out the modification and alkylation steps for 20 to 40 minutes.

15. 15. The method of any one of claims 9 to 14, wherein carrying out the pre-digestion step further comprises incubating the modified alkylated biomolecule in the presence of recombinant Lys-C protease at a pH of 5 to 5.

6.

16. The method according to any one of claims 9 to 15, further comprising carrying out the pre-digestion step at 35 to 40°C.

17. 17. The method according to any one of claims 9 to 16, further comprising carrying out the pre-digestion step for 30 to 90 minutes.

18. 18. The method of any one of claims 15 to 17, wherein the ratio of recombinant Lys-C protease to the modified alkylated biomolecule is 1:5 to 1:20, respectively.

19. 19. The method of any one of claims 9 to 18, wherein carrying out the digestion step further comprises incubating the pre-digested modified alkylated biomolecule in the presence of recombinant Lys-C protease and trypsin protease at a pH of 5 to 5.

6.

20. The recombinant Lys-C protease during the digestion step and the pre-digested modified alkylation The method according to any one of claims 9 to 19, wherein the ratio of the biomolecule to the biomolecule is about 1:5 to 1:20, respectively.

21. 21. The method of any one of claims 9 to 20, wherein the ratio of trypsin protease to the pre-digested modified alkylated biomolecule is from 1:2 to 1:10, respectively.

22. 22. The method of any one of claims 9 to 21, further comprising carrying out the digestion step at 35-40°C.

23. 23. The method of any one of claims 9 to 22, further comprising carrying out the digestion step for 2 to 4 hours.

24. 24. The method of any one of claims 1 to 23, wherein the partially reduced eluted sample components comprise one or more disulfide peptides and corresponding reduction partner peptides.

25. 25. The method of claim 24, wherein each of the one or more disulfide peptides and corresponding reduced partner peptides enters the mass spectrometer simultaneously.

26. the mass spectrometer is a tandem mass spectrometer; 26. The method of claim 25, wherein performing the mass analysis comprises obtaining an MS1 ​​spectrum and an MS2 spectrum.

27. 27. The method of claim 26, further comprising constructing a parallel reaction monitoring (PRM) inclusion list using the corresponding reduced partner peptides.

28. 28. The method of claim 26 or 27, further comprising assigning a disulfide identification confidence score to the one or more disulfide peptides based on a confidence scoring system.

29. the confidence scoring system: indicating whether the MS1 mass of the disulfide peptide has been identified by said mass spectrometry; indicating whether the MS1 mass of a first reduced partner peptide corresponding to the disulfide peptide has been identified by the mass spectrometry analysis; indicating whether the MS1 mass of a second reduced partner peptide corresponding to the disulfide peptide has been identified by the mass spectrometry analysis; indicating whether the MS2 mass of the first reduced partner peptide is identified with a score greater than a predetermined threshold; indicating whether the MS2 mass of the second reduced partner peptide is identified with a score greater than the predetermined threshold; assigning a single point for each of the displaying steps of the confidence scoring system where the corresponding peptide is identified and no point for where the corresponding peptide is not identified; summing the single points; 29. The method of claim 28, further comprising assigning the disulfide identification confidence score based on the summing, the larger the sum the more confident it is.

30. performing said mass spectrometry analysis For each of the one or more non-native disulfide bonds in the biomolecule, determining a scrambling percentage; 30. The method of any one of claims 1 to 29, wherein the disulfide scrambling percentage is the ratio of the average peak area of ​​a peptide comprising a non-native disulfide bond to the sum of the average peak area of ​​the peptide comprising the non-native disulfide bond plus the average peak areas of another two peptides comprising a native disulfide bond corresponding to the cysteine ​​residues involved in the non-native disulfide bond.

31. The method of any one of claims 1 to 30, wherein the concentration of TCEP is 20 μM to 80 μM.

32. 32. The method of claim 31, wherein the concentration of TCEP is 40 μM.

33. the biomolecule is a monoclonal antibody, 33. The method of any one of claims 1 to 32, wherein the monoclonal antibody is an IgG1, IgG2, IgG3, IgG4, or mixed isotype monoclonal antibody.

34. 34. The method of claim 33, wherein the monoclonal antibody is recombinantly produced.

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