Mass spectrometry compatibility pH gradient buffer system

Abstract

Improved mobile phase buffer compositions, methods, and kits for pH gradient LC-MS characterization of monoclonal antibodies and their charge variants are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT international patent application on June 22, 2022, and claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 213,562, filed June 22, 2021, and No. 63 / 325,392, filed March 30, 2021, the disclosures of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Monoclonal antibody (mAb) post-translational modifications (PTMs) are important quality attributes of therapeutic mAbs. Post-translational modifications can occur during production, purification, and storage. PTMs can affect the potency, activity, and stability of therapeutic mAbs. Primary amino acid sequence variations and PTMs can include C-terminal lysine truncation, N-terminal pyroglutamate formation, sialylation present during N-glycosylation, and deamidation of asparagine or glutamine residues. These modifications can alter the isoelectric point (pI) of the molecule resulting in acidic and basic charge variants of the mAb.

[0003] Monoclonal antibodies are routinely monitored during the manufacture and formulation of PTMs. Typical monitoring involves the use of ion exchange chromatography (IEC) with ultraviolet monitoring (UV) using a salt or pH gradient for elution. Monitoring mAb PTMs with IEC coupled to native high resolution mass spectrometry (HRMS) allows for rapid identification of modifications. Native MS of intact mAbs gives simpler spectra with less spectral overlap and less interference, facilitating identification of PTMs.

[0004] Improved mobile phase buffer compositions and methods for IEC-HRMS charge variant analysis of monoclonal antibodies that demonstrate improved reproducibility, resolution, sensitivity, and pH linearity are desirable. Summary of the Invention

[0005] The present disclosure provides pH gradient mobile phase buffer compositions and methods that provide good pH control over a wide range of pH values ​​(pH 5.2-10.2) with low ionic strength (about 50 mM or less, about 30 mM or less, or about 20 mM or less), with the advantages of improved MS sensitivity, "more native" mass spectra for intact proteins, lower charge states for protein isoforms, increased mass within the charge state envelope, and improved pH gradient linearity, robustness, and reproducibility.

[0006] Mobile phase pH gradient buffer compositions and methods are provided that demonstrate improved chromatographic resolution of charge variants of intact monoclonal antibodies (mAbs), including low isoelectric point (pI) mAbs (e.g., infliximab, pI 7.6), when using ion exchange chromatography.

[0007] The mobile phase buffer compositions and methods can be used in direct coupled IEC-HRMS of protein analytes and can demonstrate good MS sensitivity, for example, due to salt concentrations of less than about 50 mM, less than 30 mM, less than 25 mM, or less than 20 mM in the mobile phase buffer.

[0008] The mobile phase buffer compositions and methods may be used in IEC-HRMS of native protein analytes with improved mass spectrometry (MS) resolution due to the lower charge state of the intact protein, i.e., MS spectra showing intact protein analytes with reduced denaturation.

[0009] The buffer compositions and methods provided herein are suitable for both UV and HRMS detection of intact protein analytes.

[0010] The present disclosure provides a ready-to-use mobile phase composition for liquid chromatography comprising water, 2-50 mM ammonium carboxylate, 1-50 mM N-methylmorpholine, its isomer, or analog, and a pH in the range of about pH 4.5 to about pH 10.5 at room temperature. The ready-to-use mobile phase composition for liquid chromatography may comprise 2-25 mM ammonium carboxylate and 1-10 mM N-methylmorpholine, its isomer, or analog. The ammonium carboxylate may be ammonium acetate or ammonium formate. The N-methylmorpholine, its isomer, or analog may be N-methylmorpholine. The N-methylmorpholine isomer may be selected from the group consisting of 2-methylmorpholine, 2-methylmorpholine, 2-methyl-1,3-oxazinane, 3-methyl-1,3-oxazinane, 4-methyl-1,3-oxazinane, 5-methyl-1,3-oxazinane, and 6-methyl-1,3-oxazinane. The N-methylmorpholine analogs may be selected from the group consisting of alkyl C1-C6 morpholines, dialkyl C1-C6 morpholines, alkyl C1-C6-1,3-oxazinanes, and dialkyl C1-C6-1,3-oxazinanes.

[0011] A ready-to-use mobile phase composition for liquid chromatography is provided that includes water, 2-25 mM ammonium acetate, 1-10 mM N-methylmorpholine, and a pH in the range of about pH 4.5 to about pH 10.5 at room temperature.

[0012] The mobile phase may comprise a two-part aqueous buffer system, part A of the aqueous buffer system comprising about 10 mM to about 20 mM ammonium acetate, about 3 to about 8 mM N-methylmorpholine, and a pH in the range of pH 5.0 to 5.5, and part B of the aqueous buffer system comprising 2 to 10 mM ammonium acetate, 1 to 5 mM N-methylmorpholine, and a pH in the range of pH 9.5 to 10.5. In some embodiments, part A buffer may comprise about 14 mM to about 16 mM ammonium acetate, about 4 mM to about 6 mM N-methylmorpholine, and a pH of about pH 5.1 to about pH 5.3. In some embodiments, part B buffer may comprise about 4 mM to about 6 mM ammonium acetate, about 1 mM to about 3 mM N-methylmorpholine, and a pH of about pH 10 to about pH 10.4.

[0013] The pH of Part A buffer may be adjusted with acetic acid. The pH of Part B buffer may be adjusted with ammonium hydroxide.

[0014] The ready-to-use mobile phase composition may include an ammonium acetate concentration of 25 mM or less, 20 mM or less, or about 15 mM or less.

[0015] In some embodiments, the ready-to-use mobile phase composition may contain no more than 100 ppb of individual metal impurities.

[0016] The ready-to-use mobile phase composition may be used in a liquid chromatography method that includes a stationary phase. In some embodiments, the stationary phase may be selected from the group consisting of an ion exchange stationary phase, a size exclusion stationary phase, a hydrophilic interaction stationary phase, and a reversed phase stationary phase. In some embodiments, the ion exchange stationary phase may be a cation exchange stationary phase.

[0017] The cation exchange stationary phase may be selected from the group consisting of a strong cation exchange stationary phase and a weak cation exchange stationary phase.

[0018] The ready-to-use mobile phase composition may be used in methods involving liquid chromatography directly (on-line) coupled to a mass spectrometer.

[0019] The disclosure provides a method for separating and / or characterizing an analyte in a sample, the method comprising: flowing a mobile phase through a chromatography column, the mobile phase comprising a two-part aqueous buffer system, part A of the aqueous buffer system comprising about 10-50 mM ammonium carbonate, 3-16 mM N-methylmorpholine, or an isomer or analog thereof, and a pH in the range of about pH 5 to about pH 5.5, and part B of the aqueous buffer system comprising 2-25 mM ammonium carbonate, 1-10 mM N-methylmorpholine, or an isomer or analog thereof, and a pH in the range of about pH 9.5 to about pH 10.5; injecting a sample containing the analyte into the mobile phase; eluting the analyte from the column; and detecting the analyte in the eluate.

[0020] A method is provided for separating and / or characterizing analytes in a sample, comprising flowing a mobile phase through a chromatography column, the mobile phase comprising a two-part aqueous buffer system, part A of the aqueous buffer system comprising about 10-25 mM ammonium acetate, 3-8 mM N-methylmorpholine, and a pH in the range of about pH 5 to about pH 5.5, and part B of the aqueous buffer system comprising 2-10 mM ammonium acetate, 1-5 mM N-methylmorpholine, and a pH in the range of about pH 9.5 to about pH 10.5; injecting a sample containing the analyte into the mobile phase; eluting the analyte from the column; and detecting the analyte in the eluate.

[0021] In some embodiments, the analyte is a biomolecule. The biomolecule can be a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a charge variant thereof.

[0022] The monoclonal antibody or fragment may have a pI of about pI 6.5 to about pI 9.5.

[0023] The chromatography column may comprise a stationary phase selected from the group consisting of an ion exchange stationary phase, a size exclusion stationary phase, a hydrophilic interaction stationary phase, and a reversed phase stationary phase. The ion exchange stationary phase may be a cation exchange stationary phase. The cation exchange stationary phase may be selected from the group consisting of a strong cation exchange stationary phase and a weak cation exchange stationary phase.

[0024] In some embodiments, detecting may include determining UV absorbance of the eluate. In some embodiments, detecting may include detecting the analyte with a mass spectrometer (MS). The MS may be selected from the group consisting of sector, time-of-flight (TOF), quadrupole, ion trap, Fourier transform ion cyclotron resonance, and tandem (two or more of the above combined in a tandem or orthogonal platform) mass spectrometers.

[0025] The MS detection may include generating analyte ions. The generation may include an ionization technique selected from the group consisting of electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI), fast atom bombardment (FAB), chemical ionization (CI), electron impact (EI), atmospheric solids analytical ionization (ASAI), atmospheric pressure photoionization (APPI), desorption electrospray ionization (DESI), and atmospheric pressure vapor source (APVS). The MS detection may further include obtaining a mass spectrum of the analyte ions. The MS detection may further include determining the molecular weight of the analyte.

[0026] In some embodiments, the mobile phase buffer compositions of the present disclosure result in mass spectra of analyte ions exhibiting lower charge states for analyte isoforms than a comparable mobile phase buffer system comprising ammonium acetate without N-methylmorpholine. Use of the mobile phase buffer compositions of the present disclosure can result in lower charge states for analyte isoforms and masses of analytes within the charge state envelope than a comparable mobile phase buffer system comprising ammonium acetate without N-methylmorpholine.

[0027] In some embodiments, part A of the aqueous buffer system comprises about 10-25 mM ammonium acetate, 3-8 mM N-methylmorpholine, and a pH in the range of about pH 5 to about pH 5.5, and part B of the aqueous buffer system comprises 2-10 mM ammonium acetate, 1-5 mM N-methylmorpholine, and a pH in the range of about pH 9.5 to about pH 10.5. The pH may be adjusted to within the range of about pH 9.5 to about pH 10.5 using ammonium hydroxide. The mobile phase may contain up to about 100 ppb of individual metal impurities.

[0028] In some embodiments, elution may include forming a pH gradient. The pH gradient may include increasing the % of Buffer B over time relative to the % of Buffer A flowing through the column after injection, where % of Buffer A + % of Buffer B = 100% of the mobile phase. In some embodiments, the slope of the pH gradient is in the range of 0.1-10% B / CV of the eluate, or 0.5-5% B / column volume (CV). In some embodiments, the pH gradient may be a linear pH gradient, a segmented pH gradient, a curved pH gradient, or a step pH gradient.

[0029] The present disclosure provides a kit comprising a first container having a volume of a first concentrated liquid composition for dilution with water to obtain a buffer A comprising 10-50 mM ammonium carboxylate, 3-16 mM N-methylmorpholine, or an isomer or analog thereof, and a pH within the range of about pH 4.5 to about pH 5.5; a second container having a volume of a second concentrated liquid composition for dilution with water to obtain a buffer B comprising 2-25 mM ammonium carboxylate, 1-10 mM N-methylmorpholine, or an isomer or analog thereof, and a pH within the range of about pH 9.5 to about pH 10.5; and instructions for use.

[0030] A kit is provided that includes a first container having a volume of a first concentrated liquid composition for dilution with water to obtain a buffer A comprising 10-25 mM ammonium acetate, 3-8 mM N-methylmorpholine, and a pH in the range of about pH 4.5 to about pH 5.5, a second container having a volume of a second concentrated liquid composition for dilution with water to obtain a buffer B comprising 2-10 mM ammonium acetate, 1-5 mM N-methylmorpholine, and a pH in the range of about pH 9.5 to about pH 10.5, and instructions for use. In some embodiments, buffer A can include 10-20 mM ammonium acetate, 3-8 mM N-methylmorpholine, and a pH in the range of about pH 5 to about pH 5.5. Buffer B can include buffer B comprising 2-10 mM ammonium acetate, 1-3 mM N-methylmorpholine, and a pH in the range of about pH 9.5 to about pH 10.5. The first concentrated liquid composition and the second concentrated liquid composition may each comprise a concentration selected from the group consisting of 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x concentrated buffer A and buffer B. The kit may further comprise a chromatography column. For example, the chromatography column may be a strong ion exchange chromatography column or a weak ion exchange chromatography column. [Brief description of the drawings]

[0031] [Figure 1-1] HRMS total ion current (TIC) chromatogram from trastuzumab-anns on a Phenomenex® bioZen WCX weak cation exchange column using a new buffer system with NH4Ac+NMM. Monoclonal antibody trastuzumab-anns (Kajinti, 10mg / mL) with pI 9.1 was subjected to native WCX-HRMS using new buffers and 10uL (100ug) was injected onto a Phenomenex® bioZen 6μm WCX, 150x2.1mm weak cation exchange column using a 30-60% B gradient over 15 minutes according to Table 3, +TOF MS (2000-7000). [Figure 1-2] Figure 1 shows the total ion current (TIC) chromatogram for trastuzumab-anns on a Phenomenex® bioZen WCX column using the standard buffer system NH4Ac without NMM. Monoclonal antibody trastuzumab-anns (Kajinti, 10mg / mL) with pI 9.1 was subjected to native WCX-HRMS using optimized standard buffers using a gradient of 20-50% B over 15 minutes according to Table 2, 10uL (100ug) was injected onto a Phenomenex® bioZen 6μm WCX, 150x2.1mm weak cation exchange column according to Table 2, +TOF MS (2000-7000). [Figure 2-1] Figure 1 shows the total ion current (TIC) chromatogram for Rituximab-abbs on a Phenomenex® bioZen WCX column using a new buffer system with NH4Ac+NMM. Monoclonal antibody Rituximab-abbs (Rituxan) (10 mg / mL) with pI 9.4 was subjected to native WCX-HRMS using the new buffer. An aliquot of 10 μL (100 μg) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column and chromatographed using a 65-100% B gradient over 15 min, +TOF MS (2000-7000) as shown in Table 4. [Figure 2-2] Figure 1 shows the total ion current (TIC) chromatogram of Rituximab-abbs on a Phenomenex® bioZen WCX column using the standard buffer system NH4Ac without NMM. The monoclonal antibody Rituximab-abbs (Rituxan, 10 mg / mL) with pI 9.4 was subjected to native WCX-HRMS using the optimized standard buffer with a gradient of 60-100% B over 15 min as shown in Table 4, and 10 μL (100 μg) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column, +TOF MS (2000-7000). [Figure 3-1]Shown is the total ion current (TIC) chromatogram for the NIST reference mAb on a Phenomenex® bioZen WCX column using a new buffer system with NH4Ac+NMM. Monoclonal antibody NIST reference mAb (10 mg / mL) with pI 9.2 was subjected to native WCX-HRMS using the new buffer and 10 uL (100 ug) was injected onto a bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column using a gradient of 65-100% B over 15 minutes, +TOF MS (2000-7000). [Figure 3-2] Figure 1 shows the total ion current (TIC) chromatogram for NIST mAb on a Phenomenex® bioZen WCX column using the standard buffer system NH4Ac without NMM. The monoclonal antibody NIST Reference mAb with pI 9.2 was subjected to native WCX-HRMS using the optimized standard buffer and a 60-100% B gradient over 15 minutes as shown in Table 4, and 10 μL (100 μg) was injected onto a Phenomenex® bioZen 6 μm WCX 150×2.1 mm weak cation exchange column using the gradient shown in Table 4, +TOF MS (2000-7000). [Figure 4] Shown is the total ion current (TIC) chromatogram of infliximab on a Phenomenex® bioZen WCX column using a new buffer system with NH4Ac+NMM. Monoclonal antibody infliximab (Remicade, 10 mg / mL) with pI 7.6 was subjected to native WCX-HRMS using new buffers according to Table 5 and a gradient of 20-35% B over 15 minutes, 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column with +TOF MS (2000-7000). [Diagram 5]Shown is the total ion current (TIC) chromatogram for cetuximab on a Phenomenex® bioZen WCX column using a new buffer system with NH4Ac+NMM. Monoclonal antibody cetuximab (Erbitux, 10 mg / mL) with pI 8.8 was subjected to native WCX-HRMS using new buffers according to Table 6 and a gradient of 25-55% B over 15 minutes, 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column +TOFMS (2000-700). [Figure 6-1] Shown is a UV chromatogram monitored by Abs 280 nm Trastuzumab-anns (Kanjinti) (10 mg / mL, 4 uL inj.) The monoclonal antibody was injected onto a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) using a new buffer system containing NH4Ac+NMM and a pH gradient of 35-65% B over 10 min at a flow rate of 0.3 mL / min. [Figure 6-2] Figure 6 shows total ion current (TIC) chromatograms from SCX-HRMS analysis of trastuzumab-anns (4 uL inj, 10 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 35-65% B over 10 min at a flow rate of 0.3 mL / min, 30 °C, + TOF (2000-7000), MS temperature 500 °C. The dotted line forming a box within the main peak at RT of 4.83 min illustrates the approximate area for MS analysis as shown in Figure 6-4 and Figure 6-5. [Figure 6-3]Figure 6 shows the total ion current (TIC) chromatogram from SCX-HRMS of trastuzumab-anns (4 uL inj, 10 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 20-50% B over 10 min, 30°C, +TOFMS (2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 5.09 min illustrates the approximate area for MS analysis as shown in Figure 6-6 and Figure 6-7. [Figure 6-4] Raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 35-65% B over 10 min, 4.909-5.131 min +TOFMS (2000-7000). A mass / charge (m / z) range of 2000-7000 Da is shown. [Figure 6-5] Figure 6 shows raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) with pI 9.1, 4,858-5.217 min, using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 35-65% B over 10 min, + TOFMS (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6600 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 6-6. The same mass range is used for both spectra 6E and 6F. [Figure 6-6]Figure 6 shows raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) with pI 9.1, 4,960-5.217 min, using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with standard buffer system containing NH4Ac without NMM and pH gradient of 20-50% B / 10 min, +TOFMS (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 6-5. [Figure 6-7] Raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul, 10 mg / mL) with pI 9.1 using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 20-50% B / 10 min, +TOF (2000-7000) of 4,960-5,217 min. A mass / charge range of 2000-7000 Da is shown. [Figure 7-1] Figure 2 shows UV chromatograms monitored by Abs280nm from an SCX-UV analysis of infliximab (10mg / mL, 4uL inj) using a strong cation exchange column (trial, 3μm SCX, 100x2.1mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 25-40% B over 10 min at a flow rate of 0.3mL / min. Good separation of the variants was observed using the pH gradient with the new buffer system: five distinct peaks were observed eluting at RT of 3.357, 4.249, 5.018, 5.759, and 6.563 min. [Figure 7-2]Figure 7 shows total ion current (TIC) chromatograms from SCX-HRMS of infliximab (4ul, 5mg / mL) using a strong cation exchange LC column (trial 3μm SCX, 100x2.1mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 20-35% B over 10 minutes at a flow rate of 0.3mL / min, 30°C, +TOF(2000-7000), MS temperature 500°C. Good separation of variants was observed using the pH gradient with the new buffer system: five distinct peaks were observed eluting at RTs of 6.07, 6.57, 7.17, 7.87, and 8.57 minutes. The dotted line forming a box within the main peak at RT of 8.57 minutes illustrates the approximate area for MS analysis as shown in Figures 7-4 and 7-5. [Figure 7-3] Figure 7 shows the total ion current (TIC) chromatogram from SCX-HRMS of infliximab (4ul, 5mg / mL) using a strong cation exchange LC column (trial 3μm SCX, 100x2.1mm) with a standard buffer system containing only NH4Ac and a pH gradient of 15-25% B over 10 minutes, 30°C, +TOF(2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 5.63 minutes illustrates the approximate area of ​​the MS analysis shown in Figure 7-6 and Figure 7-7. Very poor separation of charge variants was given in the standard buffer for this low pI mAb (pI=7.6). Comparison of this figure with the previous figure (Figure 7-2) demonstrates the performance advantage gained by having good pH control at a lower pH than is routinely possible with standard buffers. [Figure 7-4] Raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) using a strong cation exchange column (trial 3μm SCX, 100x2.1mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 20-35%B / 10min, +TOF (2000-7000) of 8.621-9.032min. A mass / charge range of 2000-7000Da is shown. [Figure 7-5]Figure 7 shows the raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) at 8.621-9.032 min using a strong cation exchange column (trial 3μm SCX, 100x2.1mm) with a pH gradient of 20-35% B over 10 min, +TOF (2000-7000). The m / z range shown was narrowed down to that giving the highest intensity (~4800-6900Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 7-5, in the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen in the optimized standard NH4Ac buffer system (+25-+30) shown in Figure 7-6. [Figure 7-6] Raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) from 5.593 to 5.730 min using a strong cation exchange LC column (trial 3μm SCX, 100x2.1mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 15-25% B over 10 min, +TOF (2000-7000). The m / z range displayed was narrowed down to the one giving the highest intensity for the native analyte (~4800-6900Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 7-5. [Figure 7-7] Raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) using a strong cation exchange column (trial 3μm SCX, 100x2.1mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 15-25%B / 10min, +TOF (2000-7000) of 5.559-5.696min. A mass / charge range of 2000-6900Da is shown. [Figure 8-1]Figure 2 shows UV chromatograms with Abs280 nm from SCX-UV analysis of cetuximab (4 mg / mL) using a strong cation exchange column (trial, 3 μm SCX, 100 x 2.1 mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10 min at a flow rate of 0.3 mL / min. Multiple distinct variant peaks were observed, e.g., at RTs of 3.65, 4.03, 4.58, 5.20, and 5.93 min. [Figure 8-2] Figure 8 shows total ion current (TIC) chromatograms from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 30-60% B over 10 min at a flow rate of 0.3 mL / min, 30 °C, + TOF (2000-7000), MS temperature 500 °C. Multiple distinct variant peaks were observed at RTs of 3.33, 3.92, 4.54, and 5.22 min. The dotted line forming a box within the main peak at RT of 4.54 min illustrates the approximate area for MS analysis as shown in Figures 8-4 and 8-5. [Figure 8-3] Figure 8 shows total ion current (TIC) chromatograms from SCX-HRMS analysis of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 17-40% B / 10 min, 30°C, +TOF (2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 4.68 min illustrates the approximate area for the MS analysis shown in Figure 8-6 and Figure 8-7. [Figure 8-4] Raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10 min, +TOF (2000-7000) of 4.601-9.032 min. A mass / charge range of 2000-7000 Da is shown. [Figure 8-5] Figure 8 shows raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) at 4.601-4.892 min using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10 min, +TOF (2000-7000). The m / z range displayed was narrowed down to that giving the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 8-5, when using pH gradient elution with the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 8-6. [Figure 8-6] Raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) from 4.533 to 4.892 min using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 17-40% B over 10 min, +TOF (2000-7000). The m / z range displayed was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 8-5. [Figure 8-7] Raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) from 4.533 to 4.892 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 17-40% B over 10 min, +TOF (2000-7000). A mass / charge range of 2000-6900 Da is shown. [Figure 9-1]Figure 2 shows UV chromatogram with Abs 280 nm from SCX-HRMS of Rituximab (Rituxan) with pI 9.4 (10 mg / mL) using a strong cation exchange column (trial, 3 μm SCX, 100 x 2.1 mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 75-100% B over 10 min at a flow rate of 0.3 mL / min. A major peak at RT of 5.03 min was observed. [Figure 9-2] Figure 1 shows total ion current (TIC) chromatogram from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 65-100% B over 10 min at a flow rate of 0.3 mL / min, 30 °C, +TOFMS (2000-7000), MS temperature 500 °C. The dotted line forming a box within the main peak at RT of 5.88 min illustrates the approximate area for the MS analysis. [Figure 9-3] Figure 9 shows total ion current (TIC) chromatograms from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 55-100% B over 10 min, 30°C, +TOFMS (2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 4.65 min illustrates the approximate area for the MS analysis shown in Figures 9-6 and 9-7. [Figure 9-4] Raw MS spectrum from LC-HRMS of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 65-100% B over 10 min, 6.021-6.363 min +TOFMS (2000-7000). Mass / charge, m / z data in the range of 2000-7000 Da are shown. [Figure 9-5]Figure 9 shows raw MS spectra from an SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 65-100% B / 10 min, + TOFMS (2000-7000) from 6.021 to 6.363 min. The m / z range shown was narrowed down to that giving the highest intensity (approximately 4800-6800 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 9-5, when using a pH gradient with the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), shown in Figure 9-6. [Figure 9-6] Raw MS spectra from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) in a standard buffer system containing NH4Ac without NMM and a pH gradient of 55-100% B over 10 min, +TOFMS (2000-7000) from 4.550 to 4.772 min. The m / z range displayed was narrowed down to that giving the highest intensity (approximately 4800-6800 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. In the optimized standard buffer system, the most intense MS peak appears at a lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 9-5. [Figure 9-7] Raw MS spectra from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) from 4,550 to 4.772 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 55-100% B / 10 min, +TOFMS (2000-7000). A mass / charge range of 2000-7000 Da is shown. [Figure 10-1]Shown is a UV chromatogram monitored at Abs 280 nm from an SCX-UV analysis of NIST mAb with pI 9.2 (10 mg / mL) using a strong cation exchange column (trial, 3 μm SCX, 100 x 2.1 mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 95 to 100% B over 10 min at a flow rate of 0.3 mL / min. [Figure 10-2] Shown is a total ion current (TIC) chromatogram from SCX-HRMS of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 90-100% B over 10 min at a flow rate of 0.3 mL / min, 30 °C, +TOFMS (2000-7000), MS temperature 500 °C. The dotted line forming a box within the peak with RT of 4.39-4.82 illustrates the approximate area for MS analysis. [Figure 10-3] Shown is a total ion current (TIC) chromatogram from an SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 80-100% B / 10 min, 30° C., +TOFMS (2000-7000), MS temperature 500° C. The dotted line forming a box within the main peak at RT of 4.5 min illustrates the approximate area for the MS analysis shown in Figure 10-6 and Figure 10-7. [Figure 10-4] Raw MS spectrum from SCX-HRMS of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 90-100% B / 10 min, 5.012-5.627 min +TOFMS (2000-7000). Mass / charge (m / z) data over the range 2000-7000 Da are shown. [Figure 10-5]Raw MS spectra from SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 90-100% B / 10 min, + TOFMS (2000-7000) from 5.012 to 5.627 min. The m / z range displayed was narrowed down to that giving the highest intensity (approximately 4800-6900 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 10-6. [Figure 10-6] Raw MS spectrum from SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 80-100% B / 10 min, +TOFMS (2000-7000) from 4.259 to 4.841 min. The m / z range displayed was narrowed down to that giving the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 10-5. [Figure 10-7] Raw MS spectra from SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) from 4.259 to 4.841 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 80-100% B / 10 min, +TOFMS (2000-7000). A mass / charge range of 2000-6800 Da is shown. [Figure 11]Figure 10 shows raw MS spectrum from WCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a weak cation exchange LC column (Phenomenex® bioZen 6 μm WCX, 150 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 60-100% B, +TOFMS (2000-7000), 500 °C, 4.772-5.012 min. The m / z range displayed was narrowed to that giving the highest intensity (approximately 4800-7000 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the raw MS spectrum for NISTmAb using WCX-HRMS also exhibits the most intense MS peak at +25-+30, similar to the SCX-HRMS raw spectrum shown in Figure 10-6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Monoclonal antibodies (mAbs) are an important and rapidly growing class of therapeutic proteins. Their inherent complexity and wide variety of possible post-translational modifications (PTMs) make effective characterization and quality monitoring difficult. Charge variants of mAbs generally originate from PTMs that arise during the manufacturing process. These PTMs, such as C-terminal lysine clipping and N-glycosylation, result in the deletion / addition of basic and / or acidic residues to the native protein, thus imparting differences in the overall charge of the molecule. Typically, analysis of mAbs and other biomolecular analytes is performed using a combination of liquid chromatography (LC) and mass spectrometry (MS). Several studies have been published that show the utility of IEC for charge variant analysis (CVA) using high resolution mass spectrometry (HRMS). In this disclosure, an optimized MS-compatible buffer system was developed and used to improve the control of the pH gradient and thus the resolution of charge variants of mAbs when using ion exchange chromatography (IEC) coupled on-line to HRMS. Different cation exchanger types (strong / weak), sorbent particle size, column length, flow rate, buffer system, and gradient slope were used. Methods and reagents have been developed using commercially available mAbs spanning a wide range of pI. The present disclosure provides improved buffer systems and methods for optimal performance in routine analysis.

[0033] Approaches to address the problems of IEC-HRMS of protein analytes such as native monoclonal antibodies have been described, but each has significant drawbacks.

[0034] Bailey et al., 2018 described that charge variant native mass spectrometry benefits the mass accuracy and dynamic range of intact mass spectrometry of monoclonal antibodies. To enable IEC separation by pH gradient elution, an MS-compatible method was sought. Mobile phase buffer A was 50 mM ammonium acetate (NH4Ac), pH 6.6 without pH adjustment, and buffer B was 50 mM ammonium acetate adjusted to pH 10.1 using ammonium hydroxide. Ammonium acetate provides buffering within + / - 1 pH unit of 4.75 (acetic acid pKa) and + / - 1 pH unit of 9.25 (ammonium pKa). Due to the wide gap between the pKa values, the pH gradient using ammonium acetate may be compromised in terms of experimental linearity. Poor linearity of the pH gradient during IEC separation was exhibited. Bailey et al.,2018MABS,vol.10,No.8,1214-1225.doi.org / 10.1080 / 1940862.2018.1521131.

[0035] Fussl et al. 2018 performed charge variant analysis of monoclonal antibodies using direct-coupled pH gradient cation exchange chromatography to high-resolution native mass spectrometry. Fussl et al., 2018, Anal Chem 90, 2018-4669. A volatile pH gradient buffer system with 25 mM ammonium bicarbonate, 30 mM acetic acid, pH 5.3 (buffer A), and 10 mM ammonium hydroxide in 2 mM acetic acid, pH 10.8 (buffer B) was used using a MAbPacSCX-10RS sulfonic acid strong cation exchange column with a 5 micrometer particle size. After mixing, the buffers were allowed to rest for 24 hours at room temperature. The 24-hour rest period was found to be problematic and we were unable to reproduce the authors' results. Based on the composition and chemistry of the Fussl buffers, chemical changes (gassing, changes in the concentration of "bicarbonate" and pH) may occur during the "rest" period. In addition, pH control was difficult, especially at pH 7-8, due to poor buffering capacity over this pH range.

[0036] Fussl et al., 2019, describe the characterization of adalimumab heterogeneity via charge variant analysis hyphenated online to native resolution Orbitrap mass spectrometry. Buffer A consisted of 25 mM ammonium bicarbonate and 30 mM acetic acid in water (pH 5.3), and buffer B consisted of 10 mM ammonium hydroxide in water (pH 10.9). Buffers were prepared in 5x stocks and stored at 4°C for 2 weeks before use. The use of eluents with relatively low ionic strength, which is critical for MS detection, is said to result in low buffering capacity, which may impair the analysis of mAbs with high pI values. Fussl et al., 2019MABS vol.11, No.1, 116-128, doi.org / 10.1080 / 19420862.2018.1531664.

[0037] pH gradient buffer systems are also commercially available. For example, CX-1 pH gradient buffer (Thermo Fischer Scientific) contains four zwitterionic buffer salts, namely, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, in addition to NaCl and NaOH. This buffer system is supposed to be adjusted to provide a pH gradient of pH=5.6-10.2 with a concomitant slight ionic strength gradient. BioResolve CX pH buffer (Waters) also contains four buffer salts including succinic acid, Bis-Tris propane, triethanolamine, and N-cyclohexyl-3-aminopropanesulfonic acid, and is supposed to provide a pH gradient of pH=5.0-10.2. Farsang et al.,2020 J Chromatography A,vol.1626,30 Aug 2020,461350.

[0038] The present disclosure provides methods and compositions for separating and / or characterizing analytes in a sample. The methods may include liquid chromatography (LC). The LC may involve any suitable LC method. The LC method may include analytical LC for characterization of analytes. The LC method may include process LC for separation and / or purification of analytes. For example, the LC may include UHPLC, HPLC, ion exchange chromatography (IEC), size exclusion chromatography (SEC), hydrophilic interaction chromatography (HILIC), and / or reversed phase chromatography (RPLC). Thus, liquid chromatography columns, ultra-high performance chromatography columns, high performance chromatography, ion exchange chromatography columns, size exclusion chromatography columns, hydrophilic interaction chromatography columns, reversed phase chromatography columns may be used in the methods of the present disclosure.

[0039] The methods described herein may further include additional processes upstream or downstream, such as affinity chromatography (e.g., protein A or protein G), anion exchange chromatography (AEC), hydrophobic interaction chromatography (HIC), low pH viral inactivation, viral filtration, ultrafiltration, and / or diafiltration.

[0040] A liquid chromatograph (LC) can be coupled to a mass spectrometer. A liquid chromatograph can be directly coupled to a mass spectrometer.

[0041] The analyte may be a biomolecule such as a monoclonal antibody (mAb) and / or its charge variants. One of the most common LC techniques used to characterize charge variants in mAbs is ion exchange chromatography (IEC).

[0042] definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0043] The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0044] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0045] The term "about" when referring to a measurable value, such as an amount of a compound, a volume, a time, a temperature, and the like, is meant to encompass variations of ±10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0046] The terms "comprises" and / or "comprising", as used herein, specify the presence of stated features, integers, steps, acts, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, acts, elements, components, and / or groups thereof. Unless otherwise defined, all terms, including technical and scientific terms, used in this description have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict in terms, the present specification shall control.

[0047] The term "room temperature" refers to a temperature of 23° C. (293-298 K, 71° F.) and an absolute pressure of 1 atmosphere (14.696 psi, 101.325 kPa), unless otherwise specified.

[0048] The term "isomer" refers to two compounds that have the same chemical formula but different arrangements of the atoms in the molecule.

[0049] The terms "analog" or "structural analog" refer to a compound that has a similar molecular structure to another compound, but differs with respect to certain components.

[0050] All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety.

[0051] The embodiments described in one aspect of the present disclosure are not limited to the described aspects, and the embodiments may be applied to different aspects of the present disclosure as long as these aspects of the present disclosure do not prevent the present disclosure from functioning for its intended purpose.

[0052] Cation exchange chromatography is a form of ion exchange chromatography (IEX) that can be used to separate molecules based on net surface change. Cation exchange chromatography uses a negatively charged ion exchange resin that has an affinity for molecules that have a net positive surface charge.

[0053] The net surface charge of a protein may vary with pH as determined by the isoelectric point (pI) of the protein. At a pH equal to the pI, the protein will have no net charge. At a pH below the pI, the protein will carry a net positive charge. At a pH above the pI, the protein will carry a net negative charge. The pI of a protein may be calculated by its primary amino acid sequence. A negatively charged cation exchange resin may be selected if the protein of interest carries a net positive charge at the working pH. The buffer pH will affect the net surface charge of the protein. Proteins may not be stable at all pHs, so the stability of the protein and the choice of buffer may help determine the appropriate IEX medium for purification.

[0054] Cation exchange chromatography column resins can be classified by the pKa of the functional groups on the resin. Those resins that contain strong acids or bases are called "strong" ion exchangers, while those that contain weak acids or bases are called "weak" ion exchangers.

[0055] The term "strong cation exchange" (SCX) or "strong cation exchange resin" or "strong cation exchange stationary phase" refers to an ion exchange resin that contains sulfonic acid functional groups, such as methylsulfonate functional groups, which may be designated as S (e.g., -OCH2CH2OH-CH2-O-CH2-CHOH-CH2-SO3-), or sulfonyl functional groups, which may be designated as SP (e.g., -CH2-CH2-CH2SO3-). A typical pH range for an S strong cation exchange resin may be pH 2-12. A typical pH range for an SP strong cation exchange resin may be pH 2-14.

[0056] The term "weak cation exchange" (WCX) or "weak cation exchange resin" or "weak cation exchange stationary phase" typically refers to an ion exchange resin that contains a carboxylic acid functional group, such as a carboxymethyl functional group (e.g., -O-CH-CO-), which may be designated as CM, or other carboxylic acid functional groups (-CO-), which may be designated as C.

[0057] The term "SEC" refers to size exclusion chromatography. Size exclusion chromatography (SEC) involves separating molecules based on size, from largest to smallest, relative to the molecular size in solution. Very large molecules are excluded from the packed bed and are eluted first within the void volume. Smaller molecules may penetrate the pores to various degrees, depending on size. The smallest molecules diffuse into the furthest pores and elute last. Different mobile phases may affect the elution order due to size changes in solution, for example, due to hydrodynamic radius or radius of gyration. SEC may be used to separate proteins, such as monoclonal antibodies, from aggregates, fragments, complexes, excipients, impurities, etc. SEC may be used for size-based separation of biological compounds. For example, SEC is appropriate for aggregate and fragment analysis in the research, development, and manufacture of biological therapeutic molecules.

[0058] The term "m / z" in a mass spectrum represents the mass-to-charge ratio of individual molecules and represents the precise isotopic composition of each molecule. The most intense peaks result from the most probable isotopic compositions.

[0059] The term "charge envelope" or "charge state envelope" refers to a group of peaks in the LC / MS raw data of, for example, an intact mAb. Proteins form multiply charged ions during electrospray ionization. Large proteins such as monoclonal antibodies have a charge distribution envelope, or "charge envelope" or "charge state envelope". For example, using the new buffer system for trastuzumab, the center of the envelope is charge +24 as shown in Figure 6-5. This is compared to the standard buffer system for trastuzumab, where the center of the envelope is charge +27 as shown in Figure 6-6. Multiple peaks are observed, which are due to different glycoforms or charge variants of the mAb. The charge state of a native protein in ESI-MS is a complex function of several parameters, the relative importance of which is still under debate. Among the different parameters discussed, the conformation of the protein in solution (solvent accessible surface area), the protein sequence (number of charged AA at the "surface" of the protein, solvent accessible AA), and the gas-phase basicity of the components in the ionization environment are typically considered to be important. The charge state of the analyte is important in MS detection of biological macromolecules, since a lower charge state can give higher resolution in the deconvoluted mass spectrum. Lower charge states are generally preferred in native MS of biological molecules, since they indicate reduced protein denaturation ("more native" conformation) or fewer adducts, or both. In some embodiments, the new buffer system results in a lower charge state envelope of at least about 3 or at least about 2 charge states in the raw mass spectrum of protein analytes compared to the standard buffer system.

[0060] Detection of the LC eluate may be by any suitable method. The eluate may be monitored by UV detection, e.g., by measuring absorbance at a preselected wavelength by a UV detector and / or in the UV range, e.g., using a diode array detector (DAD). UV detection may include monitoring the eluate by absorbance at any suitable UV wavelength. For example, UV detection of protein amide bonds (220 nm), off-peak (230 nm), protein aromatic amino acid side chains of Tyr, Phe, Trp (280 nm), or nucleic acids (260 nm), or any other suitable UV wavelength value in between. The LC eluate may also be monitored by mass spectrometry (MS), refractive index (RI), laser light scattering (LS), fluorescence detector (FL), or any other suitable detection method. A pH meter may also be used to monitor the pH response of the effluent provided online by a buffer system.

[0061] The term "resolution" refers to a measure of how well two peaks are separated. Resolution can be determined by Rs = (tr,2-tr,1) / (0.5 x (w1 + w2)), where tr is the retention time of either peak 1 or peak 2, and w1 is the peak width at half height of peak 1 or peak 2. In a similar manner, resolution and peak capacity are used to refer to a measure of the number of peaks that can fit within a given separation space.

[0062] Peak capacity can be determined as Pc=1+(t / wavg), where t is the time corresponding to a given separation space, and wavg is the average peak width at half height observed for peaks in a given separation.

[0063] The total ion current ("TIC") chromatogram peak height and the signal-to-noise ratio of the TIC peak are two values ​​that can be used to define the sensitivity of an LC-MS analysis. The TIC signal-to-noise can be affected by the choice of mobile phase.

[0064] In addition to chromatographic resolution, LC-MS methods can be evaluated by the quality of the mass spectra they provide.

[0065] The term "eluent" refers to a buffer or buffer mixture used in chromatography and is synonymous with mobile phase.

[0066] The term "eluate" refers to a solution containing analytes and solvent that exits a chromatography column. The eluate is generated during the process of a separation.

[0067] The term "effluent" refers to the stream emerging from a chromatography column, regardless of whether or not a separation has taken place.

[0068] Mass spectrometry ("MS") is an analytical technique that measures the mass-to-charge ratio of charged molecules or molecular fragments formed from a sample. MS can be used to analyze the mass, chemical composition, and / or chemical structure of a sample of interest. In general, MS involves three steps: ionizing a sample to form charged molecules or molecular fragments (i.e., ions), separating the ions according to their mass-to-charge ratio, and detecting the separated ions to form a mass-to-charge signal (i.e., spectrum). The formation of ions can be achieved by a given MS ionization technique, such as, for example, electrospray ionization (ESI), fast atom bombardment (FAB), chemical ionization (CI), electron impact (EI), atmospheric solids analytical ionization (ASAI), atmospheric pressure photoionization (APPI), desorption electrospray ionization (DESI), atmospheric pressure vapor source (APVS), matrix-assisted laser desorption / ionization (MALDI), etc. There are many different types of MS devices. For example, sector, time-of-flight (TOF), quadrupole, ion trap, Fourier transform ion cyclotron resonance, and tandem (two or more of the above combined in tandem or orthogonal) mass spectrometers are all different instruments that are considered to be MS devices. Specific characterization of MS analysis includes, for example, mass accuracy, resolution, sensitivity, dynamic range, selectivity, and specificity.

[0069] Mass spectrometry (MS) can include sample preparation and introduction, ion formation, mass separation, and data processing.

[0070] MS ion formation can include, for example, electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI). In electrospray ionization (ESI), a solution containing a sample of interest is sprayed into fine droplets. The droplets dry, reduce in size, and split into smaller droplets, ultimately producing desolvated analyte ions with one or more residual charges. Proteins tend to form multiply charged ions during electrospray analysis, facilitating analysis by mass spectrometry. Mass separation can include, for example, quadrupole, TOF, orbitrap.

[0071] MS data processing can include deconvolution of raw MS spectra. For example, ESI MS mass spectra can be subjected to mathematical deconvolution to convert the raw mass spectrum, including m / z, into protein analyte abundance data. For example, m / z=mass-to-charge ratio Mr = protein mass n = number of charges for m / z mp = mass of H+ = 1.0073 Given m / z1=(Mr+nlmp) / n1 And n2=n1+1 This can be rearranged to solve for n1: n1=(m / z2-mp) / (m / z1-m / z2) and calcMr1=(n1*m / z1)-(n1*1.0073)=Mr1 calcMr2=(n2*m / z2)-(n2*1.0073)=Mr2.

[0072] See, e.g., Covey et al., The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry, Rapid Commun. Mass Spectrom. 2:11, 1988. Computer-assisted deconvolution of raw mass spectra can also be performed. Mann, M., Meng, CK, Fenn, JBInterpreting Mass Spectra of Multiply Charged Ions.Anal.Chem.1989,61,1702-1708, Labowsky, M., Whitehouse,CM, Fenn,JBThree-Dimensional Deconvolution of Multiply Charged Spectra.Rapid Commun.Mass Spectrom.1993,7,71-84, Zhang, Z., Marshall, AGA Universal Algorithm for Fast and Automated Charge State Deconvolution of Electrospray Mass-Charge Ratio Spectra.J.Am.Soc.Mass Spectrom.1998,9,225-233.

[0073] MALDI is a soft ionization technique suitable for native protein analysis, such as native monoclonal antibody MS.

[0074] The ESI and MALDI ion formation processes are competitive and different compounds can have large variations in ionization efficiency. Ion suppression can be caused by salts, detergents, polymers. Protein analytes can be separated from interfering materials before the ionization step. Acceptable concentrations of various salts and buffer components are described by the Harvard Center for Mass Spectrometry.https: / / massspec.fas.harvard.edu / files / smms / files / saltbuffer. It is important to minimize the introduction of non-volatile salts (e.g., NaCl) into the MS, as these compounds can cause suppression of intact proteins leading to poor detection sensitivity.

[0075] Traditionally, offline sample cleanup and desalting techniques such as dilution, dialysis / buffer exchange, zip tips, molecular weight cut-off (MWCO) filters, and / or desalting columns have been utilized to avoid MS ion suppression. For example, BioRad Micro Biospin P-6 gel columns, with MWCO 6000 Da, remove small molecules, salts. Disadvantages of offline sample cleanup include samples larger than 10 uL and / or samples with high concentrations, time and sample development to minimize protein loss due to precipitation, failure to adhere to or elute from the medium, and degradation of the analyte protein. If the sample is limited without prior optimization, offline desalting / cleanup may be infeasible. Typical online desalting techniques for LC-MS may include, for example, reversed-phase (RP) chromatography, size exclusion, or capillary electrophoresis. RP desalting columns may utilize, for example, phenyl, diphenyl, polyphenyl, C4, C8, or polymeric reversed phase (PLRP-S). The RP online preparation column will retain the protein allowing the salt to be washed away and discarded prior to protein elution into the mass spectrometer. However, the use of a desalting column can increase labor and cycle time by at least several minutes for intact antibodies. The present disclosure provides a single mobile phase buffer system suitable for IEC-HRMS that includes low salt concentrations to avoid MS ion suppression, provides reproducible pH gradient elution, and is compatible with both UV and MS detection. A method is provided that includes direct coupling pH gradient cation exchange chromatography to high resolution mass spectrometry.

[0076] In some embodiments, the mobile phase buffer composition of the present disclosure may contain a salt concentration of 50 mM or less, 30 mM or less, 25 mM or less, about 20 mM or less, or about 15 mM or less. In some embodiments, the composition of the present disclosure may contain a salt concentration of 2-25 mM, 2-20 mM, or 2-15 mM. The salt may be an ammonium carboxylate salt. The ammonium carboxylate may be a C1-C2 ammonium carboxylate. The ammonium carboxylate may be ammonium acetate or ammonium formate. In some embodiments, the composition of the present disclosure may contain an ammonium acetate concentration of 2 mM-25 mM, 2 mM-20 mM, or 2 mM-15 mM. In some embodiments, the composition of the present disclosure may contain an ammonium acetate concentration of 25 mM or less, 20 mM or less, or about 15 mM or less.

[0077] In some embodiments, the mobile phase buffer composition further comprises about 1 mM to about 50 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 1 mM to about 6 mM, or about 2 mM to about 5 mM N-methylmorpholine (NMM) or an isomer or analog thereof.

[0078] Isomers of N-methylmorpholine can include 2-methylmorpholine, 2-methylmorpholine, 2-methyl-1,3-oxazinane, 3-methyl-1,3-oxazinane, 4-methyl-1,3-oxazinane, 5-methyl-1,3-oxazinane, and 6-methyl-1,3-oxazinane.

[0079] The analog of N-methylmorpholine may include any suitable alkyl or dialkyl morpholine or alkyl or dialkyl 1,3-oxazinane. The analog of N-methylmorpholine may be selected from the group consisting of alkyl C1-C6 morpholine, dialkyl C1-C6 morpholine, alkyl C1-C6-1,3-oxazinane, and dialkyl C1-C6-1,3-oxazinane.

[0080] Analogs of N-methylmorpholine may include alkylmorpholine compounds. The alkylmorpholine compounds may include alkyl C1-C6 morpholine compounds, alkyl C1-C4 morpholine compounds, or alkyl C1-C3 morpholine compounds. The alkylmorpholine may be, for example, ethylmorpholine, propylmorpholine, isopropylmorpholine, butylmorpholine, or isobutylmorpholine.

[0081] Analogs of N-methylmorpholine may include N-alkylmorpholine compounds. N-alkylmorpholine compounds may include N-alkyl C1-C6 morpholine compounds, N-alkyl C1-C4 morpholine compounds, or N-alkyl C1-C3 morpholine compounds. N-alkylmorpholine may be, for example, N-ethylmorpholine, N-propylmorpholine, N-isopropylmorpholine, N-butylmorpholine, or N-isobutylmorpholine.

[0082] Analogs of N-methylmorpholine may include dialkylmorpholine. The dialkylmorpholine compound may include a dialkyl C1-C6 morpholine compound, a dialkyl C1-C4 morpholine compound, or a dialkyl C1-C3 morpholine compound. The dialkylmorpholine may be 2,2-dialkylmorpholine, 2,3-dialkylmorpholine, 2,4-dialkylmorpholine, 2,5-dialkylmorpholine, 2,6-dialkylmorpholine, 3,3-dialkylmorpholine, 3,4-dialkylmorpholine, or 3,5-dialkylmorpholine, etc.

[0083] For example, the dialkylmorpholine can be 2,2-dimethylmorpholine, 2,3-dimethylmorpholine, 2,4-dimethylmorpholine, 2,5-dimethylmorpholine, 2,6-dimethylmorpholine, 3,3-dimethylmorpholine, 3,4-dimethylmorpholine, or 3,5-dimethylmorpholine, and the like.

[0084] The dialkylmorpholine or dialkyl-1,3-oxazinane may be in any configuration. A particular dialkylmorpholine or 1,3-oxazinane compound may be in the cis configuration, the trans configuration, or a mixture of the cis and trans configurations. For example, cis, trans-2,6-dimethylmorpholine, cis-2,6-dimethylmorpholine, or trans-2,6-dimethylmorpholine, or any combination thereof.

[0085] Analogs of N-methylmorpholine can include alkyl 1,3-oxazinane compounds.

[0086] The alkyl 1,3-oxazinane compound may include an alkyl C1-6-1,3-oxazinane compound, an alkyl C1-4-1,3-oxazinane compound, an alkyl C1-3-1,3-oxazinane compound, an N-alkyl C1-6-1,3-oxazinane compound, an N-alkyl C1-4-1,3-oxazinane compound, or an N-alkyl C1-3-1,3-oxazinane compound.

[0087] For example, N-alkyl 1,3-oxazinane can include N-methyl-1,3-oxazinane, N-ethyl-1,3-oxazinane, N-propyl-1,3-oxazinane, N-isopropyl-1,3-oxazinane, N-butyl-1,3-oxazinane, N-isobutyl-1,3-oxazinane, and the like.

[0088] Analogs of N-methylmorpholine may include dialkyl 1,3-oxazinane compounds. Isomers of N-methylmorpholine may include dialkyl C1-C6-1,3-oxazinane, dialkyl C1-C4-1,3-oxazinane, or dialkyl C1-C3-1,3-oxazinane. Dialkyl 1,3-oxazinane may be 2,4-dialkyl-1,3-oxazinane, 2,5-dialkyl-1,3-oxazinane, 2,6-dialkyl-1,3-oxazinane, 3,5-dialkyl-1,3-oxazinane, 3,4-dialkyl-1,3-oxazinane, 2,2-dialkyl-1,3-oxazinane, or 3,3-dialkyl-1,3-oxazinane. For example, the dialkyl 1,3-oxazinane can be 2,4-dimethyl-1,3-oxazinane, 2,5-dimethyl-1,3-oxazinane, 2,6-dimethyl-1,3-oxazinane, 3,5-dimethyl-1,3-oxazinane, 3,4-dimethyl-1,3-oxazinane, 2,2-dimethyl-1,3-oxazinane, or 3,3-dimethyl-1,3-oxazinane.

[0089] The term "alkyl" may refer to C1-6 alkyl, or C1-4 alkyl, or C1-3 alkyl, or C1-2 alkyl. Alkyl may be straight or branched chain. For example, alkyl may be methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, pentyl, hexyl, etc.

[0090] An "analyte" can be a biomolecule. An analyte can be a protein, a peptide, a glycan, or a combination thereof. An analyte can include multiple proteins, multiple peptides, multiple glycans, or a combination thereof. A protein can be an antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate (ADC). A biomolecule analyte can be a recombinant, synthetic, or isolated biomolecule. A biomolecule can be a protein analyte.

[0091] As used herein, the term "protein" refers to a polymeric chain of amino acids called a polypeptide. Proteins can also include a number of modifications, including post-translational modifications (PTMs), phosphorylation, lipidation, prenylation, sulfation, hydroxylation, acetylation, carbohydrate addition (glycosylation and glycosylation), addition of artificial groups or cofactors, formation of disulfide bonds, proteolysis, assembly into macromolecular complexes, and the like. The antibody can be a monoclonal antibody (mAb) or a polyclonal antibody, or an antigen-binding fragment thereof. The antibody can be a full-length antibody (e.g., an IgG1 or IgG4 antibody), a bispecific antibody (bsAb), or can include only an antigen-binding portion (e.g., Fab, F(ab')2, Fab3, scFv, bis-scFv, minibody, diabody, tetrabody, triabody, fragment), and can be modified to affect functionality, can be monovalent, bivalent, trivalent, tetravalent, can have higher valency, or can be an antibody complex. Antigen-binding fragments may include, for example, (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. The biomolecule analyte may be a natural mAb and its charge variants.

[0092] composition An aqueous mobile phase buffer system is provided for use in liquid chromatography, optionally directly coupled to high resolution mass spectrometry, in the analysis of intact biomolecules such as monoclonal antibodies. In some embodiments, the LC is ion exchange chromatography (IEC) or size exclusion chromatography (SEC).

[0093] The new mobile phase buffer system provided herein is suitable for both UV and MS detection because all components are volatile and none have significant UV absorbance at UV wavelengths commonly used for protein analysis. The new mobile phase buffer system contains a low salt concentration of about 25 mM or less, making it suitable for use in pH gradient elution in IEC directly coupled to HRMS.

[0094] The new mobile phase buffer system provides optimal pH control, sensitivity, and resolution in the analysis of native biomolecules such as monoclonal antibodies (mAbs). The buffer composition and pH value are optimized for mAb charge variant analysis. The gradient range and slope can be optimized for each analyte, e.g., native mAb. MS conditions can be optimized to maximize sensitivity and / or minimize adducts. The ingredients used in the buffer components are preferably high purity, trace metal grade to improve sensitivity and reduce spectral complexity caused by metal adducts.

[0095] Buffer pH and ionic strength are important for all forms of ion exchange chromatography. Typically, the buffer pH can be adjusted after the salt concentration is adjusted. Because buffer pH and ionic strength greatly affect protein binding to the column resin, it is important to ensure that the pH is properly adjusted and that the appropriate counter ions are used.

[0096] The techniques described herein solve problems found in the prior art, such as the nonlinearity of pH gradient elution and poor chromatographic reproducibility, when using ion exchange chromatography directly coupled to high resolution mass spectrometry (online HRMS).

[0097] The techniques described herein solve problems found in the prior art, including poor MS sensitivity due to excessive salt concentration in mobile phase buffers in ion exchange chromatography using HRMS; typically, it has been discovered that salt concentrations of about 40 mM or more can reduce MS sensitivity. The present disclosure provides a buffer system with the highest buffer capacity combined with the lowest ionic strength suitable for use in HRMS. In some embodiments, an ionic strength of 25 mM salt concentration or less is used.

[0098] The technology described herein solves problems found in the prior art, such as poor chromatographic resolution of charge variants of intact monoclonal antibodies (mAbs), particularly low isoelectric point (pI) mAbs (e.g., infliximab, pI 7.6), when using ion exchange chromatography with high resolution mass spectrometry (HRMS).

[0099] The techniques described herein solve problems found in the prior art, including reduced mass spectrometry (MS) resolution due to the high charge state of intact proteins in IEC-HRMS, i.e., MS spectra indicative of significant protein denaturation.

[0100] The new mobile phase buffer systems provided herein can be used for pH gradient-based chromatographic separations, such as charge variant analysis of intact monoclonal antibodies using ion exchange chromatography, with mass spectrometry (MS) detection (i.e., MS compatibility) resulting in 1) improved pH control, 2) improved MS sensitivity, and 3) increased mass spectral resolution (higher mass spectral charge envelope for intact proteins representing a "more native" state).

[0101] Other advantages of the improved buffer system provided herein include better chromatographic and mass spectrometric resolution of intact mAb charge variants, which are often important quality attributes that must be identified and characterized during the development and manufacture of biotherapeutics.

[0102] The improved buffer system can be utilized with either weak or strong ion exchange stationary phase materials and columns.

[0103] Specifically, the new pH gradient buffer system components have pKa values ​​that span the pH range of primary interest for biomolecules such as mAbs, i.e., pKa values ​​that are within the ranges of pH 4.5-10.8, pH 4.5-10.5, pH 5-10.5, or pH 5.2-10.2. For example, acetate pKa=4.8, N-methylmorpholine pKa=7.4, ammonia pKa=9.2. One reason N-methylmorpholine was selected for use in the new buffer system is its intermediate pKa of approximately 7.4, which allows the pH gradient to be more linear and more reproducible.

[0104] The new buffer system is also UV compatible, which is an additional advantage of using N-methylmorpholine compared to other weak bases. N-methylmorpholine can be in the base form. Other potential MS-compatible weak bases with pKas close to the desired range (e.g., pyridine-based) have significant UV absorption. N-methylmorpholine has good transparency at the UV wavelengths (280 nm) commonly used for biotherapeutics, allowing the same buffer to be used for both MS and UV detection. This is beneficial because the retention times / elution order of all peaks (including impurities) are typically identified using MS, and then routine monitoring by UV detection is performed using the retention times (RT) / elution order determined by MS. Changing the buffer used for each detector can greatly complicate this task.

[0105] LC-MS of biomolecules (e.g., proteins, peptides, and / or glycans) is not simply about achieving highly sensitive detection: the performance of the method is heavily influenced by the chromatographic resolving power.

[0106] The present disclosure provides a pH gradient buffer system suitable for use in LC separations and, optionally, MS of biomolecular analytes.

[0107] For example, the development of the present pH gradient buffer system involved the analysis of five intact native monoclonal antibodies spanning a range of relevant pIs, including infliximab pI 7.6 (e.g., Remicade), cetuximab pI 8.8 (e.g., Erbitux), trastuzumab pI 9.1 (e.g., Kanjinti), NIST reference mAb pI 9.2, and rituximab pI 9.4 (e.g., Rituxan). The buffer systems provided herein are suitable for use in weak and / or strong ion exchange chromatography. For example, the buffer systems provided herein are suitable for the separation of analytes such as charge variants of native mAbs using weak ion exchange columns such as Phenomenex® bioZen 6um WCX, or strong ion exchange columns such as ThermoScientific™ mAbPac™ 5um SCX.

[0108] The buffer system of the present invention may comprise a ready-to-use buffer concentration of less than 25 mM ammonium acetate, about 2 to about 25 mM ammonium acetate, about 2 to 20 mM ammonium acetate, or about 5 mM to about 15 mM ammonium acetate. The mobile phase buffer system of the present invention may comprise a ready-to-use buffer concentration of about 1 mM to about 10 mM N-methylmorpholine, about 2 mM to about 8 mM N-methylmorpholine, or about 2 mM to about 5 mM N-methylmorpholine. The buffer system of the present invention may comprise a ready-to-use buffer concentration of less than 25 mM ammonium acetate, 20 mM or less ammonium acetate, about 2 to 25 mM ammonium acetate, about 2 to about 20 mM ammonium acetate, about 2 to about 15 mM ammonium acetate, or about 5 mM to about 15 mM ammonium acetate, and about 1 mM to about 10 mM N-methylmorpholine, about 2 mM to about 8 mM N-methylmorpholine, or about 2 mM to about 5 mM N-methylmorpholine. The buffer system may comprise a pH that is within or spans the ranges of about pH 4.5 to about pH 10.5, pH 5 to 10.5, or pH 5.2 to 10.2.

[0109] The present disclosure provides aqueous buffer systems comprising one or more, two or more, or three or more parts. The buffer system may comprise a mobile phase comprising a two-part aqueous buffer system. The two-part aqueous buffer system may comprise a part A buffer and a part B buffer. In some embodiments, part A of the aqueous buffer system may comprise about 10 mM to about 25 mM ammonium acetate and about 2 mM to about 10 mM N-methylmorpholine at a pH of about pH 4.5 to about pH 5.5 adjusted with acetic acid. In certain embodiments, part A of the aqueous buffer system may comprise about 10 mM to about 20 mM ammonium acetate and about 3 mM to about 8 mM N-methylmorpholine at a pH of about pH 5 to about pH 5.5 adjusted with acetic acid. In some embodiments, part B of the aqueous buffer system may comprise about 2 mM to about 10 mM ammonium acetate and about 1 mM to about 5 mM N-methylmorpholine at a pH of about pH 9 to about 10.5 adjusted with ammonium hydroxide. In certain embodiments, part B of the aqueous buffer system may contain about 3 mM to about 8 mM ammonium acetate and about 1 mM to about 3 mM N-methylmorpholine at a pH of 9.5 to about 10.5 adjusted with ammonium hydroxide. The two-part aqueous buffer system may be designed to span a pH range of pH 4.5 to 10.5, pH 5 to 10.5, or pH 5.2 to 10.2. A broader pH range of about pH 3.8 to 10.8 may also be used.

[0110] The mobile phase additive may contain about 100 ppb or less of any individual metal impurity. In other words, each metal impurity contained in the mobile phase additive is not present in an amount greater than about 100 ppb. In some embodiments, the mobile phase additive contains less than about 90 ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, or 10 ppb of any individual metal impurity. In some embodiments, the mobile phase additive contains less than about 95 ppb, 85 ppb, 75 ppb, 65 ppb, 55 ppb, 45 ppb, 35 ppb, 25 ppb, or 15 ppb of any individual metal impurity. A metal impurity is any metal that affects a desired feature of a mass spectrum, e.g., the quality of a mass spectrum. A metal impurity may be, for example, sodium, potassium, calcium, nickel, copper, and / or iron. These aspects of the present technology extend to the ready-to-use mobile phase or any concentrate thereof that is subsequently prepared using the mobile phase additives described above.

[0111] The buffer system may use deionized water (weight / weight or volume / volume) using LC / MS grade water, MS grade water, or Type 1 reagent grade water with a resistivity of approximately 18.2 Megaohm-cm filtered through a 0.2 micrometer filter.

[0112] The water may have trace impurities of 20 ppb or less. As with the mobile phase additive, the water may have less than about 100 ppb of any individual metal impurity, or less than about 95 ppb, 85 ppb, 75 ppb, 65 ppb, 55 ppb, 45 ppb, 35 ppb, 25 ppb, or 15 ppb of any individual metal impurity.

[0113] The mobile phase may have less than about 100 ppb, 90 ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, or 10 ppb of any individual metal impurity. The mobile phase may have less than about 50 ppb of any individual metal impurity. The mobile phase may have less than about 20 ppb of any individual metal impurity. The individual metal impurity may be, for example, aluminum, barium, cadmium, calcium, chromium, cobalt, copper, iron, lead, magnesium, manganese, nickel, potassium, silver, sodium, tin, or zinc.

[0114] The compositions of the present disclosure may be commercially available in the form of kit components, such as ready-to-use buffers, concentrated buffers for dilution, as well as combined products consisting of a buffer according to the present disclosure with an LC column or device. The ready-to-use buffers or concentrated buffers for dilution may be stored in any suitable container. The container should be clean and free of extractable materials / residues. For example, the buffer storage container may be a suitable plastic or glass storage container. In some embodiments, the use of a plastic container for buffer storage is preferred, for example, to reduce leaching of contaminants from the glass. The container may comprise any suitable plastic material. In some embodiments, the container may comprise high density polyethylene (HDPE), polypropylene (PP), polytetrafluoroethylene (PTFE), or polystyrene (PS) plastic material. The container may be sealed, for example, to exclude contaminants.

[0115] The kit may include a chromatography column, a container having a certain amount of concentrated mobile phase, and instructions for use, and / or a website address for the instructions. The chromatography column has a stationary phase material inside the column. The stationary phase material may be any stationary phase material described herein, such as an ion exchange stationary phase, a size exclusion stationary phase, a hydrophilic interaction stationary phase, and / or a reversed phase stationary phase.

[0116] The mobile phase composition may be liquid in the form of a ready-to-use buffer or in a concentrated buffer. The concentrated buffer may be 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x or more concentration for dilution before use, or any value in between. In some embodiments, the mobile phase buffer is a 10x concentrate. Optionally, organic solvents such as acetonitrile or isopropanol may be used at concentrations up to 10% v / v. The concentrate may be diluted to prepare a 1x buffer system. In some embodiments, the mobile phase buffer is a ready-to-use mobile phase buffer composition.

[0117] The instructions may instruct the user to obtain a sample containing at least one biomolecule (e.g., a protein, such as a mAb process sample or a mAb purification sample) in a sample matrix, and to dilute the mobile phase concentrate with appropriate water to obtain about 2 mM to about 25 mM ammonium acetate, about 2 mM to about 20 mM ammonium acetate, inclusive, or about 5 mM to about 15 mM ammonium acetate, and about 1 mM to 10 mM, or about 2 mM to about 5 mM N-methylmorpholine (NMM). The pH of each diluted mobile phase concentrate in the two-part buffer system may be about pH 5.2 and about pH 10.2, respectively.

[0118] The user may be instructed to run a sample with a dilute or ready-to-use mobile phase through the column to substantially resolve and retain at least one biomolecule (e.g., a protein, such as a mAb), and the instructions may also instruct the user to detect the at least one biomolecule (e.g., a protein, such as a mAb) using a detector.

[0119] Stability studies indicate acceptable shelf-life of the 10x buffer system concentrate of at least 6 months, at least 12 months, at least 18 months or more when sealed and stored at a factory refrigerated temperature of about 4°C. Optionally, to extend the shelf-life, organic solvents such as acetonitrile or isopropanol can be used at concentrations up to 10% v / v. Stability studies indicate acceptable shelf-life of the ready-to-use buffer of at least 3 months, at least 6 months, at least 12 months or more when sealed and stored at a factory refrigerated temperature of about 4°C. Preliminary shelf-life studies of the buffer at room temperature have been conducted and have shown promising factory sealed shelf-life results of the ready-to-use 10x buffer system concentrate at room temperature of at least 3 months, at least 6 months, at least 12 months or more. The room temperature shelf-life of the ready-to-use buffer is at least 1 month in appropriate containers (incoming air is filtered) to minimize microbial growth.

[0120] use A method of separating an analyte from a sample is provided, the method comprising: flowing a mobile phase through a chromatography column, the mobile phase comprising a two-part aqueous buffer system, part A of the aqueous buffer system comprising about 10-25 mM ammonium acetate, 3-10 mM N-methylmorpholine, and pH adjusted to pH 4.5-5.5 with acetic acid, and part B of the aqueous buffer system comprising 2-10 mM ammonium acetate, 1-5 mM N-methylmorpholine, and pH 9.5 to about 10.5 adjusted with ammonium hydroxide; injecting a sample containing the analyte into the mobile phase; and eluting the analyte from the column. The method may further comprise detecting the analyte in the eluate. The method may further comprise washing the column after injection before eluting the column. The method may further comprise determining the molecular weight of the analyte. The method may further comprise detecting the analyte with a mass spectrometer. The method may further comprise generating analyte ions. The method may further include obtaining a mass spectrum of the analyte ions.

[0121] In some embodiments, part A of the aqueous buffer system comprises about 15 mM ammonium acetate, about 5 mM N-methylmorpholine, adjusted to pH 5.2 with acetic acid.

[0122] In some embodiments, part B of the aqueous buffer system comprises about 5 mM ammonium acetate, about 2 mM N-methylmorpholine, adjusted to pH 10.2 with ammonium hydroxide.

[0123] In embodiments, ammonium acetate, acetic acid, ammonium hydroxide, and N-methylmorpholine may each contain less than about 100 ppb of individual metal impurities.

[0124] The analyte can be a protein. The protein can be an antibody. The protein can be selected from the group consisting of a monoclonal antibody, an antigen-binding fragment of an antibody, an isolated protein, a synthetic protein, and a recombinant protein.

[0125] In some embodiments, protein analytes may be eluted from the column using gradient elution or step isocratic elution. Gradient elution may be pH gradient elution. pH gradient elution may be selected from linear gradient, segmented gradient, curved gradient, step gradient, etc. A linear pH gradient may include a linear change in mobile phase pH from a lower pH to a higher pH over the course of performing a chromatographic separation. A step pH gradient may include a stepwise change in mobile phase pH over the course of a chromatographic separation. A segmented pH gradient may include two or more linear gradients of different slopes from a lower pH to a higher pH over the course of a chromatographic separation. A curved pH gradient may include a convex or concave curved pH gradient over the course of a chromatographic separation. Gradient elution may be a linear gradient. A linear gradient may be a linear pH gradient. The gradient may include a slope in the range of 0.1-10% B / CV of eluate, or 0.5-5% B / column volume (CV). The gradient may include a slope in the range of 1-3% B / column volume (CV) of eluate.

[0126] The chromatography column may be a liquid chromatography column. The chromatography column may comprise an ion exchange resin. The chromatography column may comprise a size exclusion chromatography resin. The ion exchange resin may be a cation exchange resin. The cation exchange resin may be a strong cation exchange resin or a weak cation exchange resin.

[0127] The results in Examples 2 and 3 have been reproduced at a contract facility (Intertek) using different equipment (Thermo Orbitrap MS vs. Sciex X500B MS). EXAMPLES

[0128] Example 1. Preparation of Buffer Solution In this example, a new buffer system was developed for pH gradient elution using reproducible MS-compatible chromatographic methods, such as cation exchange chromatography. All materials were 99.99+% pure on a trace metals basis. Total metal impurities <100 ppb. Trace metal reagents are important for reducing metal adducts observed in MS.

[0129] A standard ammonium acetate buffer system was optimized for use as the comparative buffer system. Compared to the prior art, the concentration of ammonium acetate (NH4Ac) in the comparative standard buffer was reduced to optimize MS compatibility: the NH4Ac concentration was reduced to approximately 20 mM NH4Ac or less compared to the prior art pH gradient buffer system with 40-50 mM NH4Ac. The comparative optimized standard buffer (ammonium acetate only) was prepared as follows: Buffer A = 20 mM NH4Ac adjusted to pH 5.2 with acetic acid. Buffer B = 5 mM NH4Ac adjusted to pH 10.2 with ammonium hydroxide. The comparative optimized standard NH4Ac only buffer system was found to be stable for at least approximately 4 weeks at room temperature (under an inlet air filter). Although the optimized ammonium acetate only comparative buffer can be utilized to achieve good separation of low pI mAbs, its use requires significant optimization (very shallow gradient) and daily slope adjustments (sensitive to very slight changes in buffer pH) and suffers from batch-to-batch variation.

[0130] A new pH gradient buffer system using ammonium acetate and N-methylmorpholine was prepared as follows: New buffer system 1x buffer A = 15 mM NH4Ac + 5 mM N-methylmorpholine adjusted to pH 5.2 with acetic acid, and 1x buffer B = 5 mM NH4Ac + 2 mM N-methylmorpholine adjusted to pH 10.2 with ammonium hydroxide. Procedure: Dissolve ammonium acetate in 1 L MS grade water, add N-methylmorpholine, then mix and adjust to pH 5.2 with acetic acid. Dissolve ammonium acetate in 1 L MS grade water, add N-methylmorpholine, then mix and adjust to pH 10.2 with ammonium hydroxide. A pH probe was used for pH adjustment. Preferably, the pH probe should not be immersed in the bulk buffer solution to avoid contaminating the buffer with pH probe salts. Instead, the pH of an aliquot should be tested during the pH adjustment. The new buffer with ammonium acetate and NMM gives reproducible performance with steeper, more practical gradients, as well as very shallow gradients, and does not change from day to day or batch to batch. Robustness and ease of use when performing native MS are major issues that the new NMM buffer solves. The new buffer with ammonium acetate and NMM is suitable for both UV and MS detection.

[0131] The new 1x buffer system was found to be stable for at least about 4 weeks at room temperature (inlet air filter). The inlet air filter prevents or greatly reduces the ingress of microbial contamination from the laboratory atmosphere. The buffer system uses deionized water using LC / MS grade water, MS grade water, or Type 1 reagent grade water with a resistivity of 18.2 megaohm-cm filtered through a 0.2 micrometer filter. The water may have trace impurities of 20 ppb or less.

[0132] A 10x buffer system is prepared for dilution prior to use. Buffer A = 150 mM NH4Ac + 50 mM N-methylmorpholine adjusted to pH 5.2 with acetic acid. Buffer B = 50 mM NH4Ac + 20 mM N-methylmorpholine adjusted to pH 10.2 with ammonium hydroxide. Procedure: Dissolve ammonium acetate in 1 L MS grade water, add N-methylmorpholine, then mix and adjust to pH 5.2 with acetic acid. Dissolve ammonium acetate in 1 L MS grade water, add N-methylmorpholine, then mix and adjust to pH 10.2 with ammonium hydroxide. A pH probe is used for pH adjustment.

[0133] Eluent solutions may be prepared from 10x by diluting 10x buffers A and B with deionized water (weight / weight or volume / volume) using LC / MS grade water, MS grade water, or Type 1 reagent grade water with a resistivity of approximately 18.2 megaohm-cm filtered through a 0.2 micrometer filter. The water may have trace impurities of 20 ppb or less. For example, 100 mL of 10x buffer may be diluted with 900 mL of MS grade water to provide 1 L of 1x buffer. Measure and record the pH of 1x buffer A and 1x buffer B with a standard benchtop pH meter.

[0134] Example 2. Chromatographic results for charge variant analysis of mAb using native WCX-HRMS In this example, a pH gradient using a new buffer system was developed for five mAbs of various pI values ​​using a weak cation exchange column. The mAbs are shown in Table 1A with their measured pI values. In all examples, the WCX column was directly coupled to a high-resolution mass spectrometer, i.e., an on-line native HRMS. Spectra were collected in ESI+ mode over the m / z range of 2000-7000. [Table 1]

[0135] All mAb samples were at 10 mg / mL, including infliximab pI 7.6 (Remicade), cetuximab pI 8.8 (Erbitux), trastuzumab pI 9.1 (Kanjinti), NIST reference mAb pI 9.2, and rituximab pI 9.4 (Rituxan). All five mAbs were analyzed using cation exchange chromatography in a new buffer system. Three mAbs were tested using cation exchange chromatography in a comparative standard buffer system, including trastuzumab-ann, rituximab-abb, and the NIST reference mAb. The pH gradient was optimized for each mAb, starting from the exemplary pH gradients shown in Table 1B and Table 2.

[0136] The LC system was an Agilent 1290 Infinity II UHPLC system: Binary pump: G7120A (JetWeaver V35), Multisampler: G7167B (20uL loop), Multicolumn Thermostat: G7116B, and DAD: G7117B. The mass spectrometer was a Sciex X500B quadrupole time-of-flight mass spectrometer operating in positive ion electrospray mode (ESI+). The acquired mass range was m / z 2000-7000.

[0137] The weak cation exchange column was a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm, mono-polymeric non-porous particles [—CH2CH2CH(CO2—)—CH(CO2—)—CH2—].

[0138] Chromatographic conditions included (fast equilibration method), flow rate: 0.3 mL / min, temperature: 30°C, Inj Vol: 10 uL (100 ug on column). As shown in Table 1B, the first exemplary pH gradient used for the new buffer includes 20-50% B over 15 minutes (slope = 1.2% B / CV). The flow rate during column equilibration is increased to 0.6 mL / min for fast equilibration.

[0139] The new buffer system included Buffer A = 15 mM NH4Ac + 5 mM N-methylmorpholine, pH 5.2 with acetic acid (HAc), and Buffer B = 5 mM NH4Ac + 2 mM N-methylmorpholine (NMM), pH 10.2 with ammonium hydroxide (NH4OH).

[0140] The comparative optimized standard buffer system included Buffer A = 20 mM NH4Ac, HAc at pH 5.2, and Buffer B = 5 mM NH4Ac, NH4OH at pH 10.2. As listed above, the amount of ammonium acetate (NH4Ac) in the comparative optimized standard buffer system was significantly reduced compared to the prior art references. [Table 2]

[0141] A second exemplary pH gradient using a comparative standard buffer containing 20-50% B over 15 minutes (slope = 1.2% B / CV) is shown in Table 2. The standard buffer system included Buffer A = 20 mM NH4Ac adjusted to pH 5.2 with HAc, and Buffer B = 5 mM NH4Ac adjusted to pH 10.2 with NH4OH. [Table 3]

[0142] Chromatography Results Example 2A. WCX-HRMS of trastuzumab-anns Monoclonal antibody trastuzumab-anns (Kajinti, 10 mg / mL) with pI 9.1 was subjected to native WCX-HRMS using fresh buffer and 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column using a 30-60% B gradient over 15 minutes according to Table 3, +TOF MS (2000-7000). The HRMS total ion current (TIC) chromatogram from trastuzumab-anns (Kanjinti) on a Phenomenex® bioZen WCX weak cation exchange column using the fresh buffer system with NH4Ac+NMM is shown in Figure 1-1. [Table 4]

[0143] Monoclonal antibody trastuzumab-anns (Kajinti, 10 mg / mL) with pI 9.1 was subjected to native WCX-HRMS using optimized standard buffers using a gradient of 20-50% B over 15 minutes according to Table 2, 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column according to Table 2, +TOF MS (2000-7000). Total ion current (TIC) chromatograms for trastuzumab-anns (Kanjinti) on a bioZen WCX column using the standard buffer system NH4Ac without NMM are shown in Figures 1-2.

[0144] Example 2B. WCX-HRMS of Rituximab-abbs Monoclonal antibody Rituximab-abbs (Rituxan) (10 mg / mL) with pI 9.4 was subjected to native WCX-HRMS using new buffers. Aliquots of 10 μL (100 μg) were injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column as shown in Table 4, and chromatography was performed using a 65-100% B gradient over 15 min, +TOF MS (2000-7000). The total ion current (TIC) chromatogram of Rituximab-abbs (Rituxan) on a bioZen WCX column using the new buffer system with NH4Ac+NMM is shown in Figure 2-1. [Table 5]

[0145] Monoclonal antibody Rituximab-abbs (Rituxan, 10 mg / mL) with pI 9.4 was subjected to native WCX-HRMS using standard buffers optimized with a gradient of 60-100% B over 15 min as shown in Table 4, and 10 μL (100 μg) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column, +TOF MS (2000-7000). The total ion current (TIC) chromatogram of Rituximab-abbs (Rituxan) on a bioZen WCX column using the standard buffer system NH4Ac without NMM is shown in Figure 2-2.

[0146] Example 2C. WCX-HRMS of NIST Reference mAbs Monoclonal antibody NIST reference mAb (10mg / mL) with pI 9.2 was subjected to native WCX-HRMS using the new buffer and 10uL (100ug) was injected onto a bioZen 6μm WCX, 150x2.1mm weak cation exchange column using a gradient of 65-100% B over 15 minutes +TOF MS (2000-7000). The total ion current (TIC) chromatogram of NIST reference mAb on a bioZen WCX column using the new buffer system with NH4Ac+NMM is shown in Figure 3-1.

[0147] Monoclonal antibody NIST Reference mAb with pI 9.2 was subjected to native WCX-HRMS using the optimized standard buffer and a 60-100% B gradient over 15 min as shown in Table 4, and 10 μL (100 μg) was injected onto a Phenomenex® bioZen 6 μm WCX 150×2.1 mm weak cation exchange column using the gradient shown in Table 4, +TOF MS (2000-7000). The total ion current (TIC) chromatogram of NIST mAb on a bioZen WCX column using the standard buffer system NH4Ac without NMM is shown in Figure 3-2.

[0148] Example 2D. WCX-HRMS of Infliximab Monoclonal antibody infliximab (Remicade, 10 mg / mL) with pI 7.6 was subjected to native WCX-HRMS using fresh buffer according to Table 5 and a gradient of 20-35% B over 15 minutes, 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column +TOF MS (2000-7000). The total ion current (TIC) chromatogram of infliximab on the bioZen WCX column using the fresh buffer system with NH4Ac+NMM is shown in FIG. 4. [Table 6]

[0149] Example 2E. WCX-HRMS of Cetuximab Monoclonal antibody cetuximab (Erbitux, 10 mg / mL) with pI 8.8 was subjected to native WCX-HRMS using fresh buffers according to Table 6 and a gradient of 25-55% B over 15 minutes, 10 uL (100 ug) was injected onto a Phenomenex® bioZen 6 μm WCX, 150×2.1 mm weak cation exchange column +TOF MS (2000-700). The total ion current (TIC) chromatogram of cetuximab on the bioZen WCX column using the fresh buffer system with NH4Ac+NMM is shown in FIG. 5. [Table 7]

[0150] Example 3. SCX-HRMS data using new MS-compatible buffers for improved pH gradients In this example, a method was developed for the analysis of five monoclonal antibodies and their charge variants by strong cation exchange liquid chromatography (SCX) directly coupled to high resolution mass spectrometry (HRMS) using pH gradient elution with a new buffer system (ammonium acetate + N-methylmorpholine) or an optimized standard buffer system (ammonium acetate only).

[0151] Five different mAbs were tested, including infliximab pI 7.6 (Remicade), cetuximab pI 8.8 (Erbitux), trastuzumab pI 9.1 (Kanjinti), the NIST reference mAb pI 9.2, and rituximab pI 9.4 (Rituxan).

[0152] The new buffer system was: Buffer A = 15 mM NH4Ac + 5 mM N-methylmorpholine adjusted to pH 5.2 with acetic acid. Buffer B = 5 mM NH4Ac + 2 mM N-methylmorpholine adjusted to pH 10.2 with ammonium hydroxide.

[0153] The standard buffer systems optimized for comparison were: Buffer A = 20 mM NH4Ac adjusted to pH 5.2 with acetic acid; Buffer B = 5 mM NH4Ac adjusted to pH 10.2 with ammonium hydroxide.

[0154] Both buffer systems were prepared according to Example 1 unless otherwise specified.

[0155] The strong cation exchange (SCX) column was a laboratory 3 μm SCX, 100×2.1 mm prepared by Phenomenex R&D.

[0156] The LC column eluate was monitored using UV absorbance at 280 nm or HRMS total ion current (TIC) chromatograms. Raw MS spectral data of the new and standard buffer systems were compared as follows.

[0157] The results shown in this example show that the MS signal intensity and spectral resolution using the new buffer are similar to those observed previously with the optimized standard pH gradient. However, compared to the optimized standard buffer system (NH4Ac only), the most intense MS peaks with the new buffer system (NH4Ac+NMM) are shifted to higher mass / lower charge state (+22-+27 in the new buffer vs. +25-+30 in the optimized standard buffer). This phenomenon is seen for all mAbs tested, including trastuzumab (Figure 6-5 vs. FIG. 6F), infliximab (Figure 7-5 vs. FIG. 7-6), cetuximab (Figure 8-5 vs. FIG. 8-6), rituximab (Figure 9-5 vs. FIG. 9-6), and NISTmAb (Figure 10-5 vs. FIG. 10-6). This phenomenon was also seen using the WCX column (data not shown). Using the optimized standard buffer, the raw MS data for NISTmAb in WCX-HRMS (Figure 11) shows the most intense peak at +25 to +30, similar to the raw MS data for NISTmAb in SCX-HRMS (Figure 10-6). In general, higher mass / lower charge states are preferred because the mass difference that can be differentiated in the reconstructed intact mass spectrum improves at lower charges (smaller denominators).

[0158] system: Binary pump: Agilent 1290 Infinity II UHPLC system using G7120A (JetWeaver V35), Multisampler: G7167B (20uL loop), Multicolumn Thermostat:G7116B, DAD: G7117B, +TOF MS (2000-7000).

[0159] Conditions: Flow rate: 0.3mL / min, Temperature: 30℃, Detection: UV at 280 nm, Inj Vol: 4 μL (40 μg on column), or 10 μL (100 μg on column of NISTmAb and cetuximab).

[0160] Gradient: The %B ranged from 100 to 65%B / 10 min, for which the pH gradient was optimized for each mAb using an experimental 3um SCX, 100 x 2.1 mm column pH gradient (slope = 1.2%B / CV, 5.6 CV equilibration). Table 7 shows an exemplary pH gradient of 35-65%B / 10 min. [Table 8]

[0161] Example 3A. SCX-HRMS of Trastuzumab SCX-HRMS of trastuzumab-anns (Kanjinti) was performed.

[0162] Trastuzumab-anns (Kanjinti) (10 mg / mL, 4 uL inj) was injected onto a strong cation exchange column (trial 3 μm SCX, 100 × 2.1 mm) using a fresh buffer system containing NH4Ac + NMM and a pH gradient of 35-65% B over 10 min at a flow rate of 0.3 mL / min. The UV chromatogram monitored by Abs280 nm is shown in Figure 6-1. The main peak eluted at 5.784 min RT.

[0163] Figure 6-2 shows the total ion current (TIC) chromatogram from SCX-HRMS analysis of trastuzumab-anns (4 uL inj, 10 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 35-65% B over 10 min at a flow rate of 0.3 mL / min, 30° C., +TOF (2000-7000), MS temperature 500° C. The dotted line forming a box within the main peak at RT of 4.83 min illustrates the approximate area for the MS analysis as shown in Figure 6-4 and Figure 6-5.

[0164] Figure 6-3 shows the total ion current (TIC) chromatogram from SCX-HRMS of trastuzumab-anns (4 uL inj, 10 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 20-50% B over 10 min, 30° C., +TOFMS (2000-7000), MS temperature 500° C. The dotted line forming a box within the main peak at RT of 5.09 min illustrates the approximate area for MS analysis as shown in Figure 6-6 and Figure 6-7.

[0165] Figure 6-4 shows the raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 35-65% B over 10 min, +TOFMS (2000-7000) from 4.909 to 5.131 min. A mass / charge (m / z) range of 2000-7000 Da is shown.

[0166] Figure 6-5 shows the raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) with pI 9.1, 4,858-5.217 min, using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 35-65% B over 10 min, + TOFMS (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6600 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 6-6. The same mass range is used for both spectra 6E and 6F.

[0167] Figure 6-6 shows the raw MS spectrum from SCX-HRMS of trastuzumab-anns (4 ul inj, 10 mg / mL) with pI 9.1, 4,960-5.217 min, using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 20-50% B / 10 min, +TOFMS (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 6-5.

[0168] Figures 6-7 show raw MS spectra from SCX-HRMS of trastuzumab-anns (4 ul, 10 mg / mL) with pI 9.1 using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 20-50% B / 10 min, +TOF (2000-7000) of 4,960-5,217 min. A mass / charge range of 2000-7000 Da is shown.

[0169] Example 3B. SCX-HRMS of Infliximab SCX-HRMS of infliximab (Remicade), which has a pI of 7.6, was performed.

[0170] Figure 7-1 shows a UV chromatogram monitored by Abs280 nm from an SCX-UV analysis of infliximab (10 mg / mL, 4 uL inj) using a strong cation exchange column (laboratory 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 20 to 35% B over 10 min at a flow rate of 0.3 mL / min. Good separation of the variants was observed using the pH gradient with the new buffer system: five distinct peaks were observed eluting at RTs of 3.357, 4.249, 5.018, 5.759, and 6.563 min.

[0171] Figure 7-2 shows the total ion current (TIC) chromatogram from SCX-HRMS of infliximab (4 ul, 5 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with the new buffer system containing NH4Ac+NMM and a pH gradient of 20-35% B over 10 min at a flow rate of 0.3 mL / min, 30°C, +TOF (2000-7000), MS temperature 500°C. Good separation of the variants was observed using the pH gradient with the new buffer system: five distinct peaks were observed eluting at RTs of 6.07, 6.57, 7.17, 7.87, and 8.57 min. The dotted line forming a box within the main peak at RT of 8.57 min illustrates the approximate area for MS analysis as shown in Figures 7-4 and 7-5.

[0172] Figure 7-3 shows the total ion current (TIC) chromatogram from SCX-HRMS of infliximab (4 ul, 5 mg / mL) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 15-25% B over 10 min, 30°C, +TOF (2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 5.63 min illustrates the approximate area of ​​the MS analysis shown in Figures 7-6 and 7-7. Very poor separation of charge variants was given in the standard buffer for this low pI mAb (pI=7.6). Comparison of this figure with the previous figure (Figure 7-2) demonstrates the performance advantage gained by having good pH control at a lower pH than is routinely possible with standard buffers.

[0173] Figure 7-4 shows the raw MS spectrum from SCX-HRMS of infliximab (4 ul, 5 mg / mL) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 20-35% B / 10 min, +TOF (2000-7000) of 8.621-9.032 min. A mass / charge range of 2000-7000 Da is shown.

[0174] Figure 7-5 shows the raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) from 8.621 to 9.032 min using a strong cation exchange LC column (trial 3μm SCX, 100x2.1mm) with a pH gradient of 20-35% B over 10 min, +TOF (2000-7000). The m / z range displayed was narrowed down to that giving the highest intensity (~4800-6900Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 7-5, in the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen in the optimized standard NH4Ac buffer system (+25-+30) shown in Figure 7-6.

[0175] Figure 7-6 shows the raw MS spectrum from SCX-HRMS of infliximab (4ul, 5mg / mL) at 5.593-5.730 min using a strong cation exchange LC column (trial 3μm SCX, 100x2.1mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 15-25% B over 10 min, +TOF (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (~4800-6900Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 7-5.

[0176] Figure 7 shows the raw MS spectrum from SCX-HRMS of infliximab (4 ul, 5 mg / mL) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 15-25% B / 10 min, +TOF (2000-7000) of 5.559-5.696 min. A mass / charge range of 2000-6900 Da is shown.

[0177] Example 3C. SCX-HRMS of Cetuximab SCX-HRMS of cetuximab, which has a pI of 8.8, was performed.

[0178] Figure 8-1 shows the UV chromatogram by Abs280nm from SCX-UV analysis of cetuximab (4mg / mL) using a strong cation exchange column (trial 3μm SCX, 100x2.1mm) in a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10min at a flow rate of 0.3mL / min. Multiple distinct variant peaks were observed, e.g., at RTs of 3.65, 4.03, 4.58, 5.20, and 5.93min.

[0179] Figure 8-2 shows the total ion current (TIC) chromatogram from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10 min at a flow rate of 0.3 mL / min, 30°C, +TOF (2000-7000), MS temperature 500°C. Multiple distinct variant peaks were observed at RTs of 3.33, 3.92, 4.54, and 5.22 min. The dotted line forming a box within the main peak at RT of 4.54 min illustrates the approximate area for MS analysis as shown in Figures 8-4 and 8-5.

[0180] Figure 8-3 shows the total ion current (TIC) chromatogram from SCX-HRMS analysis of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 17-40% B / 10 min, 30 °C, +TOF (2000-7000), MS temperature 500 °C. The dotted line forming a box within the main peak at RT of 4.68 min illustrates the approximate area for the MS analysis shown in Figure 8-6 and Figure 8-7.

[0181] Figure 8-4 shows the raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 30-60% B over 10 min, +TOF (2000-7000) of 4.601-9.032 min. A mass / charge range of 2000-7000 Da is shown.

[0182] Figure 8-5 shows the raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) at 4.601-4.892 min using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a pH gradient of 30-60% B over 10 min, +TOF (2000-7000). The m / z range displayed was narrowed down to that giving the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 8-5, when using pH gradient elution with the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 8-6.

[0183] Figure 8-6 shows the raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) at 4.533-4.892 min using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 17-40% B over 10 min, +TOF (2000-7000). The displayed m / z range was narrowed down to the one that gave the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 8-5.

[0184] Figure 8-7 shows the raw MS spectrum from SCX-HRMS of cetuximab (5 mg / mL, 10 uL inj) from 4.533 to 4.892 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 17-40% B over 10 min, +TOF (2000-7000). A mass / charge range of 2000-6900 Da is shown.

[0185] Example 3D. SCX-HRMS of Rituximab SCX-HRMS of Rituximab (Rituxan), which has a pI of 9.4, was carried out.

[0186] Figure 9-1 shows the UV chromatogram with Abs 280 nm from SCX-HRMS of Rituximab (Rituxan) with pI 9.4 (10 mg / mL) using a strong cation exchange column (trial, 3 μm SCX, 100 × 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 75 to 100% B over 10 min at a flow rate of 0.3 mL / min. A major peak at RT of 5.03 min was observed.

[0187] Figure 9-2 shows the total ion current (TIC) chromatogram from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 65-100% B over 10 min at a flow rate of 0.3 mL / min, 30°C, +TOFMS (2000-7000), MS temperature 500°C. The dotted line forming a box within the main peak at RT of 5.88 min illustrates the approximate area for the MS analysis.

[0188] Figure 9-3 shows the total ion current (TIC) chromatogram from an SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100×2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 55-100% B over 10 min, 30° C., +TOFMS (2000-7000), MS temperature 500° C. The dotted line forming a box within the main peak at RT of 4.65 min illustrates the approximate area for the MS analysis shown in Figures 9-6 and 9-7.

[0189] Figure 9-4 shows the raw MS spectrum from LC-HRMS of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 65-100% B over 10 min, + TOFMS (2000-7000) from 6.021 to 6.363 min. Mass / charge, m / z data in the range of 2000-7000 Da are shown.

[0190] Figure 9-5 shows raw MS spectra from an SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with the new buffer system containing NH4Ac + NMM and a pH gradient of 65-100% B / 10 min, + TOFMS (2000-7000) from 6.021 to 6.363 min. The m / z range displayed was narrowed down to that giving the highest intensity (approximately 4800-6800 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. As shown in Figure 9-5, when using a pH gradient with the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), shown in Figure 9-6.

[0191] Figure 9-6 shows raw MS spectra from an SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) in a standard buffer system containing NH4Ac without NMM and a pH gradient of 55-100% B over 10 min, +TOFMS (2000-7000) from 4.550 to 4.772 min. The m / z range displayed was narrowed down to that giving the highest intensity (approximately 4800-6800 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. In the optimized standard buffer system, the most intense MS peak appears at a lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 9-5.

[0192] Figure 9-7 shows the raw MS spectrum from SCX-HRMS analysis of rituximab (10 mg / mL, 4 uL inj) from 4,550 to 4.772 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 55-100% B / 10 min, +TOFMS (2000-7000). The mass / charge range of 2000-7000 Da is shown.

[0193] Example 3E. SCX-HRMS of NISTmAb SCX-HRMS of NISTmAb with pI 9.2 was performed.

[0194] Figure 10-1 shows a UV chromatogram monitored at Abs 280 nm from an SCX-UV analysis of NIST mAb with pI 9.2 (10 mg / mL) using a strong cation exchange column (trial, 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 95 to 100% B over 10 min at a flow rate of 0.3 mL / min.

[0195] Figure 10-2 shows the total ion current (TIC) chromatogram from SCX-HRMS of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 90-100% B over 10 min at a flow rate of 0.3 mL / min, 30 °C, + TOFMS (2000-7000), MS temperature 500 °C. The dotted line forming a box within the peak with RT of 4.39-4.82 illustrates the approximate area for the MS analysis.

[0196] Figure 10-3 shows the total ion current (TIC) chromatogram from an SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing only NH4Ac and a pH gradient of 80-100% B / 10 min, 30 °C, +TOFMS (2000-7000), MS temperature 500 °C. The dotted line forming a box within the main peak at RT of 4.5 min illustrates the approximate area for the MS analysis shown in Figures 10-6 and 10-7.

[0197] Figure 10-4 shows the raw MS spectrum from SCX-HRMS of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with a new buffer system containing NH4Ac+NMM and a pH gradient of 90-100% B / 10 min, +TOFMS (2000-7000) from 5.012 to 5.627 min. Mass / charge (m / z) data over the range 2000-7000 Da are shown.

[0198] Figure 10-5 shows raw MS spectra from an SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a new buffer system containing NH4Ac + NMM and a pH gradient of 90-100% B / 10 min, + TOFMS (2000-7000) from 5.012 to 5.627 min. The m / z range displayed was narrowed down to that giving the highest intensity (approximately 4800-6900 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the new buffer system, the most intense MS peak appears at a higher mass / lower charge (+22-+27) than seen with the optimized standard NH4Ac buffer system (+25-+30), as shown in Figure 10-6.

[0199] Figure 10-6 shows raw MS spectra from an SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a strong cation exchange LC column (trial 3 μm SCX, 100 × 2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 80-100% B / 10 min, +TOFMS (2000-7000) from 4.259 to 4.841 min. The m / z range displayed was narrowed down to that giving the highest intensity for the native analyte (approximately 4800-6900 Da) with this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the most intense MS peak appears at lower mass / higher charge (+25-+30) when compared to the new NH4Ac+NMM buffer system (+22-+27), as shown in Figure 10-5.

[0200] Figure 10-7 shows the raw MS spectrum from SCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) from 4.259 to 4.841 min using a strong cation exchange column (trial 3 μm SCX, 100 x 2.1 mm) with an optimized standard buffer system containing NH4Ac without NMM and a pH gradient of 80-100% B / 10 min, +TOFMS (2000-7000). A mass / charge range of 2000-6800 Da is shown.

[0201] FIG. 11 shows the raw MS spectrum from a WCX-HRMS analysis of NISTmAb (10 mg / mL, 10 uL inj) using a weak cation exchange LC column (Phenomenex® bioZen 6 μm WCX, 150×2.1 mm) with a standard buffer system containing NH4Ac without NMM and a pH gradient of 60-100% B, +TOFMS (2000-7000) of 4.772-5.012 min, 500°C. The displayed m / z range was narrowed to that giving the highest intensity (approximately 4800-7000 Da) of the native analyte using this buffer system. Additionally, the charge of the analyte ion within the charge state envelope is shown. With the optimized standard buffer system, the raw MS spectrum for NISTmAb using WCX-HRMS also exhibits the most intense MS peak at +25-+30, similar to the SCX-HRMS raw spectrum shown in FIG. 10-6.

[0202] References Allowable_concentrations_salt_and_buffer_components_for_MS-Harvard Center for MS-download 05-05-2021 Bailey et al.,2018,Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis,MABS,vol.10,No.8,1214-1225.doi.org / 10.1080 / 1940862.2018.1521131. Borman,1987,Eluent,Effluent,Eluate,and Eluite,Anal Chem vol.59,no.2,Jan 15,1987,99A Chen et al.,2012,Ultra-high performance liquid chromatography / tandem mass spectrometry determination of feminizing chemicals in river water,sediment and tissue pretreated using disk-type solid-phase extraction and matrix solid-phase dispersion,Talanta,Vol 89,pp.237-245.doi.org / 10.1016 / j.talanta.2011.12.020 Chirita et al.,2011,Evaluation of fused-core and monolithic versus porous silica- based C18 columns and porous graphitic carbon for ion-pairing liquid chromatography analysis of catecholamines and related compounds;Volume 879,Issues 9-10,15 March 2011,Pages 633-640;https: / / doi.org / 10.1016 / j.jchromb.2011.01.036 Fussl et al.,2018,Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry,Anal.Chem.90,4669-4676.doi 10.1021 / acs.analchem.7b05241. Fussl et al.,2019,Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry,MABS vol.11,No.1,116-128,doi.org / 10.1080 / 19420862.2018.1531664. Goyon et al.,2017,Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies,J Chrom B,vol 1065-1066,pp 119-128,http: / / dx.doi.org / 10.1016 / j.jchromb.2017.09.033 NIST Monoclonal Antibody Reference Material 8671,2021,https: / / www.nist.gov / programs-projects / nist-monoclonal-antibody-reference-material- 8671 pKa Data Compiled by R.Williams-downloaded 2021-pp.1-33 Schaefercor et al.,1996,Effect of high-performance liquid chromatography mobile phase components on sensitivity in negative atmospheric pressure chemical ionization liquid chromatography-mass spectrometry,J Am Soc Mass Spectrom,7,10,1059- 1069;https: / / doi.org / 10.1016 / 1044-0305(96)00049-9 Nortcliffe et al.,2017,Detection-of-Intact-Antibody-Impurities-using- SCIEX X500B,Application Data Sheet,AB Sciex Document number:RUO-MKT-02-6335- A Shukla et al.,2017,Evolving trends in mAb production processes,Bioengineering & Translational Medicine 2017:2,58-69,DOI 10.1002 / btm2.10061

Claims

**Claim 1** A ready-to-use mobile phase composition for liquid chromatography, comprising water, 2 to 50 mM ammonium carboxylate, 1 to 50 mM N-methylmorpholine, its isomers, or its analogs, and A ready-to-use mobile phase composition having a pH in the range of about pH 4.5 to about pH 10.5 at room temperature. **Claim 2** 2 to 25 mM of said ammonium carboxylate, and 1 to 10 mM of said N-methylmorpholine, said isomers, or said analogs, the composition according to claim 1. **Claim 3** The composition according to claim 1, wherein said ammonium carboxylate is selected from the group consisting of ammonium acetate and ammonium formate. **Claim 4** The composition according to claim 1, wherein said N-methylmorpholine isomers are selected from the group consisting of 2-methylmorpholine, 2-methylmorpholine, 2-methyl-1,3-oxazinane, 3-methyl-1,3-oxazinane, 4-methyl-1,3-oxazinane, 5-methyl-1,3-oxazinane, and 6-methyl-1,3-oxazinane. **Claim 5** The composition according to claim 1, wherein said analogs are selected from the group consisting of alkyl C1-C6 morpholine, dialkyl C1-C6 morpholine, alkyl C1-C6-1,3-oxazinane, and dialkyl C1-C6-1,3-oxazinane. **Claim 6** Said mobile phase comprises a two-part aqueous buffer system, Part A of said aqueous buffer system comprises about 10 mM to about 20 mM ammonium acetate, about 3 to about 8 mM N-methylmorpholine, and a pH in the range of pH 5.0 to 5.5, Part B of said aqueous buffer system comprises 2 to 10 mM ammonium acetate, 1 to 5 mM N-methylmorpholine, and a pH in the range of pH 9.5 to 10.5, the composition according to claim 1. **Claim 7** The composition according to claim 6, wherein said pH of the Part A buffer is adjusted with acetic acid. **Claim 8** The composition according to claim 6, wherein said pH of the Part B buffer is adjusted with ammonium hydroxide. **Claim 9** The composition according to claim 6, wherein the Part A buffer comprises about 14 to about 16 mM of said ammonium acetate, about 4 to about 6 mM of said N-methylmorpholine, and said pH is about pH 5.1 to about pH 5.

3. **Claim 10** The composition according to claim 6, wherein the partial B buffer solution contains about 4 to about 6 mM of the ammonium acetate and about 1 mM to about 3 mM of the N-methylmorpholine, and the pH is about 10.0 to about 10.

4.

11. The composition according to any one of claims 1 to 10, wherein the concentration of the ammonium acetate is 25 mM or less, 20 mM or less, or about 15 mM or less.

12. The composition according to any one of claims 1 to 10, containing individual metal impurities of 100 ppb or less.

13. The composition according to any one of claims 1 to 10, wherein the liquid chromatography includes a stationary phase.

14. The composition according to claim 13, wherein the stationary phase is selected from the group consisting of an ion exchange stationary phase, a size exclusion stationary phase, a hydrophilic interaction stationary phase, and a reverse phase stationary phase.

15. The composition according to claim 14, wherein the ion exchange stationary phase is a cation exchange stationary phase.

16. The composition according to claim 15, wherein the cation exchange stationary phase is selected from the group consisting of a strong cation exchange stationary phase and a weak cation exchange stationary phase.

17. The composition according to claim 1, wherein the liquid chromatography is directly coupled (online) to a mass spectrometer.

18. A method for separating and / or characterizing an analyte in a sample, comprising: flowing a mobile phase through a chromatography column, wherein the mobile phase includes a two-part aqueous buffer system; wherein part A of the aqueous buffer system includes about 10 to 50 mM of ammonium carboxylate, 3 to 16 mM of N-methylmorpholine, or an isomer or analog thereof, and a pH within the range of about pH 5 to about pH 5.5; wherein part B of the aqueous buffer system includes 2 to 25 mM of ammonium carboxylate, 1 to 10 mM of N-methylmorpholine, or an isomer or analog thereof, and a pH within the range of about pH 9.5 to about pH 10.5; flowing; injecting a sample containing the analyte into the mobile phase; eluting the analyte from the column; detecting the analyte in the eluate.

19. flowing a mobile phase through a chromatography column, wherein the mobile phase includes a two-part aqueous buffer system; wherein part A of the aqueous buffer system includes about 10 to 25 mM of ammonium acetate, 3 to 8 mM of N-methylmorpholine, and a pH within the range of about pH 5 to about pH 5.5; flowing the aqueous buffer portion B, which comprises ammonium acetate at 2 to 10 mM, N-methylmorpholine at 1 to 5 mM, and a pH within the range of about pH 9.5 to about pH 10.5, injecting a sample containing the analyte into the mobile phase, eluting the analyte from the column, detecting the analyte in the eluate, a method for separating and / or characterizing an analyte in a sample according to claim 18.

20. The method according to claim 18, wherein the analyte is a biomolecule.

21. The method according to claim 20, wherein the biomolecule is a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, and / or a charge variant thereof.

22. The method according to claim 21, wherein the monoclonal antibody or fragment has a pI of about pI 6.5 to about pI 9.

5.

23. The method according to any one of claims 18 to 22, wherein the chromatography column comprises a stationary phase selected from the group consisting of an ion exchange stationary phase, a size exclusion stationary phase, a hydrophilic interaction stationary phase, and a reversed phase stationary phase.

24. The method according to claim 23, wherein the ion exchange stationary phase is a cation exchange stationary phase selected from the group consisting of a strong cation exchange stationary phase and a weak cation exchange stationary phase.

25. The method according to any one of claims 18 to 22, wherein the detecting comprises determining the UV absorbance of the eluate.

26. The method according to any one of claims 18 to 22, comprising detecting the analyte with a mass spectrometer (MS).

27. The method according to claim 26, wherein the MS is selected from the group consisting of a sector, a time-of-flight (TOF), a quadrupole, an ion trap, a Fourier transform ion cyclotron resonance, and a tandem mass spectrometer, or two or more of the mass spectrometers combined within a tandem or orthogonal platform.

28. The method according to claim 26, wherein MS detection comprises generating analyte ions.

29. The method according to claim 28, wherein the generating comprises an ionization technique selected from the group consisting of electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI), fast atom bombardment (FAB), chemical ionization (CI), electron impact (EI), atmospheric solids analysis ionization (ASAI), atmospheric pressure photoionization (APPI), desorption electrospray ionization (DESI), and atmospheric pressure vapor source (APVS).

30. The method according to claim 28, wherein the MS detection further comprises obtaining a mass spectrum of the analyte ions.

31. The method according to claim 30, wherein the mass spectrum of the analyte ions exhibits a lower charge state for the analyte isoform than an equivalent method using a mobile phase buffer system containing ammonium acetate that does not contain N-methylmorpholine, its isomers, or analogs.

32. The method according to claim 31, wherein the lower charge state for the analyte isoform results in an increased amount of analyte mass within the charge state envelope compared to an equivalent method using a mobile phase buffer system containing ammonium acetate that does not contain N-methylmorpholine, its isomers, or analogs.

33. The method according to claim 25, wherein the MS detection further comprises determining the molecular weight of the analyte.

34. Part A of the aqueous buffer system contains about 15 mM ammonium acetate and about 5 mM N-methylmorpholine adjusted to pH 5.2 with acetic acid, Part B of the aqueous buffer system contains about 5 mM ammonium acetate and about 2 mM N-methylmorpholine adjusted to pH 10.2 with ammonium hydroxide, according to the method of claim 19.

35. The method according to claim 19, wherein the mobile phase contains individual metal impurities of about 100 ppb or less.

36. Elution is increasing buffer B% over time with respect to buffer A% flowing through the column after the injection, wherein buffer A% + buffer B% = 100% of the mobile phase, including forming a pH gradient, according to the method of claim 19.

37. The method according to claim 36, wherein the slope of the gradient is in the range of 0.1 - 10% B / CV or 0.5 - 5% B / column volume (CV) of the eluate.

38. The method according to claim 36 or 37, wherein the pH gradient is a linear pH gradient, a segmented pH gradient, a curved pH gradient, or a step pH gradient.

39. A kit comprising: a first container having a volume of a first concentrated liquid composition for obtaining a buffer solution A containing 10 to 50 mM ammonium carboxylate, 3 to 16 mM N-methylmorpholine, or an isomer or analog thereof, and a pH in the range of about pH 4.5 to about pH 5.5 by diluting with water; a second container having a volume of a second concentrated liquid composition for obtaining a buffer solution B containing 2 to 25 mM ammonium carboxylate, 1 to 10 mM N-methylmorpholine, or an isomer or analog thereof, and a pH in the range of about pH 9.5 to about pH 10.5 by diluting with water; instructions for use; and a kit.

40. a first container having a volume of a first concentrated liquid composition for obtaining a buffer solution A containing 10 to 25 mM ammonium acetate, 3 to 8 mM N-methylmorpholine, and a pH in the range of about pH 4.5 to about pH 5.5 by diluting with water; a second container having a volume of a second concentrated liquid composition for obtaining a buffer solution B containing 2 to 10 mM ammonium acetate, 1 to 5 mM N-methylmorpholine, and a pH in the range of about pH 9.5 to about pH 10.5 by diluting with water; instructions for use; and a kit according to claim 39.

41. The kit according to claim 39 or 40, wherein the first concentrated liquid composition and the second concentrated liquid composition each contain a concentration selected from the group consisting of the buffer solution A and the buffer solution B concentrated 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

42. The kit according to claim 39, further comprising a chromatography column.

43. The kit according to claim 42, wherein the chromatography column is a strong ion exchange chromatography column or a weak ion exchange chromatography column.

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