HPLC quantification of excipients

Abstract

The present invention provides an analytical method for separating and optionally quantifying two or more buffers or excipients in a sample in a single assay.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 948,357, filed on December 16, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] High performance liquid chromatography (HPLC) is a common analytical method for separating and quantifying components of liquid samples. Sample components eluted from the HPLC column are passed through a detection cell and detected using any one or more than one method, including but not limited to ultraviolet detection, mass spectrometry, refractive index detection, evaporative light scattering detection (ELSD), charged aerosol detection (CAD) and condensation nucleation light scattering detection (CNLSD).

[0004] Pharmaceutical formulations typically contain one or more than one active ingredient in combination with a variety of physiologically acceptable excipients and / or carriers. These excipients and / or carriers can help improve the stability, dilution or expansion of the active ingredient, promote its absorption, reduce its viscosity and / or increase its solubility. Preclinical and clinical studies often require analysis of the physicochemical properties of pharmaceutical formulations, which always includes quantification of the excipients and / or carriers used therein. Since the physicochemical properties of the component excipients may vary greatly, multiple analyses are often performed by HPLC using multiple columns filled with different stationary phases.

[0005] There is a need for improved methods of separating, detecting and quantifying two or more excipients having different physicochemical properties in a pharmaceutical formulation that are capable of resolving and detecting the two or more excipients in a single HPLC run using a single stationary phase chromatographic column. Summary of the invention

[0006] The present invention generally relates to methods for analytically separating and optionally quantifying two or more buffers or excipients in a single HPLC assay.

[0007] In one aspect, the method of the present invention comprises: performing chromatography on a pentafluorophenyl (PFP) high performance liquid chromatography (HPLC) column on a test sample comprising two or more buffers or excipients to separate the two or more buffers or excipients; detecting the separated two or more buffers or excipients in the HPLC column effluent; and generating a chromatogram having peaks corresponding to the separated two or more buffers or excipients.

[0008] In some embodiments, two or more buffers or excipients present in the test sample are selected from sodium phosphate, sodium citrate, potassium phosphate, histidine and sugar or glycosyl molecules. For example, in some embodiments, sugar or glycosyl molecules are selected from 2-hydroxypropyl-β-cyclodextrin (hpβCD), sucrose, trehalose and mannitol.

[0009] In some embodiments, the method of the present invention further comprises: obtaining standard calibration chromatographic data of the two or more excipients running on the same HPLC column; and calculating the concentration or amount of the two or more buffers or excipients in the test sample by: determining the integrated peak area of ​​the two or more buffers or excipients from the chromatogram, and converting the integrated area into a concentration or amount based on the obtained standard calibration chromatographic data. In certain embodiments, the conversion comprises a linear regression fit to the standard calibration chromatographic data.

[0010] In some embodiments, two or more buffers or excipients in the test sample are detected using an evaporative light scattering detector (ELSD). In certain embodiments, the evaporation temperature of the ELSD is set to 40 to 70°C, the pressure is set to 30 to 70 psi, the gain is set to 0.5 to 2, and the filter is set to 0.5 to 1.

[0011] In other embodiments, two or more buffers or excipients in the test sample are detected using a charged aerosol detector (CAD). In certain embodiments, the CAD has an evaporation temperature set to 25 to 35°C, a frequency set to 4 to 6 Hz, a filter set to 4 to 6 seconds, a power function set to 1.78 for the first two thirds of the HPLC run, and a power function set to 1.68 for the last third of the HPLC run.

[0012] In some embodiments of the invention, HPLC is run using two mobile phases: mobile phase A and mobile phase B. In some embodiments, mobile phase A is 100% HO. In other embodiments, mobile phase A comprises HO and formic acid. In other embodiments, mobile phase A comprises HO and trifluoroacetic acid. In some embodiments, mobile phase B comprises acetonitrile.

[0013] In some embodiments, mobile phase A is H2O and 0.5% formic acid. In other embodiments, mobile phase A comprises H2O and 0.05% trifluoroacetic acid. In some embodiments, mobile phase B is 100% acetonitrile.

[0014] In some embodiments, performing the chromatography comprises a balancing step of flowing 100% mobile phase A through the HPLC column at a rate of 0.1 ml / min to 1.0 ml / min. In some embodiments, the balancing step flow rate is 0.25 ml / min or 0.5 ml / min. In some embodiments, the balancing step is between 0.5 and 10 minutes. In certain embodiments, the balancing step is 3.0 or 4.0 minutes.

[0015] In some embodiments, performing chromatography includes a gradient change of 60% mobile phase A and 40% mobile phase B flowing through an HPLC column. In some embodiments, performing chromatography includes a gradient change of 40% mobile phase A and 60% mobile phase B flowing through an HPLC column. In some embodiments, performing chromatography includes a gradient change of 100% mobile phase A flowing through an HPLC column. In some embodiments, the gradient change flow rate is between 0.1ml / min and 1.0ml / min. In certain embodiments, the gradient change flow rate is 0.25ml / min or 0.5ml / min. In some embodiments, the gradient change is between 0.5min and 10min. In certain embodiments, the gradient change is 0.5min, 2.0min or 4.0min.

[0016] In some embodiments, performing chromatography includes a maintenance step of flowing 40% mobile phase A and 60% mobile phase B through an HPLC column. In some embodiments, performing chromatography includes a maintenance step of flowing 100% mobile phase A through an HPLC column. In some embodiments, the maintenance step flow rate is between 0.1 ml / min and 1.0 ml / min. In certain embodiments, the maintenance step flow rate is 0.5 ml / min or 1.0 ml / min. In some embodiments, the maintenance step is between 0.5 and 10 minutes. In certain embodiments, the maintenance step is 2.5 minutes or 4.0 minutes.

[0017] In some embodiments, performing the chromatography includes a re-equilibration step of flowing 100% mobile phase A through the HPLC column at a rate of 0.1 ml / min to 1.0 ml / min. In certain embodiments, the re-equilibration flow rate is 0.25 ml / min or 0.5 ml / min. In some embodiments, the re-equilibration step is between 0.5 and 10 minutes. In some embodiments, the re-equilibration step is 3.0 minutes.

[0018] In some embodiments of the present invention, performing chromatographic analysis includes: (i) equilibration with 100% mobile phase A at a flow rate of 0.25 ml / min for 3.0 minutes; (ii) changing the gradient to 60% mobile phase A and 40% mobile phase B at a flow rate of 0.25 ml / min over 0.5 minutes; (iii) changing the gradient to 40% mobile phase A and 60% mobile phase B at a flow rate of 1.0 ml / min over 4.0 minutes; (iv) maintaining at 40% mobile phase A and 60% mobile phase B at a flow rate of 1.0 ml / min for 2.5 minutes; (v) changing the gradient to 100% mobile phase A at a flow rate of 1.0 ml / min over 2 minutes; and (vi) re-equilibration with 100% mobile phase A at a flow rate of 0.25 ml / min for 3.0 minutes.

[0019] In other embodiments of the invention, performing a chromatographic analysis comprises: (i) equilibration with 100% mobile phase A at a flow rate of 0.5 ml / min for 4.0 minutes; (ii) gradient changing to 40% mobile phase A and 60% mobile phase B at a flow rate of 0.5 ml / min over 2.0 minutes; (iii) gradient changing to 100% mobile phase A at a flow rate of 0.5 ml / min over 2.0 minutes; and (iv) maintaining at 100% mobile phase A at a flow rate of 0.5 ml / min for 4 minutes.

[0020] In other embodiments of the invention, performing a chromatographic analysis comprises: (i) equilibration with 100% mobile phase A at a flow rate of 0.5 ml / min for 4.0 minutes; (ii) gradient changing to 40% mobile phase A and 60% mobile phase B at a flow rate of 0.5 ml / min over 2.0 minutes; (iii) gradient changing to 100% mobile phase A at a flow rate of 0.5 ml / min over 2.0 minutes; and (iv) maintaining at 100% mobile phase A at a flow rate of 0.5 ml / min for 4 minutes.

[0021] In some embodiments of the invention, 1 μl to 100 μl of the test sample is injected into the HPLC column. In certain embodiments, 10 μl to 4 μl of the test sample is injected into the HPLC column.

[0022] In certain embodiments of the invention, the PFP HPLC column is a 2.6 μm 150×4.6 mm column. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A Shown is a chromatogram of a formulation buffer containing histidine, sucrose and mannitol resolved on a C18 column.

[0024] Figure 1BShown is a chromatogram of a formulation buffer containing tris, sucrose and hydroxypropyl beta-cyclodextrin (hpβCD) subjected to chromatography on a C18 column.

[0025] Figure 2A Shown is a chromatogram of a formulation buffer containing histidine, sucrose and mannitol subjected to chromatography on an anion exchange column.

[0026] Figure 2B Shown is a chromatogram of a formulation buffer containing Tris, sucrose and hpβCD resolved on an anion exchange column.

[0027] Figure 3 Shown is a chromatogram of a formulation buffer containing hpβCD and sucrose resolved on a pentafluorophenyl (PFP) column.

[0028] Figure 4 Shown is a chromatogram of a formulation buffer containing Tris resolved on a PFP column.

[0029] FIG5 shows a chromatogram of a formulation buffer containing sodium phosphate (A), sodium citrate (B), potassium phosphate (C), histidine (D), and trehalose (E) resolved on a PFP column.

[0030] Figure 6 Shown is a chromatogram of a formulation buffer containing mannitol, sucrose and histidine resolved on a PFP column. DETAILED DESCRIPTION

[0031] Disclosed herein is a method for analytically separating and optionally quantifying two or more buffers or excipients in a sample using a pentafluorophenyl (PFP) high performance liquid chromatography (HPLC) column in a single assay. The advantage of the method is that it can resolve two or more buffers or excipients in a sample, whereas conventional methods known in the art cannot resolve due to solvent interference and / or suboptimal column retention time and peak shape.

[0032] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0033] As used herein, the terms "a" or "an" mean "one or more" and include the plural unless the context otherwise requires.

[0034] As used herein, the term "about" refers to a value at or near the stated amount. For example, "about" can mean less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, or such as less than or equal to ±0.05%.

[0035] As used herein, the term "analyte" means a substance or chemical component that is identified and / or measured.

[0036] As used herein, the term "standard calibration sample series" means two or more samples, each sample having a different, known concentration of an analyte, and wherein the concentration range of the different samples covers or is close to the expected concentration of the analyte in the test sample.

[0037] The term "calibration curve" as used herein means a graph based on the analyte signal detected and measured by an HPLC instrument for each known concentration of a sample including a series of standard calibration samples.

[0038] As used herein, the term "linear regression fit" means a mathematical algorithm that plots a line along which a set of signal data has a minimum measured value. The graph produced by the linear regression fit has a slope, a y-intercept, and an R-squared value that is used to measure how well the signal data fits the line.

[0039] As used herein, the term "integrated peak area" means the quantified area under a chromatographic peak corresponding to a detected analyte signal of an analyte in a test sample.

[0040] As used herein, the term "mobile phase" means a liquid or gas that flows through a chromatographic instrument, wherein the liquid or gas moves one or more analytes in a sample at different rates over a stationary phase.

[0041] When used in connection with a mobile phase description, the terms percentage (or %) are expressed as volume percentages unless the context clearly dictates otherwise.

[0042] The term "stationary phase" as used herein means a solid or liquid in a chromatographic instrument on which one or more than one analytes are separated or selectively adsorbed.

[0043] Buffers and excipients

[0044] The present invention provides an analytical method for quantifying two or more buffers or excipients in a sample in a single assay. In some embodiments, the sample can be a pharmaceutical composition comprising two or more buffers or excipients formulated with an effective amount of an active ingredient, as described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For example, in some embodiments, the two or more buffers or excipients may be selected from, but are not limited to, carbohydrates (e.g., glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, fructose, maltose, cellobiose, lactose, deoxyribose, hexose); glycosyl molecules (e.g., mannitol, sorbitol, ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, fucitol, iditol, inositol, heptyl alcohol, lactitol, isomalt, maltitol, maltotriitol, and polyethylene glycol); ; Cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl β-cyclodextrin (hpβCD), and sulfobutyl ether β-cyclodextrin); lipids (e.g., lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), heptadecanoic acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0)); minerals (e.g., chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium); vitamins (e.g., vitamin C, vitamin A, vitamin E, vitamin B1 2. Vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid and biotin); buffers (e.g. sodium citrate, potassium phosphate, histidine, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate); preservatives (e.g. alpha-tocopherol, ascorbate, parabens, chlorobutanol and phenol); binders (e.g. starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyl oxazolidinone, polyvinyl alcohol, C 12 -C 18fatty acid alcohols, polyethylene glycol, polyols, sugars, oligosaccharides); lubricants (e.g. magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate and light mineral oil); dispersants (e.g. starch, alginic acid, polyvinyl pyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate (isoamorphous silicate) disintegrants (e.g., corn starch, potato starch, pregelatinized starches and modified starches thereof, sweeteners, clays (e.g., bentonite), microcrystalline cellulose, alginates, sodium starch glycolate, gums (e.g., agar, guar gum, locust bean gum, karaya gum, pectin, tragacanth gum), combinations of sodium bicarbonate and citric acid, and combinations of sodium bicarbonate and tartaric acid); flavorings; sweeteners; and coloring agents.

[0045] In certain embodiments, the analytical methods of the invention quantify two or more buffers or excipients selected from sucrose, hpβCD, sucrose, mannitol, histidine, sulfobutyl ether β-cyclodextrin, sodium phosphate, sodium citrate, potassium phosphate, trehalose, and Tris.

[0046] High performance liquid chromatography

[0047] The analytical method of the present invention quantifies two or more buffers or excipients by resolving the two or more buffers or excipients in a test sample on a high performance liquid chromatography (HPLC) column. For example, in certain embodiments, the HPLC column is a pentafluorophenyl (PFP) column.

[0048] In some embodiments, the PFP column can facilitate rapid, high-resolution separation of sample analytes at low back pressures. In some embodiments, the PFP column can be filled with particles having a diameter of about 0.8 μm to about 8.0 μm. For example, in some embodiments, the PFP column can be filled with particles having a diameter of about 1.7 μm, about 2.6 μm, or about 5.0 μm. In some embodiments, the PFP column can be filled with particles having a diameter of about 2.4 μm to about 2.6 μm. In some embodiments, the PFP column can be filled with particles having a diameter of about 2.6 μm.

[0049] In some embodiments, the particles of the PFP column may have a particle size of about to about In certain embodiments, the particles of the PFP column may have a pore size of about to about In certain embodiments, the PFP column may have a pore size of about of aperture.

[0050] As used herein, "size distribution" refers to a relative measure of the distribution of particle sizes. For example, the ratio of the particle size at 10% of the total size distribution to the particle size at 90% of the total size distribution can be used as a relative measure of the particle size distribution. The closer the ratio is to a value of 1, the more uniform the particle size distribution. In some embodiments, the particles of the PFP column may have a size distribution of less than or equal to about 1.5. For example, in some embodiments, the particles of the PFP column may have a size distribution of less than or equal to about 1.4, less than or equal to about 1.3, or less than or equal to about 1.2.

[0051] In some embodiments, the PFP column can have a diameter of about 2.0 mm to about 5.0 mm. For example, in some embodiments, the PFP column can have a diameter of about 2.1 mm, about 3 mm, or about 4.6 mm.

[0052] In some embodiments, the PFP column can have a length of about 10 mm to about 150 mm. For example, in some embodiments, the PFP column can have a length of about 10 mm, about 30 mm, about 50 mm, about 100 mm, or about 150 mm.

[0053] In some embodiments of the invention, the two or more buffers or excipients are detected using an evaporative light scattering detector (ELSD). ELSD uses a laser beam to measure reflected light scattered to a photomultiplier tube, wherein the larger the size / mass of the particles, the greater the degree of light scattering.

[0054] In some embodiments, the evaporation temperature of the ELSD is set to about 10°C to about 100°C. For example, in some embodiments, the evaporation temperature of the ELSD is set to about 20°C to about 100°C, about 30°C to about 100°C, about 40°C to about 100°C, about 50°C to about 100°C, about 10°C to about 90°C, about 20°C to about 90°C, about 30°C to about 90°C, about 40°C to about 90°C, about 50°C to about 90°C, about 10°C to about 80°C, about 20°C to about 80°C, about 30°C to about 80°C, about 40°C to about 80°C, about 50°C to about 80°C, about 10°C to about 70°C, about 20°C to about 70°C, about 30°C to about 70°C, about 40°C to about 70°C, about 50°C to about 70°C, about 10°C to about 60°C, about 20°C to about 60°C, about 30°C to about 60°C, about 40°C to about 60°C, or about 50°C to about 60°C. In certain embodiments, the evaporation temperature of the ELSD is set to about 50°C.

[0055] In some embodiments, the pressure of the ELSD is set to about 10 psi to about 100 psi. For example, in some embodiments, the pressure of ELSD is set to about 20psi to about 100psi, about 30psi to about 100psi, about 40psi to about 100psi, about 50psi to about 100psi, about 10psi pressure about 90psi, about 20psi to about 90psi, about 30psi to about 90psi, about 40psi to about 90psi, about 50psi to about 90psi, about 10psi to about 80psi, about 20psi to about 80psi, about 30psi to about 80psi, about 40psi to about 80psi, about 50psi to about 80psi, about 10psi to about 70psi, about 20psi to about 70psi, about 30psi to about 70psi, about 40psi to about 70psi, about 50psi to about 70psi, about 10psi to about 60psi, about 20psi to about 60psi, about 30psi to about 60psi, about 40psi to about 60psi, about 50psi to about 60psi. In certain embodiments, the pressure of ELSD is set to about 50psi.

[0056] In some embodiments, the gain of the ELSD is set to 0.1 to 5. For example, in some embodiments, the gain of the ELSD is set to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0057] In some embodiments, the filter setting of the ELSD is 0.1-1.0. For example, in some embodiments, the filter setting of the ELSD is 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0.

[0058] In some embodiments, the evaporation temperature of the ELSD is set to about 10°C to about 100°C. For example, in some embodiments, the evaporation temperature of the ELSD is set to about 15°C to about 100°C, about 20°C to about 100°C, about 25°C to about 100°C, about 30°C to about 100°C, about 35°C to about 100°C, about 40°C to about 100°C, about 45°C to about 100°C, about 50°C to about 100°C, about 55°C to about 100°C, about 10°C to about 90°C, about 15°C to about 90°C, about 20°C to about 90°C, about 25°C to about 90°C, about 30°C to about 90°C, about 35°C to about 90°C, about 40°C to about 90°C, about 45°C to about 90°C, about 50°C to about 90°C, about 55°C to about 90°C, about 10°C to about 80°C, about 15°C to about 80°C, about 20°C to about 80°C, about 25°C to about 90°C, ℃, about 30 ℃ to about 80 ℃, about 35 ℃ to about 80 ℃, about 40 ℃ to about 80 ℃, about 45 ℃ to about 80 ℃, about 50 ℃ to about 80 ℃, about 55 ℃ to about 80 ℃, about 10 ℃ to about 70 ℃, about 15 ℃ to about 70 ℃, about 20 ℃ to about 70 ℃, about 25 ℃ to about 70 ℃, about 30 ℃ to about 70 ℃, about 35 ℃ to about 70 ℃, about 40 ℃ to about 70 ℃, about 45 ℃ to about 70 ℃, about 50 ℃ to about 70 ℃, about 55 ℃ to about 70 ℃, about 10 ℃ to about 60 ℃, about 15 ℃ to about 60 ℃, about 20 ℃ to about 60 ℃, about 25 ℃ to about 60 ℃, about 30 ℃ to about 60 ℃, about 35 ℃ to about 60 ℃, about 40 ℃ to about 60 ℃, about 45 ℃ to about 60 ℃, about 50 ℃ to about 60 ℃, or about 55 ℃ to about 60 ℃.

[0059] In other embodiments, the two or more buffers or excipients are detected using a charged aerosol detector (CAD).CAD uses a high voltage corona needle to charge nitrogen gas, which collides with analyte particles to produce charged particles.

[0060] In some embodiments, the frequency of CAD is set to about 1 Hz to about 10 Hz. For example, in some embodiments, the frequency of CAD is set to about 2 Hz to about 10 Hz, about 3 Hz to about 10 Hz, about 4 Hz to about 10 Hz, about 5 Hz to about 10 Hz, about 6 Hz to about 10 Hz, about 7 Hz to about 10 Hz, about 8 Hz to about 10 Hz, about 8 Hz to about 10 Hz, about 1 Hz to about 9 Hz, about 2 Hz to about 9 Hz, about 3 Hz to about 9 Hz, about 4 Hz to about 9 Hz, about 5 Hz to about 9 Hz, about 6 Hz to about 9 Hz, about 7 Hz to about 9 Hz, about 8 Hz to about 10 Hz, about 9 Hz, about 1 Hz to about 8 Hz, about 2 Hz to about 8 Hz, about 3 Hz to about 8 Hz, about 4 Hz to about 8 Hz, about 5 Hz to about 8 Hz, about 6 Hz to about 8 Hz, about 7 Hz to about 8 Hz, about 1 Hz to about 7 Hz, about 2 Hz to about 7 Hz, about 3 Hz to about 7 Hz, about 4 Hz to about 7 Hz, about 5 Hz to about 7 Hz, about 6 Hz to about 7 Hz, about 1 Hz to about 6 Hz, about 2 Hz to about 6 Hz, about 3 Hz to about 6 Hz, about 4 Hz to about 6 Hz, or about 5 Hz to about 6 Hz.

[0061] In some embodiments, the filter setting of CAD is about 1 second to about 10 seconds. For example, in some embodiments, the filter setting of CAD is about 2 seconds to about 10 seconds, about 3 seconds to about 10 seconds, about 4 seconds to about 10 seconds, about 5 seconds to about 10 seconds, about 6 seconds to about 10 seconds, about 7 seconds to about 10 seconds, about 8 seconds to about 10 seconds, about 8 seconds to about 10 seconds, about 1 second to about 9 seconds, about 2 seconds to about 9 seconds, about 3 seconds to about 9 seconds, about 4 seconds to about 9 seconds, about 5 seconds to about 9 seconds, about 6 seconds to about 9 seconds, about 7 seconds to about 9 seconds, about From about 8 seconds to about 9 seconds, from about 1 second to about 8 seconds, from about 2 seconds to about 8 seconds, from about 3 seconds to about 8 seconds, from about 4 seconds to about 8 seconds, from about 5 seconds to about 8 seconds, from about 6 seconds to about 8 seconds, from about 7 seconds to about 8 seconds, from about 1 second to about 7 seconds, from about 2 seconds to about 7 seconds, from about 3 seconds to about 7 seconds, from about 4 seconds to about 7 seconds, from about 5 seconds to about 7 seconds, from about 6 seconds to about 7 seconds, from about 1 second to about 6 seconds, from about 2 seconds to about 6 seconds, from about 3 seconds to about 6 seconds, from about 4 seconds to about 6 seconds, or from about 5 seconds to about 6 seconds.

[0062] In some embodiments, the power function of CAD is set to about 1.0 to about 2.0. For example, in some embodiments, the power function of CAD is set to about 1.1 to about 2.0, about 1.2 to about 2.0, about 1.3 to about 2.0, about 1.4 to about 2.0, about 1.5 to about 2.0, about 1.6 to about 2.0, about 1.0 to about 1.9, about 1.1 to about 1.9, about 1.2 to about 1.9, about 1.3 to about 1.9, about 1.4 to about 1.9, about 1.5 to about 1.9, about 1.6 to about 1.9, about 1.1 to about 1.8, about 1.2 to about 1.8, about 1.3 to about 1.8, about 1.4 to about 1.8, about 1.5 to about 1.8, or about 1.6 to about 1.8. In certain embodiments, the power function of CAD is set to about 1.78. In certain embodiments, the power function of CAD is set to about 1.68. In some embodiments, the power function of CAD is set so that the power function in the first two thirds of the HPLC run is different from the power function in the last one third of the HPLC run. For example, in some embodiments, for the first two thirds of the HPLC run, the power function of CAD is set to 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79 or 1.80, and for the last one third of the HPLC run, the power function is set to 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69 or 1.70. In certain embodiments, for the first two thirds of the HPLC run, the power function of CAD is set to 1.78, and for the last one third of the HPLC run, the power function is set to 1.68.

[0063] In yet other embodiments, the two or more buffers or excipients are detected using a condensation nucleation light scattering detector (CNLSD). CNLSD utilizes water condensation to increase the particle size of the analyte, and then places the particles in a laser beam to measure the reflected light scattered to a photomultiplier tube.

[0064] In some embodiments, the evaporation temperature of the CNLSD is set at about 10°C to about 100°C. For example, in some embodiments, the evaporation temperature of the CNLSD is set to about 20°C to about 100°C, about 30°C to about 100°C, about 40°C to about 100°C, about 50°C to about 100°C, about 10°C to about 90°C, about 20°C to about 90°C, about 30°C to about 90°C, about 40°C to about 90°C, about 50°C to about 90°C, about 10°C to about 80°C, about 20°C to about 80°C, about 30°C to about 80°C, about 40°C to about 80°C, about 50°C to about 80°C, about 10°C to about 70°C, about 20°C to about 70°C, about 30°C to about 70°C, about 40°C to about 70°C, about 50°C to about 70°C, about 10°C to about 60°C, about 20°C to about 60°C, about 30°C to about 60°C, about 40°C to about 60°C, or about 50°C to about 60°C. In certain embodiments, the evaporation temperature of the CNLSD is set to about 50°C.

[0065] In some embodiments, the pressure of the CNLSD is set at about 10 psi to about 100 psi. For example, in some embodiments, the pressure of the CNLSD is set to about 20psi to about 100psi, about 30psi to about 100psi, about 40psi to about 100psi, about 50psi to about 100psi, about 10psi pressure about 90psi, about 20psi to about 90psi, about 30psi to about 90psi, about 40psi to about 90psi, about 50psi to about 90psi, about 10psi to about 80psi, about 20psi to about 80psi, about 30psi to about 80psi, about 40psi to about 80psi, about 50psi to about 80psi, about 10psi to about 70psi, about 20psi to about 70psi, about 30psi to about 70psi, about 40psi to about 70psi, about 50psi to about 70psi, about 10psi to about 60psi, about 20psi to about 60psi, about 30psi to about 60psi, about 40psi to about 60psi, about 50psi to about 60psi. In certain embodiments, the pressure of the CNLSD is set to about 50psi.

[0066] In certain embodiments, the gain of the CNLSD is set to 0.1 to 5. For example, in some embodiments, the gain of the CNLSD is set to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0067] In some embodiments, the filter setting of CNLSD is 0.1-1.0. For example, in some embodiments, the filter setting of CNLSD is 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.0.

[0068] In some embodiments, the analytical method of the present invention uses a mobile phase selected from 100% H2O, H2O containing 0.5% formic acid, H2O containing 0.05% trifluoroacetic acid, and 100% acetonitrile to quantify two or more buffers or excipients in a test sample by HPLC. In some embodiments, the HPLC method uses more than one mobile phase: for example, 1, 2, 3, 4, 5, 7, 8, 9 or 10 mobile phases. In certain embodiments, the HPLC method uses two mobile phases: mobile phase A and mobile phase B. In certain embodiments, mobile phase A is selected from 100% H2O, H2O containing formic acid, H2O containing trifluoroacetic acid, or a combination thereof, and mobile phase B comprises acetonitrile. In some embodiments, the formic acid concentration is about 0.5% to about 5%, such as about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, or about 5%. In some embodiments, the trifluoroacetic acid concentration is about 0.05% to about 0.5%, such as about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, or about 0.5%.

[0069] In some embodiments, the test sample is injected into the HPLC column at a volume of about 1 μl to about 50 μl. For example, in some embodiments, the HPLC injection volume is about 1 μl to about 45 μl, about 1 μl to about 40 μl, about 1 μl to about 35 μl, about 1 μl to about 30 μl, about 1 μl to about 25 μl, about 1 μl to about 20 μl, about 1 μl to about 15 μl, about 1 μl to about 10 μl, about 5 μl to about 50 μl, about 5 μl to about 45 μl, about 5 μl to about 40 μl, about 5 μl to about 35 μl, about 5 ... 0 μl, about 5 μl to about 25 μl, about 5 μl to about 20 μl, about 5 μl to about 15 μl, about 5 μl to about 10 μl, about 10 μl to about 50 μl, about 10 μl to about 45 μl, about 10 μl to about 40 μl, about 10 μl to about 35 μl, about 10 μl to about 30 μl, about 10 μl to about 25 μl, about 10 μl to about 20 μl, about 10 μl to about 15 μl, about 15 μl to about 50 μl, about 15 μl to about l to about 45μl, about 15μl to about 40μl, about 15μl to about 35μl, about 15μl to about 30μl, about 15μl to about 25μl, about 15μl to about 20μl, about 20μl to about 50μl, about 20μl to about 45μl, about 20μl to about 40μl, about 20μl to about 35μl, about 20μl to about 30μl, about 20μl to about 25μl, about 25μl to about 50μl, about 25μl to about 4 In some embodiments, the injection volume is 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl or 10 μl.

[0070] In some embodiments, the HPLC analysis comprises one or more than one step. For example, in some embodiments, the HPLC analysis comprises 1, 2, 3, 4, 5, 6, 8, 9, or 10 steps. In some embodiments, each step lasts from about 1.0 minute to about 10 minutes.For example, in some embodiments, each step lasts from about 1.5 minutes to about 10 minutes, from about 2 minutes to about 10 minutes, from about 2.5 minutes to about 10 minutes, from about 3 minutes to about 10 minutes, from about 3.5 minutes to about 10 minutes, from about 4 minutes to about 10 minutes, from about 4.5 minutes to about 10 minutes, from about 5 minutes to about 10 minutes, from about 5.5 minutes to about 10 minutes, from about 6 minutes to about 10 minutes, from about 6.5 minutes to about 10 minutes, from about 7 minutes to about 10 minutes, from about 7.5 minutes to about 10 minutes, from about 8 minutes to about 10 minutes, from about 8.5 minutes to about 10 minutes, from about 9 minutes to about 10 minutes, from about 9.5 minutes to about 10 minutes, from about 1.0 minute to about 9 minutes, From about 1.5 minutes to about 9 minutes, from about 2 minutes to about 9 minutes, from about 2.5 minutes to about 9 minutes, from about 3 minutes to about 9 minutes, from about 3.5 minutes to about 9 minutes, from about 4 minutes to about 9 minutes, from about 4.5 minutes to about 9 minutes, from about 5 minutes to about 9 minutes, from about 5.5 minutes to about 9 minutes, from about 6 minutes to about 9 minutes, from about 6.5 minutes to about 9 minutes, from about 7 minutes to about 9 minutes, from about 7.5 minutes to about 9 minutes, from about 8 minutes to about 9 minutes, from about 8.5 minutes to about 9 minutes, from about 1.0 minute to about 8 minutes, from about 1.5 minutes to about 8 minutes, from about 2 minutes to about 8 minutes, from about 2.5 minutes to about 8 minutes, from about 3 minutes to about 8 minutes, from about 3.5 minutes to about 8 minutes, from about 4 minutes to about 8 minutes, about 4.5 minutes to about 8 minutes, about 5 minutes to about 8 minutes, about 5.5 minutes to about 8 minutes, about 6 minutes to about 8 minutes, about 6.5 minutes to about 8 minutes, about 7 minutes to about 8 minutes, about 7.5 minutes to about 8 minutes, about 1.0 minute to about 7 minutes, about 1.5 minutes to about 7 minutes, about 2 minutes to about 7 minutes, about 2.5 minutes to about 7 minutes, about 3 minutes to about 7 minutes, about 3.5 minutes to about 7 minutes, about 4 minutes to about 7 minutes, about 4.5 minutes to about 7 minutes, about 5 minutes to about 7 minutes, about 5.5 minutes to about 7 minutes, about 6 minutes to about 7 minutes, about 6.5 minutes to about 7 minutes, about 1.0 minute to about 6 minutes, about 1 .5 minutes to about 6 minutes, about 2 minutes to about 6 minutes, about 2.5 minutes to about 6 minutes, about 3 minutes to about 6 minutes, about 3.5 minutes to about 6 minutes, about 4 minutes to about 6 minutes, about 4.5 minutes to about 6 minutes, about 5 minutes to about 6 minutes, about 5.5 minutes to about 6 minutes, about 1.0 minute to about 4 minutes, about 1.5 minutes to about 4 minutes, about 2 minutes to about 4 minutes, about 2.5 minutes to about 4 minutes, about 3 minutes to about 4 minutes, about 3.5 minutes to about 4 minutes, about 1.0 minute to about 3 minutes, about 1.5 minutes to about 3 minutes, about 2 minutes to about 3 minutes, about 2.5 minutes to about 3 minutes, about 1.0 minute to about 2 minutes or about 1.5 minutes to about 2 minutes.

[0071] In some embodiments, the flow rate for each step of the HPLC analysis is about 0.1 ml / min to about 5.0 ml / min. For example, in some embodiments, the flow rate for each step is about 0.25 ml / min to about 5.0 ml / min, 0.5 ml / min to about 5.0 ml / min, 0.75 ml / min to about 5.0 ml / min, about 1.0 ml / min to about 5.0 ml / min, about 1.25 ml / min to about 5.0 ml / min, about 1.5 ml / min to about 5.0 ml / min, about 2.0 ml / min to about 5.0 ml / min, about 2.5 ml / min to about 5.0 ml / min, about 3.0 ml / min to about 5.0 ml / min, about 3.5 ml / min to about 5.0 ml / min, about 4.0 ml / min to about 5.0 ml / min, about 5.0 ml / min to about 5.0 ml / min, about 6.0 ml / min to about 5.0 ml / min, about 7.0 ml / min to about 5.0 ml / min, about 8.0 ml / min to about 5.0 ml / min, about 9.0 ml / min to about 5.0 ml / min, about 10.0 ml / min to about 15.0 ml / min, about 11.0 ml / min to about 15.0 ml / min, about 12.0 ml / min to about 15.0 ml / min, about 13.0 ml / min to about 15.0 ml / min, about 14.0 ml / min to about 15.0 ml / min, about 15.0 ml / min to about 15.0 ml / min, about 16.0 ml / min to about 16.0 ml / min, about 17.0 ml / min to about 17.0 ml / min, about 18.0 ml / min to about 18 l / min to about 5.0ml / min, about 4.0ml / min to about 5.0ml / min, about 4.5ml / min to about 5.0ml / min, about 0.1ml / min to about 4.0ml / min, about 0.25ml / min to about 4.0ml / min, 0.5ml / min to about 4.0ml / min, 0.75ml / min to about 4.0ml / min, about 1.0ml / min to about 4.0ml / min, about 1.25ml / min to about 4.0ml / min, about 1.5ml / min to about 4.0ml / min, about 2.0ml / min to about 4.0ml / minute, about 2.5ml / minute to about 4.0ml / minute, about 3.0ml / minute to about 4.0ml / minute, about 3.5ml / minute to about 4.0ml / minute, about 0.1ml / minute to about 3.0ml / minute, about 0.25ml / minute to about 3.0ml / minute, 0.5ml / minute to about 3.0ml / minute, 0.75ml / minute to about 3.0ml / minute, about 1.0ml / minute to about 3.0ml / minute, about 1.25ml / minute to about 3.0ml / minute, about 1.5ml / minute to about 3.0ml / minute, about 2.0 ml / minute to about 3.0 ml / minute, about 2.5 ml / minute to about 3.0 ml / minute, about 0.1 ml / minute to about 2.0 ml / minute, about 0.25 ml / minute to about 2.0 ml / minute, 0.5 ml / minute to about 2.0 ml / minute, 0.75 ml / minute to about 2.0 ml / minute, about 1.0 ml / minute to about 2.0 ml / minute, about 1.25 ml / minute to about 2.0 ml / minute, about 1.5 ml / minute to about 2.0 ml / minute or about 0.5 ml / minute to about 1.0 ml / minute.

[0072] In some embodiments, each step of the HPLC analysis includes: an equilibrium step; a gradient change step, in which the relative percentages of two or more different mobile phases are adjusted; a holding step, in which the relative percentages of two or more different mobile phases are kept constant; or a reequilibrium step.

[0073] In an embodiment in which the HPLC analysis includes the use of two mobile phases, each step may include relative percentages of 100% mobile phase A, 0% mobile phase B; 90% mobile phase A, 10% mobile phase B; 80% mobile phase A, 20% mobile phase B; 70% mobile phase A, 30% mobile phase B; 60% mobile phase A, 40% mobile phase B; 50% mobile phase A, 50% mobile phase B; 40% mobile phase A, 60% mobile phase B; 30% mobile phase A, 70% mobile phase B; 20% mobile phase A, 80% mobile phase B; 10% mobile phase A, 90% mobile phase B; or 0% mobile phase A, 100% mobile phase B.

[0074] Example

[0075] The present disclosure, now generally described, will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the disclosure and are not intended to limit the scope of the disclosure in any way.

[0076] Example 1: C18 column and anion exchange column cannot separate excipients and / or buffers in a single sample

[0077] Standard HPLC-based methods as known to those skilled in the art are not able to resolve the constituent excipients and / or buffers in samples comprising histidine, sucrose and mannitol, or tris, sucrose and hpβCD.

[0078] C18 column HPLC and detector conditions

[0079] C18 column HPLC conditions are summarized in Table 1 below. Briefly, the sample was loaded onto an Agilent Zorbax EclipsePlus C18, 5 μm, 4.6×150 mm column. The column was balanced at 90% mobile phase A, 10% mobile phase B. At 6.0 min, the mobile phase was changed to 60% mobile phase A, 40% mobile phase B over 1 minute. From 7.0 min to 9.0 min, the column was maintained at 60% mobile phase A, 40% mobile phase B. At 9.0 min, the mobile phase was changed back to 90% mobile phase A, 10% mobile phase B over 1 minute, and maintained at 90% mobile phase A, 10% mobile phase B for 2 minutes to rebalance the column. The flow rate was always maintained at 1.0 ml / min. The total cycle time for each run was 12 minutes.

[0080] Light scattering signals were acquired from the target excipients using an Agilent 1200 series evaporative light scattering detector (ELSD). The ELSD parameters are also summarized in Table 1 below. Briefly, the evaporation temperature was set to 50°C and the pressure was set to 50 psi. The gain and filter were set to 1 and 0.5, respectively.

[0081] Table 1

[0082]

[0083] As shown in Figure 1, the C18 column was unable to separate the excipient and buffer peaks in the sample containing histidine, sucrose, and mannitol ( Figure 1A ), nor could they separate samples containing tris, sucrose, and hpβCD ( Figure 1B ).

[0084] Anion exchange column HPLC and detector conditions

[0085] Anion exchange column HPLC conditions are summarized in Table 2 below. Briefly, the sample was loaded onto a Water Oasis MAX 2.1×20 mm, 30 μm column. The column was equilibrated in 90% mobile phase A, 10% mobile phase B for 1 minute. From 1.0 minutes to 3.4 minutes, the gradient was changed to 80% mobile phase A, 20% mobile phase B. From 3.4 minutes to 3.5 minutes, the gradient was changed to 100% mobile phase B and maintained for 1 minute. At 4.5 minutes, the gradient was changed back to 90% mobile phase A, 10% mobile phase B. At 4.6 minutes, the gradient was maintained at 90% mobile phase A, 10% mobile phase B for 2 minutes. The flow rate was always maintained at 1.0 ml / min. The total cycle time for each run was 6.6 minutes.

[0086] Light scattering signals were acquired from the target excipients using an Agilent 1200 series evaporative light scattering detector (ELSD). The ELSD parameters are also summarized in Table 2 below. Briefly, the evaporation temperature was set to 85°C and the pressure was set to 3.5 bar ± 0.1. The gain and filter settings were variable, with nominal values ​​of 4 and 4 seconds, respectively.

[0087] Table 2

[0088]

[0089]

[0090] As shown in Figure 2, the anion exchange column was unable to separate the excipient and buffer peaks in the sample containing histidine, sucrose, and mannitol ( Figure 2A ), nor could they separate samples containing tris, sucrose, and hpβCD ( Figure 2B ).

[0091] Example 2: HPLC quantification of hpβCD and sucrose using a PFP column

[0092] A HPLC-based method was developed to separate and quantify hpβCD and sucrose in test samples in a single assay.

[0093] HPLC and detector conditions

[0094] The HPLC conditions are summarized in Table 3 below. Briefly, a Kinetex pentafluorophenyl (PFP) 2.6 μm 150×4.6 mm column was used to separate the target excipients. The column was equilibrated in 100% mobile phase A. At 3.5 min, the mobile phase gradient was changed to 60% mobile phase A, 40% mobile phase B. From 3.5 to 7.5 min, the gradient was gradually changed to 40% mobile phase A, 60% mobile phase B, and then maintained at this gradient for 2.5 minutes. At 12 min, the mobile phase gradient was changed back to 100% mobile phase A. During the excipient separation, the flow rate was started at 0.25 ml / min and then gradually increased to 1.0 ml / min to achieve good separation between the buffer substance and sucrose. Sucrose and hpβCD were eluted between 4 and 10 minutes. At the end of the run, the flow rate was reduced to 0.25 mg / mL and the column was re-equilibrated with a mobile phase containing 100% water. The total cycle time for each run was 15 minutes.

[0095] Light scattering signals were acquired from the target excipients using an Agilent 1200 series evaporative light scattering detector (ELSD). The ELSD parameters are also summarized in Table 3 below. Briefly, the evaporation temperature was set to 50°C and the pressure was set to 50 psi. The gain and filter were set to 1 and 0.5, respectively.

[0096] Table 3

[0097]

[0098] Calibration curve preparation and test sample preparation

[0099] To quantify the excipients, a calibration curve of known concentrations of the excipients was prepared according to Table 4 below. The calibration curve range was determined based on the detector capability.

[0100] Table 4

[0101]

[0102]

[0103] If the expected concentration is within the calibration curve concentration range, the test sample is injected into the HPLC column as a neat sample. If the test sample is expected to contain analytes at a concentration outside the calibration curve range, the test sample is diluted with H2O so that the concentration is within the standard curve.

[0104] HPLC data analysis

[0105] The peak of each analyte was integrated to obtain the peak area. A linear regression fit was performed on the peak area and concentration to calculate the slope (m) and intercept (b) of the calibration curve. The concentration of each analyte in the unknown sample was calculated using the following equation using the calibration curve.

[0106]

[0107] in,

[0108] c = concentration of excipient in unknown sample

[0109] Peak Area = Peak area measured by integrating the excipient peak in the unknown sample chromatogram

[0110] b = y-intercept of the equation calculated using the calibration curve

[0111] m = slope of the equation calculated using the calibration curve

[0112] like Figure 3 As shown, HPLC runs using a PFP column under the conditions described in Table 3 resolved the sucrose and hpβCD present in the individual samples as defined peaks. Figure 4 As shown, an HPLC run performed under the same conditions using a PFP column clearly resolved Tris.

[0113] Example 3: Quantification of sucrose, hpβCD and other excipients

[0114] Based on the work described in Example 2, a general HPLC method was developed to separate and quantify excipients using similar HPLC conditions but using a charged aerosol detector (CAD). This method can be used to quantify hpβCD and sucrose as well as several other excipients.

[0115] HPLC and detector conditions

[0116] The HPLC conditions are summarized in Table 5 below. Briefly, a Kinetex pentafluorophenyl (PFP) 2.6 μm 150×4.6 mm column was used to separate the target excipients. The column was equilibrated in 100% mobile phase A, which consisted of water containing 0.5% formic acid (FA). FA is used to improve the peak shape of buffer substances (e.g., Tris buffer). From 4 to 6 min, the gradient was gradually changed to 40% mobile phase A, 60% mobile phase B. Then, from 6 to 8 min, the gradient was gradually returned to 100% mobile phase A. This gradient was maintained for another 4 minutes to allow the column to equilibrate. During the run, the flow rate was maintained at 0.5 ml / min. Sucrose eluted at around 3 minutes, hpβCD eluted at around 9.5 minutes, and Tris buffer eluted at around 2.8 minutes. The total cycle time for each run was 12 minutes.

[0117] Using the ThermoFisher Corona TM Veo TM RS was charged with an aerosol detector (CAD) to obtain signals from the target excipient. The CAD parameters are also summarized in Table 5 below. In brief, the evaporation temperature was set to 35°C, the frequency was set to 5 Hz, and the filter was set to 5 seconds. The power function was set to 1.78 for the first 8 minutes and 1.68 for the last 4 minutes. In the first 1.5 minutes, the stream was diverted directly to waste.

[0118] Table 5

[0119]

[0120]

[0121] Calibration curve preparation and test sample preparation

[0122] To quantify the excipients, a calibration curve of excipients of known concentrations was prepared according to Table 6 below. The calibration curve range is determined based on the detector capability. Compared with the ELSD detector in Example 2, the CAD detector is able to establish a linear curve over a wider range.

[0123] Table 6

[0124]

[0125]

[0126] If the expected concentration is within the calibration curve concentration range, the test sample is injected into the HPLC column as a neat sample. If the sample is expected to contain analytes at a concentration outside the calibration curve range, the test sample is diluted with H2O so that the concentration is within the standard curve.

[0127] Data analysis

[0128] Data analysis was performed as described in Example 1.

[0129] As shown in FIG5 , an HPLC run using a PFP column under the conditions described in Table 5 was able to separate the sodium phosphate ( Figure 5A )、Sodium citrate( Figure 5B )、Potassium phosphate( Figure 5C ), histidine ( Figure 5D ) and trehalose ( Figure 5E ) were resolved into definite peaks.

[0130] Example 4: Sucrose and mannitol quantification

[0131] Based on the work described in Example 2, a HPLC-based method was developed to separate and quantify the contents of histidine, sucrose, and mannitol.

[0132] HPLC conditions are summarized in Table 6 below. Briefly, a Kinetex pentafluorophenyl (PFP) 2.6 μm 150×4.6 mm column was used to separate the target excipients. The column was equilibrated in 100% mobile phase A, which consisted of water containing 0.05% trifluoroacetic acid (TFA). TFA was required to improve the separation of mannitol, sucrose, and histidine. From 4 to 6 min, the gradient was gradually changed to 40% mobile phase A, 60% mobile phase B. Then, from 6 to 8 min, the gradient was gradually returned to 100% mobile phase A. This gradient was maintained for another 4 minutes to allow the column to equilibrate. During the run, the flow rate was maintained at 0.5 ml / min. Sucrose and mannitol eluted between 2.5 and 3.5 minutes, and histidine eluted between 3.5 and 4.5 minutes. The total cycle time for each run was 12 minutes.

[0133] Using the ThermoFisher Corona TM Veo TM RS CAD was used to acquire signals from the target excipient. CAD parameters were as described in Example 2.

[0134] Table 6

[0135]

[0136] Calibration curve preparation and test sample preparation

[0137] To quantify the excipients, a calibration curve of known excipient concentrations was prepared according to Table 7 below. The calibration curve range was determined based on the detector capability.

[0138] Table 7

[0139]

[0140]

[0141] If the expected concentration is within the calibration curve concentration range, the test sample is injected into the HPLC column as a neat sample. If the sample is expected to contain analytes at a concentration outside the calibration curve range, the test sample is diluted with H2O so that the concentration is within the standard curve.

[0142] Data analysis

[0143] Data analysis was performed as described in Example 1.

[0144] like Figure 6 As shown, the HPLC run using the PFP column under the conditions described in Table 6 was able to resolve mannitol, sucrose, and histidine present in a single sample as defined peaks.

Claims

1. A method for analytically separating two or more buffers or excipients in a single assay, wherein the two or more buffers or excipients are sugars or sugar-based molecules, the method comprising: Chromatographically analyzing a test sample, i.e., a sample comprising the two or more buffers or excipients, on a pentafluorophenyl (PFP) high performance liquid chromatography (HPLC) column to separate the two or more buffers or excipients; detecting the separated two or more buffers or excipients in the HPLC column effluent; and A chromatogram is generated having peaks corresponding to the separated two or more buffers or excipients.

2. The method of claim 1, wherein the two or more buffers or excipients are sugars.

3. The method of claim 1, wherein the two or more buffers or excipients are selected from the group consisting of 2-hydroxypropyl-β-cyclodextrin, sucrose, trehalose and mannitol.

4. The method according to any one of claims 1 to 3, further comprising: obtaining standard calibration chromatographic data for the two or more excipients run on the same HPLC column; as well as The concentration or amount of the two or more buffers or excipients in the test sample is calculated by determining the integrated peak areas of the two or more buffers or excipients from the chromatogram and converting the integrated peak areas into concentrations or amounts based on the obtained standard calibration chromatogram data.

5. The method of claim 4, wherein the transforming comprises a linear regression fit to the standard calibration chromatographic data.

6. The method of any one of claims 1 to 3, wherein the two or more buffers or excipients are detected using an evaporative light scattering detector (ELSD).

7. The method of claim 6, wherein the ELSD has an evaporation temperature set to 40 to 70°C, a pressure set to 30 to 70 psi, a gain set to 0.5 to 2, and a filter set to 0.5 to 1.

8. The method of any one of claims 1 to 3, wherein the two or more buffers or excipients are detected using a charged aerosol detector (CAD).

9. The method of claim 8, wherein the CAD has an evaporation temperature set to 25 to 35°C, a frequency set to 4 to 6 Hz, a filter set to 4 to 6 seconds, a power function set to 1.78 for the first two thirds of the HPLC run, and a power function set to 1.68 for the last one third of the HPLC run.

10. The method according to any one of claims 1 to 3, wherein the HPLC is run using mobile phase A and mobile phase B.

11. The method according to claim 10, wherein mobile phase A is selected from the group consisting of 100% H2O, H2O containing formic acid, and H2O containing trifluoroacetic acid.

12. The method of claim 11, wherein mobile phase A is selected from the group consisting of H2O containing 0.5% formic acid and H2O containing 0.05% trifluoroacetic acid.

13. The method of claim 10, wherein mobile phase B comprises acetonitrile.

14. The method of claim 13, wherein mobile phase B is 100% acetonitrile.

15. The method of claim 10, wherein performing chromatography comprises an equilibration step of flowing 100% mobile phase A through the HPLC column at a rate of 0.1 ml / min to 1.0 ml / min.

16. The method of claim 15, wherein the flow rate of the equilibration step is 0.25 ml / min.

17. The method of claim 15, wherein the flow rate of the equilibration step is 0.5 ml / min.

18. The method of claim 15, wherein the equilibration step is between 0.5 minutes and 10 minutes.

19. The method of claim 18, wherein the equilibration step is 3.0 minutes.

20. The method of claim 18, wherein the equilibration step is 4.0 minutes.

21. The method of claim 10, wherein performing chromatography comprises a gradient step of flowing 60% mobile phase A and 40% mobile phase B through the HPLC column.

22. The method of claim 10, wherein performing chromatography comprises a gradient step of flowing 40% mobile phase A and 60% mobile phase B through the HPLC column.

23. The method of claim 10, wherein performing chromatography comprises a gradient step of flowing 100% mobile phase A through the HPLC column.

24. The method of claim 21, wherein the flow rate of the gradient change step is between 0.1 ml / min and 1.0 ml / min.

25. The method of claim 24, wherein the flow rate of the gradient change step is 0.25 ml / min.

26. The method of claim 24, wherein the flow rate of the gradient change step is 0.5 ml / min.

27. The method of claim 21, wherein the gradient step is between 0.5 minutes and 10 minutes.

28. The method of claim 27, wherein the gradient change step is 0.5 minutes.

29. The method of claim 27, wherein the gradient change step is 2.0 minutes.

30. The method of claim 27, wherein the gradient step is 4.0 minutes.

31. The method of claim 10, wherein performing chromatography comprises a maintenance step of flowing 40% mobile phase A and 60% mobile phase B through the HPLC column.

32. The method of claim 10, wherein performing chromatography comprises a maintenance step of flowing 100% mobile phase A through the HPLC column.

33. The method of claim 31 , wherein the flow rate of the maintaining step is between 0.1 ml / min and 1.0 ml / min.

34. The method of claim 33, wherein the flow rate of the maintaining step is 0.5 ml / min.

35. The method of claim 33, wherein the flow rate of the maintaining step is 1.0 ml / min.

36. The method of claim 31 , wherein the maintaining step is between 0.5 minutes and 10 minutes.

37. The method of claim 36, wherein the maintaining step is 2.5 minutes.

38. The method of claim 36, wherein the maintaining step is 4.0 minutes.

39. The method of claim 10, wherein performing chromatography comprises a re-equilibration step of flowing 100% mobile phase A through the HPLC column at a rate of 0.1 ml / min to 1.0 ml / min.

40. The method of claim 39, wherein the flow rate of the re-equilibration step is 0.25 ml / min.

41. The method of claim 39, wherein the re-equilibration step is between 0.5 minutes and 10 minutes.

42. The method of claim 41, wherein the re-equilibration step is 3.0 minutes.

43. The method of claim 10, wherein performing a chromatographic analysis comprises: (i) equilibrate with 100% mobile phase A at a flow rate of 0.25 ml / min for 3.0 minutes; (ii) gradient change to 60% mobile phase A and 40% mobile phase B at a flow rate of 0.25 ml / min over 0.5 minutes; (iii) gradient change to 40% mobile phase A and 60% mobile phase B at a flow rate of 1.0 ml / min over 4.0 minutes; (iv) maintain at 40% mobile phase A and 60% mobile phase B at a flow rate of 1.0 ml / min for 2.5 minutes; (v) gradient change to 100% mobile phase A at a flow rate of 1.0 ml / min over 2 minutes; and (vi) re-equilibrate with 100% mobile phase A at a flow rate of 0.25 ml / min for 3.0 minutes.

44. The method of claim 10, wherein performing chromatographic analysis comprises: (i) equilibrate with 100% mobile phase A at a flow rate of 0.5 ml / min for 4.0 min; (ii) gradient change to 40% mobile phase A and 60% mobile phase B over 2.0 min at a flow rate of 0.5 ml / min; (iii) gradient change to 100% mobile phase A over 2.0 min at a flow rate of 0.5 ml / min; and (iv) maintain at 100% mobile phase A for 4 min at a flow rate of 0.5 ml / min.

45. The method according to any one of claims 1 to 3, wherein 1 μl to 100 μl of the test sample is injected into the HPLC column.

46. ​​The method of claim 45, wherein 10 μl of the test sample is injected into the HPLC column.

47. The method of claim 45, wherein 4 μl of the test sample is injected into the HPLC column.

48. The method of any one of claims 1 to 3, wherein the pentafluorophenyl (PFP) high performance liquid chromatography (HPLC) column is a 2.6 μm 150×4.6 mm column.

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