Improving the Chromatographic Performance of RP-LC-Based Peptide Mapping by Using Metal Chelators as Mobile Phase Additives

Abstract

The present technology relates to a method for analyzing a sample containing an analyte. The method includes injecting the sample containing the analyte into a mobile phase. The mobile phase contains a metal chelating agent additive at a concentration between about 1 ppm and about 10 ppm. The method further includes separating the analyte using liquid chromatography and analyzing the analyte using a mass spectrometer, an ultraviolet detector, or a combination thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 62 / 858,380, filed on June 7, 2019, entitled "RPLC - BASED PEPTIDE MAPPING CHROMATOGRAPHIC PERFORMANCE USING METAL CHELATORS AS MOBILE PHASE ADDITIVES", the entire content of which is hereby incorporated by reference. This application also claims the priority and benefit of U.S. Provisional Patent Application No. 62 / 883,182, filed on August 6, 2019, entitled "RPLC - BASED PEPTIDE MAPPING CHROMATOGRAPHIC PERFORMANCE USING METAL CHELATORS AS MOBILE PHASE ADDITIVES", the entire content of which is hereby incorporated by reference. Technical field

[0003] The present disclosure relates to improving the chromatographic performance of reversed - phase liquid chromatography (RPLC) - based peptide mapping using metal chelators as mobile - phase additives. More specifically, the present disclosure relates to a method for analyzing a sample containing intact proteins or fatty acids for peptide mapping, the method comprising using citric acid at a concentration between about 1 ppm and about 10 ppm as a mobile - phase additive. Background art

[0004] Analysis of protein - modified peptide levels is one of the major analytical methods routinely performed throughout the life cycle of therapeutic proteins. It has proven to be a very valuable tool for elucidating structural information during the discovery and / or characterization phases of protein - based therapeutic agents and for assessing product - and / or process - related impurities in the development of control strategies as part of process development. As a monitoring tool, peptide mapping is routinely deployed in a manufacturing environment as an identity assay and a batch release assay to ensure drug product safety. Recently, peptide mapping has been deployed as a multi - attribute monitoring (MAM) technique to improve productivity and data quality by simultaneously and effectively monitoring multiple potential critical quality attributes (pCQAs).

[0005] The peptide - mapping workflow relies on reducing intact proteins to their secondary structures, straight chains of amino acids, where they are enzymatically treated to cleave the amino - acid chain at specific amino - acid residues to produce a peptide mixture. The individual peptide physicochemical properties (e.g., pKa, hydrophobicity, etc.) are determined by the cumulative properties of their constituent amino acids. As Figures 1A - 1DAs shown, amino acids can exhibit charged groups, polar groups, neutral groups (hydrophobic), and unique groups. After digestion, peptide-level analysis of protein-based therapeutic agents is typically performed using reversed-phase liquid chromatography (RPLC) on an LC-UV or LC-UV / MS platform. SUMMARY OF THE INVENTION

[0006] In RPLC, mobile phase additives such as formic acid (FA) and trifluoroacetic acid (TFA) are commonly used to improve analyte retention and peak shape. However, FA and TFA are not necessarily complementary to each other as ion-pairing reagents in the peptide mapping workflow. While FA produces favorable mass spectral intensities compared to TFA in MS-based assays, as a weak ion-pairing reagent, FA has increased baseline noise and broader peaks compared to TFA in UV-based assays. In addition, trace metal contamination in LC systems has been previously documented to affect the recovery and separation performance of compounds exhibiting specific charged moieties such as phosphorylated groups. Trace metal contamination is associated with increased mass spectral adducts in oligonucleotide analysis, which can have a negative impact on chromatographic separation.

[0007] The present technology solves the problems of the prior art by using a metal chelator as a mobile phase additive, specifically citric acid as a mobile phase additive. Using citric acid as a mobile phase additive improves chromatographic performance (e.g., better peak resolution), and the chromatographic performance is maintained over an extended period (e.g., within 2 - 3 days or 64 - 100 consecutive injections). Previous attempts to use citric acid to enhance the performance of LC / MS systems for phosphopeptide analysis did not change the mobile phase (solvent) composition to avoid interfering with chromatographic performance (Winter et al., “Citrate Boosts the Performance of Phosphopeptide Analysis by UPLC-ESI-MS / MS”, J. of Proteome Research, 2009, 8, 418 - 424). However, the present technology changes the mobile phase by adding citric acid as an additive to the mobile phase, which is shown herein to enhance the chromatographic performance of peptides and fatty acids.

[0008] In one aspect, the present technology relates to a method for analyzing a sample comprising an analyte. The method includes injecting a sample comprising the analyte into a mobile phase comprising a metal chelator additive at a concentration between about 1 ppm and about 10 ppm. The method further includes separating the analyte using liquid chromatography and analyzing the analyte using a mass spectrometer, an ultraviolet detector, or a combination thereof. The method can include one or more of the embodiments described herein.

[0009] In some embodiments, the metal chelator additive is selected from the group consisting of citric acid, sodium citrate, isocitrate, diammonium citrate, and triammonium citrate. The metal chelator additive can be citric acid. In some embodiments, the concentration of citric acid in the mobile phase is from about 1 ppm to about 10 ppm. In some embodiments, the concentration of citric acid in the mobile phase is about 10 ppm. In some embodiments, the concentration of citric acid in the mobile phase is about 1 ppm.

[0010] In another aspect, the present technology relates to a method for analyzing a sample containing intact proteins for peptide mapping. The method includes reducing the intact proteins in the sample to a linear chain of amino acids. The method further includes enzymatically treating the linear chain of amino acids to produce a peptide mixture. The sample containing the peptide mixture is injected into a mobile phase containing citric acid. The peptide mixture is separated using reversed-phase liquid chromatography. The separated peptides are analyzed using a mass spectrometer, an ultraviolet detector, a fluorescence detector, or a combination thereof. The method can include one or more of the embodiments described herein.

[0011] In some embodiments, at least one peptide in the peptide mixture contains charged amino acid residues. At least one peptide in the peptide mixture can have a net charge. In some embodiments, the net charge is negative. In some embodiments, the net charge is positive. The net charge of a peptide refers to the sum of the charges at a specific pH. Figures 1A - 1D As can be seen, the side chains have different pKas, and thus depending on the pH of the mobile phase, the amino acids can have a positive or negative charge. The net charge sums up the charge states, for example, 5 positive charges and 6 negative charges will have a net "negative charge".

[0012] The protein can be a monoclonal antibody (mAb). In some embodiments, the protein is a synthetic protein or a recombinant protein.

[0013] In some embodiments, the amino acids are aspartic acid, glutamic acid, or isoaspartic acid. The peptide mixture can include PENNYK peptides and deamidated forms of PENNYK peptides. The amino acids can be enzymatically treated with trypsin, Lys-C, Asp-N, or a combination thereof.

[0014] In some embodiments, the chromatographic performance is maintained over a period of about 2 days to about 3 days, wherein the average USP tailing value is from about 0.95 to about 1.30. In some embodiments, the chromatographic performance is maintained over about 48 consecutive injections, wherein the average USP tailing value is from about 0.95 to about 1.30. In some embodiments, the USP tailing value is between about 0.98 and about 1.20, or about 1.00 and about 1.10. In some embodiments, the USP tailing value is about 1.00.

[0015] In some embodiments, the peptide mixture contains peptides and deamidated species of the peptides.

[0016] The concentration of citric acid in the mobile phase can be from about 1 ppm to about 10 ppm. The concentration of citric acid in the mobile phase can be about 1 ppm. In some embodiments, the concentration of citric acid in the mobile phase is about 10 ppm.

[0017] The mobile phase may further include formic acid, acetic acid, water, acetonitrile, methanol, isopropanol, n-propanol, trifluoroacetic acid, difluoroacetic acid, or a combination thereof.

[0018] In some embodiments, the method further includes passivating a reverse-phase chromatography column with citric acid before separating the peptide mixture.

[0019] In another aspect, the present technology relates to a method for analyzing a sample containing fatty acids. The method includes injecting a sample containing fatty acids into a mobile phase containing citric acid. The method further includes separating the fatty acids using reverse-phase liquid chromatography. The separated fatty acids are separated using a mass spectrometer, an ultraviolet detector, or a combination thereof. The method may include one or more of the embodiments described herein.

[0020] In some embodiments, the fatty acid includes lauric acid. The concentration of citric acid in the mobile phase can be from about 1 ppm to about 10 ppm.

[0021] In another aspect, the present technology relates to a kit for analyzing a sample. The kit includes a liquid chromatography column, a vial containing a mobile phase additive of citric acid, and instructions for using the mobile phase additive of citric acid in a method for analyzing a sample containing an analyte. The method includes adding the mobile phase additive of citric acid to the mobile phase, injecting a sample containing the analyte into the mobile phase containing citric acid, separating the analyte using liquid chromatography, and analyzing the separated analyte using a mass spectrometer, an ultraviolet detector, or a combination thereof. The kit may include one or more of the embodiments described herein.

[0022] In some embodiments, the liquid chromatography column is a reverse-phase liquid chromatography column. The mobile phase additive of citric acid can be added to the mobile phase at a concentration of from about 1 ppm to about 10 ppm. The analyte can be a fatty acid. In some embodiments, the analyte is a complete protein. In some embodiments, the concentration of citric acid is about 1 ppm. In some embodiments, the concentration of citric acid is about 10 ppm.

[0023] In some embodiments, the method further includes reducing the complete protein to an amino acid straight chain and enzymatically treating the amino acid straight chain to produce a peptide mixture.

[0024] In another aspect, the present technology relates to a kit for analyzing a sample. The kit includes a liquid chromatography column, a vial containing a metal chelator mobile phase additive, and instructions for using a citric acid mobile phase additive in a method for analyzing a sample containing an analyte. The method includes adding the metal chelator mobile phase additive to the mobile phase to obtain a metal chelator mobile phase additive at a concentration between about 1 ppm and about 10 ppm, injecting the sample containing the analyte into the mobile phase containing the metal chelator, separating the analyte using liquid chromatography, and analyzing the separated analyte using a mass spectrometer, an ultraviolet detector, a fluorescence detector, or a combination thereof. The kit may include one or more of the embodiments described herein.

[0025] In some embodiments, the metal chelator mobile phase additive is selected from the group consisting of citric acid, sodium citrate, isocitrate, diammonium citrate, triammonium citrate, and ammonium formate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0027] Figure 1A It is shown that the physicochemical properties of a peptide (e.g., pKa, hydrophobicity, etc.) are determined by the cumulative properties of its constituent amino acids. Figure 1A Amino acids with charged side chains are shown.

[0028] Figure 1B It is shown that the physicochemical properties of a peptide (e.g., pKa, hydrophobicity, etc.) are determined by the cumulative properties of its constituent amino acids. Figure 1B Amino acids with polar uncharged side chains are shown.

[0029] Figure 1C It is shown that the physicochemical properties of a peptide (e.g., pKa, hydrophobicity, etc.) are determined by the cumulative properties of its constituent amino acids. Figure 1C Amino acids in special cases are shown.

[0030] Figure 1D It is shown that the physicochemical properties of a peptide (e.g., pKa, hydrophobicity, etc.) are determined by the cumulative properties of its constituent amino acids. Figure 1D Amino acids with hydrophobic side chains are shown.

[0031] Figure 2A Is a representative total ion chromatogram (TIC) of tryptic digests of NIST mAb RM according to an illustrative embodiment of the present technology. Figure 2A Is an example of a peptide map that shows all peptides from tryptic digests via MS ionization and detection.

[0032] Figure 2BRepresentative extracted ion chromatogram (XIC) of the PENNYK peptide according to an illustrative embodiment of the present technology.

[0033] Figure 2C is a representative single ion recording (SIR) chromatogram of the PENNYK peptide according to an illustrative embodiment of the present technology, which shows the tight elution of the native and deamidated forms of the PENNYK peptide as key pairs under formic acid conditions using a moderate gradient (see Figure 2D ).

[0034] Figure 2D lists the gradients and other chromatographic conditions for the representative chromatograms for Figure 2A , Figure 2B and Figure 2C according to an illustrative embodiment of the present technology.

[0035] Figure 3 is a graph depicting the variables for calculating the figure of merit according to an illustrative embodiment of the present technology.

[0036] Figure 4A is a chromatogram according to an illustrative embodiment of the present technology, which shows the baseline performance of a system for RPLC / MS-based separation of the PENNYK peptide directly after a cleaning procedure with a 30% phosphoric acid solution.

[0037] Figure 4B is a chromatogram according to an illustrative embodiment of the present technology, which shows the baseline performance of a system for RPLC / MS-based separation of the PENNYK peptide 12 hours after a cleaning procedure with a 30% phosphoric acid solution.

[0038] Figure 4C is a chromatogram according to an illustrative embodiment of the present technology, which shows the baseline performance of a system for RPLC / MS-based separation of the PENNYK peptide 24 hours after a cleaning procedure with a 30% phosphoric acid solution.

[0039] Figure 5A is a chromatogram according to an illustrative embodiment of the present technology, which shows separation using a mobile phase prepared with a metal chelator citric acid at a concentration of 0.001% at the initial injection.

[0040] Figure 5B is a chromatogram according to an illustrative embodiment of the present technology, which shows separation using a mobile phase prepared with a metal chelator citric acid at a concentration of 0.001% at the 24th injection at 32 hours.

[0041] Figure 5CIs a chromatogram according to an illustrative embodiment of the present technology, which shows separation using a mobile phase prepared with the metal chelator citric acid at a concentration of 0.001% at the 48th injection in 64 hours.

[0042] Figure 5D Is a graph showing the USP tailing factor according to an illustrative embodiment of the present technology.

[0043] Figure 6A Is a chromatogram of an RPLC / MS-based separation of a tryptic digest of an NIST mAb of the "PENNYK" peptide with an identified peptide of interest containing an anionic residue according to an illustrative embodiment of the present technology.

[0044] Figure 6B Is for using a mobile phase composed of H 2 O:MeCN (0.1% FA v / v) to monitor the USP tailing factor of the PENNYK peptide and related impurities in 72 hours at W 0.05 The single-ion recording (SIR). According to an illustrative embodiment of the present technology, the SIR overlays are time-aligned for easy comparison.

[0045] Figure 7 Is a graph showing the rate of deterioration of chromatographic performance monitored by plotting at intervals of 7.5 hours according to an illustrative embodiment of the present technology in the case of using a mobile phase composed of H 2 O:MeCN (0.1% FA v / v) for the tailing factor at W 0.05 And below. When the peak width approaches the baseline, the tailing factor is observed to have an exponential-like behavior.

[0046] Figure 8A Is a chromatogram showing the SIR overlay of the PENNYK peptide for 3 injections over time without adding a chelating agent to the mobile phase according to an illustrative embodiment of the present technology. The tailing factor and relative peak area calculations are reported in the table. The SIR overlays are time-aligned for easy comparison.

[0047] Figure 8B Is a chromatogram showing the SIR overlay of the PENNYK peptide for 3 injections over time with 1 ppm citric acid added to the mobile phase according to an illustrative embodiment of the present technology. The tailing factor and relative peak area calculations are reported in the table. The SIR overlays are time-aligned for easy comparison.

[0048] Figure 8CIt is a chromatogram showing the SIR overlay of 3 injections over time of the PENNYK peptide in the case of adding 5 μM (0.9 ppm) methylenediphosphonic acid to the mobile phase according to an illustrative embodiment of the present technology. The tailing factor and relative peak area calculations are reported in the table. The SIR overlay is time-aligned for easy comparison.

[0049] Figure 9A It is an extracted ion chromatogram (XIC) showing the main peptide species eluting in the PENNYK peptide (T37) region in the case of not adding a chelating agent to the mobile phase according to an illustrative embodiment of the present technology. The corresponding baseline mass spectrum under initial conditions is used to evaluate mass spectrometry contaminants and is shown in the inset.

[0050] Figure 9B It is an extracted ion chromatogram (XIC) showing the main peptide species eluting in the PENNYK peptide (T37) region in the case of adding 1 ppm citric acid to the mobile phase according to an illustrative embodiment of the present technology. The corresponding baseline mass spectrum under initial conditions is used to evaluate mass spectrometry contaminants and is shown in the inset.

[0051] Figure 9C It is an extracted ion chromatogram (XIC) showing the main peptide species eluting in the PENNYK peptide (T37) region in the case of adding 5 μM (0.9 ppm) methylenediphosphonic acid to the mobile phase according to an illustrative embodiment of the present technology. The corresponding baseline mass spectrum under initial conditions is used to evaluate mass spectrometry contaminants and is shown in the inset.

[0052] Figure 9D It is a graph showing the MS responses of citric acid and methylenediphosphonic acid normalized to the chromatogram without a chelating agent according to an illustrative embodiment of the present technology and plotted as % intensity change. Asterisks indicate that no peak for methylenediphosphonic acid was detected in the XIC.

[0053] Figure 10 It is a chromatogram showing the effect of column loading on tailing under RPLC / MS conditions using a reduced mass load according to an illustrative embodiment of the present technology. 0.01 The tailing factor at W is calculated to be 1.05 and fits well with the Gaussian fit, indicating a minimal presence of tailing due to column loading effects.

[0054] Figure 11A It is a chromatogram showing the experiment (1110) and Gaussian fit (1115) of T37(HC)GFYPSDIAVEWESNGQPENNYK at 60 °C for 1 ppm chelating agent according to an illustrative embodiment of the present technology. For the peptide segment T37( Figure 11A -C) and T14( Figure 11D-F), the influence of temperature on tailing was evaluated at 60 °C and 40 °C in the presence and absence of a chelating agent (citric acid) in the mobile phase. For both peptides, in the presence of the chelating agent, the tailing factor remained almost constant at the lower temperature, with only a 5% increase in tailing. In the absence of the chelating agent, T14 showed a 2.35-fold increase in tailing compared to T37, which had a 1.10-fold increase in tailing. The asterisks indicate the T f values.

[0055] Figure 11B is a chromatogram showing the experiment (1120) and Gaussian fit (1125) of T37(HC)GFYPSDIAVEWESNGQPENNYK at 40 °C for 1 ppm chelating agent according to an illustrative embodiment of the present technology.

[0056] Figure 11C is a chromatogram showing the experiment (1130) and Gaussian fit (1135) of T37(HC)GFYPSDIAVEWESNGQPENNYK at 40 °C without chelating agent according to an illustrative embodiment of the present technology.

[0057] Figure 11D is a chromatogram showing the experiment (1140) and Gaussian fit (1145) of T14(LC)VDNALQSGNSQESVTEQDSK at 60 °C for 1 ppm chelating agent according to an illustrative embodiment of the present technology.

[0058] Figure 11E is a chromatogram showing the experiment (1150) and Gaussian fit (1155) of T14(LC)VDNALQSGNSQESVTEQDSK at 40 °C for 1 ppm chelating agent according to an illustrative embodiment of the present technology.

[0059] Figure 11F is a chromatogram showing the experiment (1160) and Gaussian fit (1165) of T14(LC)VDNALQSGNSQESVTEQDSK at 40 °C without chelating agent according to an illustrative embodiment of the present technology.

[0060] Figure 12 is a diagram showing the use of H 2 O:NeCN (0.1% FA v / v) to monitor the tailing factor of the PENNYK peptide at W 0.01 over a 67-hour period with and without a chelating agent (citric acid) as a mobile phase additive. In the presence of the chelating agent, the chromatographic performance was stable, with an average tailing factor of 0.96% (%RSD = 0.43%).

[0061] Figure 13A This is the peptide map of the tryptic digest of the NIST mAb RM in the absence of a chelating agent. The same method as outlined for Figure 2 was used.

[0062] Figure 13B This is the peptide map of the tryptic digest of the NIST mAb RM in the presence of a chelating agent according to an illustrative embodiment of the present technology. The same method as outlined for Figure 2 was used.

[0063] Figure 14 This figure shows the improvement in the recovery of lauric acid (the major fatty acid component of polysorbate 20 (PS-20)) by adding a chelating agent to the mobile phase to obtain a linear MS response according to an illustrative embodiment of the present technology.

[0064] Figure 15 This figure shows the system conditions used in testing several different mobile phase additives as shown in Figures 16 - 21 the following.

[0065] Figure 16 A is the chromatogram of the PENNYK peptide and the deamidated species (inset) in the presence of a 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology.

[0066] Figure 16 B is the chromatogram of the PENNYK peptide and the deamidated species (inset) in the presence of a 1 ppm sodium citrate mobile phase additive according to an illustrative embodiment of the present technology.

[0067] Figure 16 C is the combined QDa MS scan (top mass spectrum) of the PENNYK peptide and the combined mass spectrum of the baseline chromatogram shown in Figure 16 A and Figure 16 B in the presence of a 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology ( Figure 16 bottom mass spectrum of C).

[0068] Figure 16 D is the combined QDa MS scan (top mass spectrum) of the PENNYK peptide and the combined mass spectrum of the baseline chromatogram shown in Figure 16 A and Figure 16 B in the presence of a 1 ppm sodium citrate mobile phase additive according to an illustrative embodiment of the present technology ( Figure 16 bottom mass spectrum of D).

[0069] Figure 17 A is the chromatogram of the PENNYK peptide and the deamidated species (inset) in the presence of a 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology.

[0070] Figure 17 B is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm isocitrate mobile phase additive according to an illustrative embodiment of the present technology.

[0071] Figure 17 C is the combined QDa MS scan (top mass spectrum) and the combined mass spectrum of the baseline chromatogram (bottom mass spectrum) of the PENNYK peptide with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 17 A and Figure 17 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0072] Figure 17 D is the combined QDa MS scan (top mass spectrum) and the combined mass spectrum of the baseline chromatogram (bottom mass spectrum) of the PENNYK peptide with 1 ppm isocitrate mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 17 A and Figure 17 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0073] Figure 18 A is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology.

[0074] Figure 18 B is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm diammonium citrate mobile phase additive according to an illustrative embodiment of the present technology.

[0075] Figure 18 C is the combined QDa MS scan (top mass spectrum) and the combined mass spectrum of the baseline chromatogram (bottom mass spectrum) of the PENNYK peptide with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 18 A and Figure 18 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0076] Figure 18D is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of the PENNYK peptide and the baseline chromatogram with 1 ppm ammonium citrate mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 18 A and Figure 18 B, the same time region used for Figure 16 A and Figure 16 B was used for the baseline.

[0077] Figure 19 A is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology.

[0078] Figure 19 B is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm ammonium citrate mobile phase additive according to an illustrative embodiment of the present technology.

[0079] Figure 19 C is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of the PENNYK peptide and the baseline chromatogram with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 19 A and Figure 19 B, the same time region used for Figure 16 A and Figure 16 B was used for the baseline.

[0080] Figure 19 D is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of the PENNYK peptide and the baseline chromatogram with 1 ppm ammonium citrate mobile phase additive according to an illustrative embodiment of the present technology. Although no baseline mass spectrum was noted in Figure 19 A and Figure 19 B, the same time region used for Figure 16 A and Figure 16 B was used for the baseline.

[0081] Figure 20 A is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm citric acid mobile phase additive according to an illustrative embodiment of the present technology.

[0082] Figure 20 B is the chromatogram of the PENNYK peptide and the deamidated species (inset) with 1 ppm methylenediphosphonic acid mobile phase additive according to an illustrative embodiment of the present technology.

[0083] Figure 20 C is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of PENNYK peptide with 1 ppm citric acid mobile phase additive and the baseline chromatogram according to the illustrative embodiments of the present technology. Although no baseline mass spectrum was noted in Figure 20 A and Figure 20 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0084] Figure 20 D is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of PENNYK peptide with 1 ppm methylenediphosphonic acid mobile phase additive and the baseline chromatogram according to the illustrative embodiments of the present technology. Although no baseline mass spectrum was noted in Figure 20 A and Figure 20 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0085] Figure 21 A is the chromatogram of PENNYK peptide and the deamidated species (inset) with 1 ppm citric acid mobile phase additive according to the illustrative embodiments of the present technology.

[0086] Figure 21 B is the chromatogram of PENNYK peptide and the deamidated species (inset) with 5 μM Agilient InfinityLab mobile phase additive according to the illustrative embodiments of the present technology.

[0087] Figure 21 C is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of PENNYK peptide with 1 ppm citric acid mobile phase additive and the baseline chromatogram according to the illustrative embodiments of the present technology. Although no baseline mass spectrum was noted in Figure 21 A and Figure 21 B, the same time region used for Figure 16 A and Figure 16 B is used for the baseline.

[0088] Figure 21 D is the combined mass spectrum (bottom mass spectrum) of the combined QDa MS scan (top mass spectrum) of PENNYK peptide with 5 μM Agilient InfinityLab mobile phase additive and the baseline chromatogram according to the illustrative embodiments of the present technology. Although no baseline mass spectrum was noted in Figure 21 A and Figure 21Baseline mass spectrometry was not noted in B, but the same time region as used for Figure 16 A and Figure 16 B was used for the baseline. DETAILED DESCRIPTION

[0089] Mobile phase additives such as formic acid (FA) and trifluoroacetic acid (TFA) are commonly used in RPLC to improve analyte retention and peak shape. However, FA and TFA are not necessarily complementary to each other as ion-pairing reagents in the peptide mapping workflow. While FA produces favorable mass spectrometry intensities compared to TFA in MS-based assays, as a weak ion-pairing reagent, FA has increased baseline noise and broader peaks compared to TFA in UV-based assays. In addition to ionization efficiency, the selected ion-pairing reagent has a significant impact on retention, separation efficiency, and chromatographic resolution, which can affect peak shape and width to varying degrees. Complicating the effects of ion-pairing reagent selection is the presence of non-specific interactions of proteins and / or peptides with the wetted surfaces of the analytical flow path, which can affect chromatographic performance.

[0090] Trace metal contamination in LC systems has been previously documented to affect the recovery and separation performance of compounds exhibiting specific charged moieties such as phosphorylated groups. More recently, trace metal contamination has been associated with increased mass spectrometry adducts in oligonucleotide analysis. Given this evidence, it is reasonable to assume that peptides with charged moieties may also interact with trace metals, which can adversely affect chromatographic performance.

[0091] Trace metal sources have been identified as mobile phases containing impurities, metal surfaces such as instrument tubing, and even column hardware containing metal such as frits and the column housing itself. In these cases, metal impurities such as iron can leach or desorb from the metal surface and embed themselves along the flow path into frits, filters, and even the stationary phase, where they can negatively interact with the target analyte under study. This presents a challenge in developing a peptide mapping workflow where the chromatographic resolution of impurities requires methods that can deliver consistent and accurate measurements in a robust manner, yet the entire flow path is at risk of trace metal contamination that can impede the analysis.

[0092] Applying a metal chelator such as EDTA or CDTA to “clean” an LC system is a method to minimize trace metal contamination. However, the washing of these systems is often long and tedious, resulting in significant instrument downtime. Engineering an LC system with inert materials such as PEEK or low iron content metal alloys is another method; however, process complexity and material availability can increase the cost and delay the deployment of such systems. An alternative method to mitigate metal contamination is to directly add a trace metal chelator to the mobile phase to act as a metal scavenger to dissolve and minimize the contamination of wetted surfaces, as well as to act as a protecting group for any surface containing insoluble contaminants. Although the literature has shown the effect of such additives on the recovery and a certain degree of chromatographic performance of small phosphorylated compounds, there is little evidence, if any, regarding the potential application to biomolecules such as peptides. The aim of the current technology is to demonstrate the applicability of adding a metal chelator to the mobile phase as an additive to improve the chromatographic performance of RPLC-based peptide separation.

[0093] The technology includes a method for analyzing a sample containing an analyte. The technology includes injecting the sample into a mobile phase containing a metal chelator additive at a concentration between about 1 ppm and about 10 ppm. The concentration can be, for example, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, or 10 ppm. These values can be used to form a range, for example, between about 1 ppm and about 10 ppm or between about 2 ppm and about 5 ppm. The analyte is separated from the sample using liquid chromatography (LC). The separated analyte is analyzed using a mass spectrometer, an ultraviolet (UV) detector, or a combination thereof. The metal chelator can be citric acid at a concentration of about 1 ppm to about 10 ppm. The concentration of citric acid can be, for example, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, or 10 ppm. These values can be used to form a range, for example, between about 1 ppm and about 10 ppm or between about 2 ppm and about 5 ppm. The metal chelator can also be sodium citrate, isocitrate, diammonium citrate, triammonium citrate, or a combination thereof.

[0094] As used herein, the term “about” means that the value is approximate and that minor variations will not significantly affect the practice of the disclosed embodiments. In the case of using numerical limitations, unless the context indicates otherwise, “about” means that the value can vary by ±10% and still be within the scope of the disclosed embodiments.

[0095] The technique also includes a method for analyzing a sample containing intact proteins for peptide mapping. The method includes reducing the intact proteins in the sample to a linear chain of amino acids. The method also includes enzymatically treating the linear chain of amino acids to produce a peptide mixture. The sample containing the peptide mixture is injected into a mobile phase containing citric acid. The peptide mixture is separated using RPLC. Individual peptides are analyzed using a mass spectrometer, an ultraviolet detector, a fluorescence detector, or a combination thereof. In some embodiments, instead of or in addition to citric acid, sodium citrate, isocitrate, diammonium citrate, or triammonium citrate is used.

[0096] At least one peptide in the peptide mixture includes a charged amino acid residue or has a net charge. The net charge can be negative. The net charge can be positive. Table 1 shows the PENNYK peptide charges at different pH values. The formic acid pH is about 3.0, so the net charge is slightly positive. Without being bound by theory, the interaction with metal ions most likely occurs between the negative charges on the peptide chain. The protein can be synthetic or recombinant. The protein can be a monoclonal antibody (mAb). The amino acid can be aspartic acid, glutamic acid, or isoaspartic acid. The peptide mixture can include peptides and deamidated forms of the peptides. For example, the peptide mixture can include PENNYK peptides and deamidated forms of PENNYK peptides (discussed in detail below). The amino acids can be enzymatically treated with trypsin, Lys-C, Asp-N, or a combination thereof. The amino acids can be enzymatically treated with other enzymes known to those skilled in the art.

[0097] Table 1: Charge of PENNYK Peptide at Different pH Values

[0098] pH Charge 1.00 2.0 1.50 2.0 2.00 1.9 2.50 1.7 3.00 1.4 3.50 0.8 4.00 -0.0 4.50 -1.2 5.00 -2.2 5.50 -2.7 6.00 -2.9 6.50 -3.0 7.00 -3.1 7.50 -3.2 8.00 -3.5 8.50 -3.9 9.00 -4.2 9.50 -4.7 10.00 -5.5

[0099] The USP tailing value can be from about 0.95 to about 1.30 and can be maintained over a period of about two to about three days. The USP tailing value can be maintained within about 1 day, 2 days, 3 days, 4 days, or 5 days. The USP tailing value can be maintained within about 48 consecutive injections. For example, the USP tailing value can be maintained between about 40 and about 100 consecutive injections. In some embodiments, the USP tailing value is between about 0.98 and about 1.20, or between about 1.00 and about 1.10. In some embodiments, the USP tailing value is about 1.00.

[0100] The concentration of citric acid can be, for example, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, or 10 ppm. These values can be used to form a range, for example, between about 1 ppm and about 10 ppm or between about 2 ppm and about 5 ppm. The mobile phase can also include formic acid, acetic acid, water, acetonitrile, methanol, isopropanol, n-propanol, or a combination thereof.

[0101] The method may further include passivating the RPLC column with citric acid before separating the peptide mixture.

[0102] The methods described herein can also be used to separate analyte fatty acids (e.g., lauric acid). The method can include injecting a sample containing the fatty acid into a mobile phase containing citric acid. RPLC can be used to separate the fatty acids. A mass spectrometer, ultraviolet detector, fluorescence detector, or a combination thereof can be used to analyze the separated fatty acids.

[0103] The concentration of citric acid can be, for example, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, or 10 ppm. These values can be used to form a range, for example, between about 1 ppm and about 10 ppm or between about 2 ppm and about 5 ppm. Other embodiments described for peptide separation can also be used when separating fatty acids.

[0104] The techniques described herein also include kits. The kit can include a liquid chromatography column (e.g., an RPLC column) and a vial containing a citric acid mobile phase additive. The kit also includes instructions for using the citric acid mobile phase additive in a method for analyzing a sample containing an analyte (e.g., a peptide, PENNYK peptide, fatty acid, lauric acid). The instructions can be any of the methods described herein. The instructions can include adding the citric acid mobile phase additive to the mobile phase. The instructions can also include detailed information about a specific concentration of the citric acid mobile phase additive, e.g., obtaining a citric acid mobile phase additive of about 1 ppm to about 10 ppm. The instructions can also state injecting the sample containing the analyte into the mobile phase containing citric acid. The analyte is separated using the liquid chromatography column (or RPLC column) provided in the kit. The separated analyte can then be analyzed using a mass spectrometer, ultraviolet detector, or a combination thereof. The analyte analyzed with the kit can be a fatty acid, lauric acid, protein, peptide, PENNYK peptide, intact protein, synthetic protein, recombinant protein, or a combination thereof.

[0105] When the analyte is a protein, intact protein, synthetic protein, or recombinant protein, the method can further include reducing the intact protein to a straight chain of amino acids and enzymatically treating the straight chain of amino acids to produce a peptide mixture.

[0106] As another example of a kit of the present technology, the kit can include a liquid chromatography column, a vial containing a metal chelator mobile phase additive, and instructions for using the metal chelator additive in any of the methods described herein. For example, the method can include adding the metal chelator mobile phase additive to the mobile phase to obtain a metal chelator mobile phase additive at a concentration between about 1 ppm and about 10 ppm, injecting a sample containing an analyte into the mobile phase having the metal chelator, separating the analyte using LC, and analyzing the separated analyte using a mass spectrometer, an ultraviolet detector, a fluorescence detector, or a combination thereof. The metal chelator additive can be citric acid. The metal chelator can also be sodium citrate, isocitrate, diammonium citrate, triammonium citrate, or ammonium formate.

[0107] For initial studies, identifying representative peptides and associated quality factors is necessary to evaluate the impact of chelators on chromatographic performance. To this end, the peptides studied should be important for the target application prospect and contain charged amino acid residues that can interact with metal substances. Deamidation of asparagine to aspartic acid and isoaspartic acid is a common post-translational modification of monoclonal antibodies (mAbs). Due to its direct correlation with drug efficacy, deamidation has been considered a critical quality attribute (CQA) of mAb-based therapeutics. Therefore, biopharmaceutical companies have invested a large amount of resources in controlling and monitoring impurities such as deamidation. Among the deamidated substances monitored, the PENNYK peptide with SEQ ID NO 1 is of concern because it contains 7 hydrophobic amino acids and 4 charged amino acids after enzymatic treatment of intact mAb with trypsin. These characteristics make its separation particularly challenging (see Figure 2A , Figure 2B and Figure 2C ). Using RPLC-based separation, the native and deamidated forms of the PENNYK peptide elute closely as a key pair under formic acid conditions using a moderate gradient (see Figure 2D ). As shown by the SIR trace in Figure 2C , peak tailing can hinder the accurate integration of the deamidated form. For LC-based separation, an ideal peak is defined as a Gaussian peak with a tailing factor of 1.0 and a narrow peak width. For initial studies, the USP tailing factor at 5% will be used as a quality factor to evaluate the impact of using chelators as mobile phase additives in RPLC-based peptide separation.

[0108] SEQ ID NO 1: G FY PSD IAV E W ESNGQPENN Y K

[0109] The underlined portion of SEQ ID NO 1 is hydrophobic, the bold portion is charged, and the italicized portion is anionic (note that some are both charged and anionic as denoted by both bold and italic). The molecular formula is C 114 H 158 N 28 O 39 , the monoisotopic mass is 2543.12 Da, and the average mass is 2544.67 Da.

[0110] Regarding the quality factor, in RPLC-based methods, peak tailing is usually accepted due to the C-term contribution to the overall chromatographic performance. The presence of electrostatic interactions of charged groups may manifest as tailing and the interaction of charged peptides with trace metal contaminants in the wetted flow path. For these reasons, the USP tailing factor can be used as a quality factor to determine the effect of metal chelators on peptides (see EQ.1)

[0111]

[0112] In EQ.1, T f is the tailing factor, W 0.05 is the peak width at 5% peak height, R t is the retention time, and F is the time from the start of the width at 5% peak height to the retention time (R t ) (see Figure 3 ). The tailing factor, T f , establishes the maximum allowable asymmetry of the peak. For pharmaceutical purposes, T f is defined as the distance between the front and back edges of the peak at the width at 5% peak height divided by twice the distance F between the peak maximum and the front edge of the peak at 5% peak height. For a symmetric peak, T f is 1.0, and the value of T f increases as the tailing becomes more pronounced. When T f is less than 1.0, there is a "front" of the peak, which may be related to mass loading or potential overloading.

[0113] From previous work studying adduct formation during oligonucleotide isolation, it was found that an initial system wash with 30% phosphoric acid was able to reduce trace metal contamination in the LC system. Using this same protocol, the LC system was cleaned to determine the baseline performance of the PENNYK peptide using an RPLC-based separation method. The protocol involved washing and passivating the LC / MS system using a solution containing 30% phosphoric acid v / v% before each experiment. The acid was prepared from stock reagents using LC / MS grade water. The system modules cleaned and passivated included the BSM, AS-FTN, and CM-A, where a stainless-steel two-way replaced the column. After the two-way bypassed the TUV and mass detector, the flow was directed to waste. For each treatment, 250 mL of the acid solution was prepared and passed through the LC / MS system at 1.0 mL / min for 250 minutes. After the cleaning and passivation protocol, the system was rinsed overnight or for at least 12 hours or until the eluent reached a pH of approximately 7.0 using 1 L of LC / MS grade water at a flow rate of 1 mL / min. As Figure 4A shown, after purifying the system with the 30% phosphoric acid solution, the RPLC-based method was able to directly resolve the native form of PENNYK from its deamidated form with minimal tailing. However, using the same sample and mobile phase, the same method was run for 12 hours ( Figure 4B ) and 24 hours ( Figure 4C ), and chromatographic performance deterioration was observed according to the USP tailing factor and visual assessment of the resolution between the native and deamidated peptide forms. This indicates that an accumulation of trace metal contamination occurred and negatively affected the method's ability to resolve the native and deamidated forms of the PENNYK peptide. To maintain chromatographic performance, the mobile phase was prepared with the metal chelator citric acid at a concentration of 0.01%. Using the same method, 48 consecutive injections were made from the form of the tryptic digest of the NIST mAb reference material. As Figures 5A - 5C shown, the chromatographic performance of the PENNYK peptide was maintained over a 64-hour period, with an average USP tailing value of 1.00 ( Figure 5D ).

[0114] This indicates that the chelator, as a mobile phase additive, can increase and maintain the chromatographic performance of key substances over an extended period.

[0115] Example 1 - PENNYK Peptide

[0116] Metal ion-mediated adsorption in liquid chromatography has been identified as a factor contributing to poor peak shape, tailing, and reduced recovery of compounds that readily undergo cation-exchange-like interactions with metal-based active sites. When using a weak acid (e.g., formic acid) as a mobile phase additive, peptides carrying negatively charged amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal ion-mediated adsorption in RPLC / MS-based separations. In an RPLC / MS-based peptide mapping assay, the ability of citric acid and methylenediphosphonic acid as metal-complexing mobile phase additives to mitigate metal ion-mediated adsorption was evaluated. In this example, chromatographic performance was stable, with up to a 40% reduction in peak tailing of the peptide of interest in the presence of a chelating agent at a mobile phase concentration of 1 ppm. Over a 67-hour time study, the performance gain was observed to be stable, with an average USP tailing factor of 1.00% and %RSD = 0.64. The stabilizing effect of the chelating agent improved the robustness of the peptide mapping assay, with relative peak areas of target impurities of 2.32% (%R.S.D. = 2.23) and 2.57% (%R.S.D. = 3.97). This example demonstrates that chelating agents as mobile phase additives provide a means to improve the chromatographic performance of biomolecules sensitive to metal ion-mediated adsorption under formic acid-based RPLC conditions.

[0117] In LC-based assays, metal ion-mediated adsorption has been recognized as a factor contributing to poor peak shape, tailing, and reduced recovery of compounds that readily interact based on charge. Without being bound by theory, the proposed mechanism is that trace mobile phase contaminants in the form of metal impurities are adsorbed by the wetted mobile path or active sites within the column, after which they exhibit cation-exchange properties towards negatively charged solute molecules. The strong binding properties exhibited by these high-energy active sites result in peak tailing and reduced recovery, where desorption of the adsorbed solute molecules (if present) occurs towards the rear region of the elution band. This immobilized metal ion affinity chromatography (IMAC)-like phenomenon has been shown to be particularly problematic in the separation and recovery of phosphorylated compounds, where the inherent chelating properties of the phosphoryl group enhance metal ion binding.

[0118] Combining mobile phase additives in chromatography is a well-known strategy to suppress the adsorption characteristics of high-energy active sites. In terms of metal ion-mediated adsorption, early work using MALDI-MS technology showed that the recovery of phosphorylated compounds was improved by adding diammonium citrate or phosphoric acid to the matrix. In LC-ESI-MS analysis using EDTA and phosphoric acid as sample additives, the beneficial effects of metal complexing agents on improving the recovery of phosphorylated peptides were further investigated. Improvements to these techniques were demonstrated by Winter et al. using a more MS-compatible chelating agent in the form of citrate to overcome the clogging of the ESI spray needle under RPLC conditions when using EDTA. (D. Winter, J. Seidler, Y. Ziv, Y. Shiloh, W. D. Lehmann, Citrate boosts the performance of phosphopeptide analysis by UPLC-ESI-MS / MS, Journal of proteome research, 8 (2009) 418-424.) Further studies by Siedler et al. showed that using metal complexing agents as sample additives achieved performance gains across various LC / MS configurations. (J. Seidler, N. Zinn, E. Haaf, M. E. Boehm, D. Winter, A. Schlosser, W. D. Lehmann, Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS, Amino acids, 41 (2011) 311-320.)

[0119] Recently, Hsiao et al. investigated the recovery and peak shape of anions and phosphorylated compounds under HILIC conditions in the presence of metal ion complexing agents. (J.J. Hsiao, O.G. Potter, T.W. Chu, H. Yin, Improved LC / MS Methods for the Analysis of Metal-Sensitive Analytes Using Medronic Acid as a Mobile Phase Additive, Analytical chemistry, 90(2018)9457-9464.) Notably, it was found that direct addition of methylene diphosphonic acid (medronic acid) to the mobile phase did not result in significant ion suppression or residual LC adsorption compared to more traditional chelating agents such as EDTA. Since metal impurities are ubiquitously present at trace levels in makeup solvents, reagents, and LC equipment, metal impurities may be introduced during the execution of experiments, and such direct methods are more attractive in developing robust LC-based assays because tailing and poor peak shape of anions or phosphorylated compounds may affect the sensitivity and accuracy of the assay, just like therapeutic proteins.

[0120] Peptide-based analysis is one of the primary analytical methods routinely performed throughout the life cycle of therapeutic proteins. It has proven to be a very valuable tool for the characterization and quality control of protein-based therapeutics to elucidate structural information and assess protein modifications. Recently, MS-based peptide mapping has been deployed in a manufacturing setting to improve productivity and data quality by simultaneously and effectively monitoring multiple potential critical quality attributes (pCQAs). As part of its composition, peptides inherently contain negatively charged amino acids in the form of aspartic acid and glutamic acid, which may be affected by metal ion-mediated adsorption. Interestingly, a search of the literature revealed limited information available when evaluating metal complexing agents and their effects on peptides containing these charge motifs. This is not surprising considering that ion-pairing agents such as trifluoroacetic acid (TFA) as a mobile phase additive can suppress adsorption artifacts to an acceptable level in RPLC-based peptide separations. However, as the number of MS-based methods implemented in the development and manufacture of therapeutic biologics increases and the use of weaker mobile phase additives such as formic acid (FA) is favored for sensitivity rather than chromatographic performance, there are concerns that metal ion-mediated adsorption may affect the robustness and performance of the assay.

[0121] The objective of this example is to evaluate the effect of metal complexing agents on peptides containing negatively charged residues (such as aspartic acid and glutamic acid) under RPLC-MS conditions. Chromatographic performance in terms of peak tailing and MS response will be used as a measure of the evaluation results, and the optimal strategies for reducing metal ion-mediated adsorption of non-phosphorylated anionic peptides will be discussed.

[0122] Materials and Methods

[0123] Materials

[0124] The NIST monoclonal antibody reference material 8671 was purchased from the National Institute of Standards and Technology (Gaithersburg, MD) and stored at -80 °C before use. Tris-hydrochloride (Tris-HCl), guanidine hydrochloride, dithiothreitol (DTT), iodoacetamide (IAA), citric acid (≥99.5%), sodium citrate (pure), ammonium citrate dibasic (≥99.0%), ammonium citrate tribasic (≥97%), DL-sodium isocitrate hydrate (≥93%), and ammonium formate (MS grade) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. The deactivator additive (methylene diphosphonic acid) was purchased from Agilent Scientific Instruments (Santa Clara, CA) and used according to the manufacturer's instructions. Sequencing grade modified trypsin was purchased from Promega (Madison, WI) and stored at -20 °C before use. LC / MS grade water, acetonitrile, and nitric acid were purchased from Honeywell (Charlotte, NC). LC / MS grade formic acid and orthophosphoric acid (85%) were purchased from Fisher Scientific (Hampton, NH). Micro-P6 centrifugal columns were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA) and stored at 4 °C.

[0125] Sample Preparation

[0126] To control variability across the entire example, attention was paid to the selection, preparation, and data acquisition of the samples. A commercially available mAb standard (NIST RM 8671) was zymogenized using a protocol adapted from Ren et al. (An improved Trypsin Digestion Method Minimizes Digesterion-Induced Modifications on Proteins, Analytical Biochemistry, 392(2009)12-21) to reduce digestion artifacts. Briefly, intact mAb was digested with sequence-grade trypsin using the following protocol. Stock solutions were prepared with applicable LC / MS-grade water. 20 μL of NIST mAb was added at 10 mg / mL to 60 μL of a denaturing buffer consisting of 0.1 M Tris-HCl (pH 7.5) and 8.25 M guanidine. 2.5 μl of DTT prepared in 0.1 M Tris-HCl (pH 7.5) was added to the solution at a concentration of 400 mM. The sample was incubated at 37 °C for 60 minutes in an MJ PTC-100 thermal cycler (commercially available from MJ Research, Inc., Waltham, MA). After incubation, 5 μL of 400 mM IAA prepared in 0.1 M Tris-HCl (pH 7.5) was added to the mixture, followed by incubation at room temperature for 45 minutes in the dark. The Micro-P6 centrifugal column (commercially available from Bio-Rad Laboratories, Inc., Hercules, CA) for sample desalting was prepared by washing with 500 μL of 0.1 M Tris-HCl (pH 7.5) buffer loaded onto the column and centrifuging at 1000 g for 1 minute into a waste bottle using a 5415R centrifuge (commercially available from Eppendorf AG, Hauppauge, NY). This step was repeated for a total of 4 washing cycles. Then the sample was loaded onto the center of the column bed and centrifuged at 1000 g for 4 minutes into a collection bottle. After desalting, 10 μL of 1 mg / mL trypsin prepared in the resuspension buffer provided by the manufacturer was added to the sample at an enzyme-to-substrate ratio of 1:20. The mixture was incubated at 37 °C for 60 minutes. After incubation, the digest was diluted 1:1 with LC / MS-grade water containing 0.1% formic acid. All analyses were performed using an injection volume of 10 μL. To minimize sample degradation, the sample was homogenized into a master pool and aliquoted into ppendorf tubes (commercially available from Eppendorf AG, Hauppauge, NY) and stored at -80 °C after enzymatic digestion and before use.

[0127] LC / MS System Configuration

[0128] Use I-Class PLUS series LC instruments (commercially available from Waters Technologies Corporation, Milford, MA) were used for data acquisition to minimize system dispersion and take advantage of the impact of gradient accuracy on chromatographic performance. The system was configured with a binary solvent manager (BSM) equipped with a 380 μL mixer (PN 205000705), a flow-through needle sample manager (SM-FTN), and a column manager (CM-A). Optical data were acquired at 214 nm wavelength using a tunable ultraviolet detector (TUV) equipped with a 10 mm analytical flow cell. Online configuration after the TUV a single quadrupole mass detector (commercially available from Waters Technologies Corporation, Milford, MA) to facilitate the monitoring of key peptide species. At Reverse-phase separation was performed at 60 °C on a CSH C18 column (1.7 μm, 2.1×100 mm) (commercially available from Waters Technologies Corporation, Milford, MA). After an initial isocratic hold at 1% B for 5 minutes, a 60-minute gradient was applied to 35% B. The column was then cleaned by increasing the %B composition to 70% in 3 minutes and holding at 70% for an additional 2 minutes. After returning to the initial conditions in 1 minute, column equilibration was carried out for 6 minutes. All gradients were run at a flow rate of 0.200 ml / min, and the total run time was 87 minutes. The mobile phase was prepared with LC / MS grade water (MP A) and acetonitrile (MP B) with 0.1% FA v / v. Citric acid and methylenediphosphonic acid of the metal complexing agent were prepared at 1 ppm or 5 μM in the two mobile phases, respectively. To ensure chelator solubility, the mobile phase was prepared with 3% B in MP A and 3% A in MP B. MS quadrupole data were acquired in positive mode at a sampling rate of 2 Hz using a full scan range of 450 - 1250 m / z and single ion recording at 849.2 m / z and 849.6 m / z to monitor the native and deamidated forms of [M+3H] of peptide T37(HC) of NIST RM, respectively +3 charge state. The cone voltage, capillary voltage, and probe temperature were set to 10 V, 1.5 kV, and 600 °C, respectively.

[0129] System Cleaning and Passivation

[0130] Before each experiment, the LC / MS was cleaned and passivated using a solution of 30% (v / v%) phosphoric acid prepared from stock reagents with LC / MS grade water. The system modules cleaned and passivated included the BSM, AS-FTN, and CM-A, where a stainless-steel two-way replaced the column. After the two-way bypass TUV and mass detector, the flow was directed to waste. For each treatment, a solution of 250 mL phosphoric acid solution was prepared at the desired concentration and passed through the LC / MS system at 1.0 mL / min for 250 minutes. Following the cleaning and passivation protocol, the system was rinsed overnight or for at least 12 hours with 1 L of LC / MS grade water at a flow rate of 1 mL / min or until the eluate reached a pH of approximately 7.0. The pH was determined by collecting approximately 10 mL of eluate in a plastic conical tube and measuring it using a Fisher Scientific Acument XL250 pH / mV / ISE meter (Pittsburgh, PA).

[0131] Results and Discussion

[0132] For this example, identifying representative peptides was necessary to evaluate the impact of chelating agents on chromatographic performance. To this end, the peptides studied should be important for the target application and contain charged amino acid residues that can interact with metal ions. Deamidation of asparagine to aspartic acid and isoaspartic acid is a common post-translational modification of monoclonal antibodies (mAbs) that has been associated with drug efficacy. Therefore, biopharmaceutical companies have invested significant resources in controlling and monitoring impurities such as deamidation. Among the deamidated species monitored, the "PENNY" peptide (SEQ ID NO 1) is of interest because it contains 7 hydrophobic amino acids and 4 negatively charged amino acids after enzymatic treatment of intact mAb with trypsin, making it an ideal candidate for metal ion-mediated adsorption. Additionally, the PENNYK sequence is located in the constant region of the Fc domain in all humanized mAbs, which increases its relevance as peptides are routinely monitored as a critical quality attribute across the biopharmaceutical industry.

[0133] Before evaluating metal complexing agents, it was necessary to establish baseline system performance to facilitate comparison of results. From previous work, it was observed that a 30% phosphoric acid solution was capable of displacing trace metal impurities adsorbed on the entire wetted flow path surface. Following a similar procedure, the system fluid path was cleaned using a phosphoric acid solution. After neutralizing the system, a series of 10 injections of mAb digest were performed using a conventional RPLC mobile phase (H 2 O:MeCN, 0.1% FA) commonly used in RPLC / MS peptide mapping assays, with 4 blank injections occurring between protein injections.

[0134] As Figure 6AAs shown, the preliminary characterization of native PENNYK peptide (T37) using RPLC separation with enzymatically treated NIST mAb reference material showed that the PENNYK peptide eluted approximately in the middle of the 60-minute separation gradient with a retention time of 34.9 minutes. Due to the peak density of the chromatogram and the close elution nature of the impurities, the distribution of native and deamidated forms was monitored using single ion recording (SIR), as Figure 6B shown. The USP tailing factor (T f = W 0.05 / 2f) at 5% peak width was used to evaluate the tailing of PENNYK during the injection series. After phosphoric acid wash, the initial chromatographic performance (0 hours) was observed to be acceptable, with the tailing of the native peak calculated as 1.12. However, an increase in tailing was observed after 72 hours of continuous instrument use, with a calculated tailing factor of 1.21. Traditionally, in terms of chromatographic performance, a tailing value below 1.2 is generally considered acceptable. However, as shown in the chromatogram, the increased tailing can significantly affect the distribution and integration of closely eluting low-abundance peaks. In this injection series, during the 72-hour period, the leading impurity peak at 35.35 minutes showed a 12% decrease in relative area from 1.58% to 1.40%, while the trailing impurity peak at 35.55 minutes showed a 25% increase from 2.20% to 2.76%. Although subtle, these observations demonstrate that in the presence of a relatively low degree of tailing, closely eluting low-abundance impurities are more prone to exhibit increased variability, and it is impractical to avoid this due to the increased tailing as the trailing peak edge approaches the baseline.

[0135] As Figure 7 shown, the tailing factor at lower peak widths exhibits pseudo-exponential behavior, making shallower gradients a controversial issue at the expense of longer run times and sensitivity. Upon careful observation Figure 7 , the observed deterioration of chromatographic performance over time indicates that the cause of tailing is introduced in the form of contaminants with adsorption characteristics rather than as a pre-existing condition, as the system's performance immediately following a cleaning protocol with phosphoric acid was acceptable. In our previous study, it was observed that metal adduct species such as Na + and K + were able to adsorb onto metal surfaces within the LC fluid path. However, the likelihood of active sites being associated with Na + and K + is negligible, considering that it was shown that acidic LC conditions induced by formic acid were sufficient to displace weakly adsorbed metal ions such as Na + and K +。If metal ion-mediated adsorption is the cause of the tailing, these observations would suggest that metal ions with strong adsorption properties such as Fe II / III may be present in the wetting flow path as adsorption sites and lead to the observed deterioration of the chromatographic performance of the PENNYK peptide, similar to the observations made by Seidle et al. (Metal Ion-Mobilizing Additives for Comprehensive Detection of Femtomole Amounts of Phosphopeptides by Reversed Phase LC-MS, Amino acids, 41 (2011) 311-320). With this concept in mind, mitigation strategies using metal chelators were explored.

[0136] Citric acid and methylenediphosphonic acid were selected as metal chelators to evaluate their effects on PENNYK tailing and area response. Using a simplified injection series, the same cleaning method and separation as before were employed. The mobile phase was prepared in the same way, with the corresponding chelating agent added at a concentration of 1 ppm. As Figures 8A - 8C shown, compared to the previous experiment without the chelating agent, both chelating agents had a significant effect on stabilizing the chromatographic performance of the PENNYK peptide ( Figure 8A ). The observed performance gain was maintained from the initial injection, greatly reducing experimental variability and confirming the concept of metal ion-mediated adsorption. Without being bound by theory, the chelating agent complexes with metal ion impurities present in the mobile phase and acts as a solubilizer to prevent metal ions from adsorbing onto the fluid surface or as a protecting group to mitigate metal ion-mediated adsorption. As Figure 8B and Figure 8C shown, at peak widths as low as 1% of the peak height, citric acid and methylenediphosphonic acid were able to reduce peak tailing by up to 40% within 22 hours. Mitigating tailing improved the peak area consistency of low-abundance impurities (%R.S.D. < 3%), with methylenediphosphonic acid showing a peak area closer to the initial separation ( Figure 8A , 0 h). However, it should be noted that methylenediphosphonic acid was observed to have a negative impact on the overall chromatographic performance and exhibited increased ionization efficiency differences and / or ion suppression artifacts.

[0137] As Figures 9A - 9D shown, the extracted ion chromatograms (XICs) of the major peptide species eluting in the vicinity of the PENNYK peptide (from 32 minutes to 54 minutes) were used to evaluate the MS responses of these peptides. Examination of the MS response of the PENNYK peptide (T37) showed that both citric acid ( Figure 9B ) and methylenediphosphonic acid ( Figure 9C ) had increased MS responses compared to the same run without the chelating agent (Figure 9A )。 In this case, the citric acid MS-response was 35% higher than the initial run, while methylenediphosphonic acid only showed an MS-response 10% higher than the initial run. The lower MS-response exhibited by methylenediphosphonic acid relative to citric acid may be due in part to foreign contaminants in the mobile phase. As Figures 9A - 9C shown in the inset of, the combined MS mass spectra under initial conditions indicated a higher degree of mass spectrometry contamination in the mobile phase with chelating agents, particularly in the presence of methylenediphosphonic acid. In the absence of prior knowledge of the impurity spectra of commercial chelating agents, it can only be speculated that these foreign components contribute to differences in ionization efficiency across experiments, as Figure 9D shown. Contrary to the findings of Hsiao et al., this example shows that methylenediphosphonic acid has the unexpected effect of increasing peak tailing in later-eluting peptides, most notably in Figure 9C peak 3 as shown in. The study of peak 3 identified it as the alkylated hinge region peptide T20(HC): THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO 2). In contrast, the citric acid peak profiles were generally retained or improved across the same peptide species, most notably in the later-eluting species (peaks 7-11). Interestingly, peak 3 did not show a significant increase in tailing in the absence of chelating agents, although the peptide T14(LC): VDNALQSGNSQESVTEQDSK (SEQ ID NO 3), which contains multiple glutamate and aspartic acid residues, exhibited tailing behavior similar to that of the PENNYK peptide (data not shown). Without being bound by theory, the possible reason for the observed differences may be due to changes in the acidic properties of the peptides, where the carboxylic acid group of citric acid acts as a better mimic of the peptide compared to the diphosphate structure of methylenediphosphonic acid, or the chelator affinity / coordination properties. Given this, further studies are needed to fully understand the role played by the physicochemical properties of chelating agents and peptides in the adsorption / coordination properties of peptides. However, the chromatographic performance gains provided by chelating agents in peptide mapping assays are evident, particularly for targeted assays of CQAs or routine monitoring scenarios where key analytes are not affected by secondary interactions in the presence of chelating agents.

[0138] In the case where supportive evidence for metal ion-mediated adsorption was established, the effects of derivatives of citric acid, including sodium citrate, diammonium citrate, and triammonium citrate, on the US / MS response and whether chromatographic performance could be further improved were evaluated. Interestingly, all of the derivatives explored showed increased fronts compared to citric acid and methylenediphosphonic acid. Assuming that the mobile phase was prepared with the same concentration of chelating agent and the same sample was used across the chelating agent groups, the observed fronts seemed to be related to the chelating agent rather than the sample or the sample matrix. It should be noted, however, that the chelating agents did not have comparable purity, which could have contributed to some of the observed differences. Regarding chromatographic performance, the alternative chelating agents showed increased resolution between the native peak and impurities of the PENNYK peptide but could not improve the resolution between impurity peaks and, in some cases, the resolution decreased. As expected, the Na + -containing derivatives produced more salt adducts in the MS-mass spectrum compared to the ammonium-based derivatives; however, in terms of chromatographic performance, neither type offered advantages over citric acid or methylenediphosphonic acid. Additive concentrations up to 50 ppm were also evaluated, but no additional beneficial effects were observed. It should be noted that in the case of citric acid, 1 ppm seemed to be the minimum amount of chelating agent required to have an observable effect on chromatography. Given these results, citric acid was selected, considering its optimal MS-response, selectivity comparable to native conditions, and the absence of secondary interactions.

[0139] To determine Figure 8B whether the residual tailing observed was the result of column overloading, peptide mapping was performed at reduced mass loads by diluting the sample with an equal volume of sample buffer. As Figure 10 shown, the peak tailing was only reduced by an additional 7% (T f = 1.05), where the peak shape had an ideal Gaussian behavior at the peak width at 1% of the peak height. In addition to the mass load, the thermodynamic contribution to tailing was also evaluated. As Figures 11A - 11C shown, for separations performed at a lower temperature of 40 °C in the presence of a chelating agent, the T f value was 1.13 and the peak tailing of the PENNYK peptide was retained. Although some peak broadening was observed, the chromatographic profile and relative intensities were highly similar to the 60 °C data. In the absence of a chelating agent, T37 did show a marginal increase in tailing of 11% at 40 °C (T f = 1.26), and the relative intensities were more similar to the chromatographic profiles shown in Figure 6A and Figure 6B . These observations suggest that for the PENNYK peptide, in the presence of a chelating agent, the contributions of mass load and heat to residual peak tailing are minimal. In cases showing similar behavior to that in Figures 11D - 11FConfirmatory data was observed in the peptide T14 with similar behavior as shown. As a peptide containing acidic residues, the chromatographic profile of T14 was generally retained at lower temperatures, with only a 6% increase in tailing in the presence of a chelating agent, as opposed to more than a 2-fold increase in tailing when the chelating agent was removed from the mobile phase. The lower sensitivity to tailing observed in T37 in the presence and absence of a chelating agent, compared to T14, further supports the concept that metal ion-mediated adsorption is the major contributing factor to tailing of PENNYK peptides in this study. To evaluate the long-term stability determined under more severe conditions, peptide mapping was performed on a series of 48 consecutive injections using a modified gradient. As Figure 12 shown, the peak tailing of the newly prepared peptide digest was stable within 67 hours of continuous instrument use, with an average tailing factor of 0.96 (%R.S.D. = 0.43) at W 0.01 . It should be noted that the peak width at W 0.05 was experimentally determined to be Gaussian, with a calculated tailing factor of 1.00 (%R.S.D. = 0.64). The stabilizing effect of the chelating agent improved the assay robustness, with relative peak areas calculated as 2.32% (%R.S.D. = 2.23) and 2.57% (%R.S.D. = 3.97) for the leading and trailing impurity peaks, respectively. In summary, these results demonstrate that the use of a chelating agent as a mobile phase additive provides a method to improve the chromatographic performance of biomolecules sensitive to metal ion-mediated adsorption under formic acid-based RPLC conditions.

[0140] Conclusion

[0141] Metal-ion mediated adsorption has emerged as an area of ongoing research interest due to its impact on chromatographic performance and the recovery of problematic compounds. In the biopharmaceutical industry, peptide-level separation of protein-based therapeutics is particularly challenging for closely eluting critical pairs because peak tailing and recovery directly affect assay robustness in terms of accuracy and sensitivity. Metal-ion mediated adsorption of critical peptides further complicates the issue, especially in manufacturing environments where limited resources are available for developing and validating conventional monitoring assays. In such cases, there is a great need for assays that are robust and capable of delivering accurate and consistent results. This example demonstrates that metal complexing agents as mobile phase additives can significantly reduce metal-ion mediated adsorption of sensitive compounds to improve chromatographic performance in RPLC / MS-based separations. Specifically, in the presence of citric acid at a concentration of 1 ppm in the mobile phase, peak tailing of the PENNYK peptide was reduced by up to 40% at a peak width of as low as 1% peak height. The reduction in metal-ion mediated adsorption allowed for the accurate quantification of low-abundance deamidated impurities associated with the PENNYK peptide, a known critical quality attribute related to the efficacy of the drug product, with % R.S.D. ≤ 2.4%. The stabilizing property of the chelating agent on chromatographic performance was demonstrated by an average peak tailing value of 1.0 (R.S.D. of 0.64%) for native peaks calculated over a series of 48 injections spanning 67 hours, in contrast to performance deterioration in the absence of the chelating agent, where peak tailing increased to 1.85 over the same time period. Additionally, when citric acid was used as a mobile phase additive, the MS response of the PENNYK peptide was improved, with an increase in MS signal intensity of 35%, thus increasing the assay sensitivity for detecting low-abundance critical impurities. In summary, these results demonstrate the use of chelating agents as mobile phase additives for separation conditions to improve the chromatographic performance of peptides sensitive to metal-ion mediated adsorption in RPLC / MS-based separations. These conditions are particularly suitable for manufacturing environments where robust and consistent assays are inherently required to ensure drug safety and efficacy.

[0142] Example 2 - Lauric Acid

[0143] Test the effect of mobile phase chelating agents on different analyte types such as lauric acid. Use the same method as outlined for Figure 2. Start a serial dilution of lauric acid at 4,000 ppm. Run 10 μL injections moderately in a mobile phase of 75:25 acetonitrile:water and 1 ppm citric acid. Figure 14 A graph is shown that adding a chelating agent to the mobile phase improves the recovery of lauric acid (the major fatty acid component of polysorbate 20 (PS-20)) to obtain a linear MS response. This is useful for quantifying impurities present in the drug substance of a drug product after a finishing stage (cleaning or refining). In the absence of a linear response in the calibration curve, it is not possible to accurately quantify fatty acids using a multi-point calibration curve or a single-point calibration curve.

[0144] Example 3 - Comparison of Mobile Phase Additives

[0145] In this example, several different mobile phase additives were compared with 1 ppm citric acid. The system conditions are shown in Figure 15 .

[0146] Figure 16 A- Figure 16 D shows the comparison of 1 ppm citric acid with 1 ppm sodium citrate. From the comparison between Figure 16 A and Figure 16 , it can be seen that the deamidated species of the PENNYK peptide showed poor resolution and some fronts were seen when 1 ppm sodium citrate was used. In addition, Figure 16 the top mass spectrum of C compared with the top mass spectrum of Figure 16 D shows that several adducts of the main peak that appeared in Figure 16 C (1 ppm citric acid) were absent or present at significantly lower levels in Figure 16 D (1 ppm sodium citrate) (e.g., Na and K). Figure 16 The bottom mass spectra of C and Figure 16 D were used to view the baseline region of the chromatogram to evaluate the baseline noise intensity.

[0147] Figure 17 A- Figure 17 D shows the comparison of 1 ppm citric acid with 1 ppm isocitrate. From the comparison between Figure 17 A and Figure 17 B, it can be seen that the deamidated species of the PENNYK peptide showed poor resolution and some fronts were seen when 1 ppm isocitrate was used. In addition, Figure 17 the top mass spectrum of C compared with the top mass spectrum of Figure 17 D shows that Figure 17 several adducts of the main peak that appeared in Figure 17 C (1 ppm citric acid) were absent or present at significantly lower levels in Figure 17 D (1 ppm isocitrate) (e.g., Na and K). Figure 17 The bottom mass spectra of C and

[0148] Figure 18 A- Figure 18 D shows the comparison of 1 ppm citric acid with 1 ppm diammonium citrate. From the comparison between Figure 18 A and Figure 18 B, it can be seen that the deamidated species of the PENNYK peptide showed poor resolution and some fronts were seen when 1 ppm diammonium citrate was used. In addition, Figure 18 the top mass spectrum of C compared withFigure 18 Comparison of the top mass spectra of D shows that Figure 18 several adducts of the main peaks that appear in C (1 ppm citric acid) are absent or present at significantly lower levels in Figure 18 D (1 ppm diammonium citrate) (e.g., Na and K). Figure 18 C and Figure 18 the bottom mass spectra of D view the baseline region of the chromatogram to evaluate the baseline noise intensity.

[0149] Figure 19 A - Figure 19 D shows a comparison of 1 ppm citric acid with 1 ppm ammonium citrate tribasic. From Figure 19 A and Figure 19 B comparison, it can be seen that the deamidated species of the PENNYK peptide shows poor resolution and some fronting is seen when using 1 ppm ammonium citrate tribasic. In addition, Figure 19 the top mass spectrum of C and Figure 19 the top mass spectrum of D comparison shows that Figure 19 several adducts of the main peaks that appear in C (1 ppm citric acid) are absent or present at significantly lower levels in Figure 19 D (1 ppm ammonium citrate tribasic) (e.g., Na and K). Figure 19 C and Figure 19 the bottom mass spectra of D view the baseline region of the chromatogram to evaluate the baseline noise intensity.

[0150] Figure 20 A - Figure 20 D shows a comparison of 1 ppm citric acid with 1 ppm methylenediphosphonic acid. From Figure 20 A and Figure 20 B comparison, it can be seen that when using 1 ppm methylenediphosphonic acid, the deamidated species of the PENNYK peptide shows comparable shoulder peak resolution. In addition, Figure 20 the top mass spectrum of C and Figure 20 the top mass spectrum of D comparison shows that Figure 20 several adducts of the main peaks that appear in C (1 ppm citric acid) are absent in Figure 20 D (1 ppm methylenediphosphonic acid). Figure 20 C and Figure 20 the bottom mass spectra of D view the baseline region of the chromatogram to evaluate the baseline noise intensity.

[0151] Figure 21 A - Figure 21 D shows a comparison of 1 ppm citric acid with 5 μM Agilent InfinityLab additive. From Figure 21 A and Figure 21From the comparison of B, the deamidated species of the PENNYK peptide showed reasonable resolution but lower intensity of the impurities, but when using 5 μM Agilent InfinityLab additive, better resolution of the main peak was seen. This may be an artifact of suppression as only the top part of the peak was seen and thus it was a narrower peak. Additionally, Figure 21 The comparison of the top mass spectra of C with Figure 21 the top mass spectra of D showed that Figure 21 several adducts of the main peak that appeared in C (1 ppm citric acid) were absent in Figure 21 D (5 μM Agilent InfinityLab additive) (the baseline increased due to strong mass spectrometry contamination - see Example 2). Figure 21 The bottom mass spectra of C and Figure 21 D were examined in the baseline region of the chromatogram to evaluate the baseline noise intensity.

[0152] Those skilled in the art will recognize or be able to determine many equivalent forms of the specific procedures described herein using only routine experimentation. Such equivalent forms are considered to be within the scope of the present technology and are covered by the following claims. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference.

Claims

1. A method for analyzing a sample containing intact proteins for peptide mapping, the method comprises: reducing the intact proteins in the sample to a straight chain of amino acids; enzymatically treating the straight chain of amino acids to produce a peptide mixture; injecting the sample containing the peptide mixture into a mobile phase containing citric acid at a concentration of 1 ppm to 10 ppm; separating the peptide mixture using reversed-phase liquid chromatography; and analyzing the separated peptides using a mass spectrometer, an ultraviolet detector, a fluorescence detector, or a combination thereof.

2. The method according to claim 1, wherein at least one peptide in the peptide mixture contains a charged amino acid residue.

3. The method according to claim 1, wherein at least one peptide in the peptide mixture has a net charge.

4. The method according to claim 3, wherein the net charge is negative.

5. The method according to claim 1, wherein the protein is a monoclonal antibody.

6. The method according to claim 1, wherein the protein is a synthetic protein or a recombinant protein.

7. The method according to claim 1, wherein the amino acid is aspartic acid, glutamic acid, or isoaspartic acid.

8. The method according to claim 1, wherein the peptide mixture contains the peptide of SEQ ID No:1 and a deamidated form of the peptide of SEQ ID No:

1.

9. The method according to claim 1, wherein the amino acids are enzymatically treated with trypsin, Lys-C, Asp-N, or a combination thereof.

10. The method according to claim 1, wherein the chromatographic performance is maintained over a period of 2 days to 3 days, and the average USP tailing value is 0.95 to 1.

30.

11. The method according to claim 1, wherein the chromatographic performance is maintained within 48 consecutive injections, and the average USP tailing value is 0.95 to 1.

30.

12. The method according to claim 1, wherein the peptide mixture contains a peptide and a deamidated substance of the peptide.

13. The method according to claim 1, wherein the concentration of citric acid in the mobile phase is 1 ppm.

14. The method according to claim 1, wherein the mobile phase further comprises formic acid, acetic acid, water, acetonitrile, methanol, isopropanol, n-propanol, trifluoroacetic acid, difluoroacetic acid, or a combination thereof.

15. The method according to claim 1, the method further comprises passivating the reversed-phase chromatographic column with citric acid before separating the peptide mixture.

16. A kit for analyzing a sample containing intact proteins for peptide mapping, the kit comprises: a liquid chromatography column; a vial containing a citric acid mobile phase additive, the additive containing 1 ppm to 10 ppm of citric acid; and instructions for using the citric acid mobile phase additive in a method for analyzing the sample, wherein the method comprises: reducing the intact proteins in the sample to a straight chain of amino acids; enzymatically treating the straight chain of amino acids to produce a peptide mixture; adding the citric acid mobile phase additive to the mobile phase; Inject the sample containing the peptide mixture into the mobile phase containing citric acid; separate the peptide mixture using liquid chromatography; and Analyze the separated peptide mixture using a mass spectrometer, an ultraviolet detector, or a combination thereof.

17. The kit according to claim 16, wherein the liquid chromatography column is a reversed-phase liquid chromatography column.

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