Method of measuring polysorbate levels

Abstract

The present disclosure provides methods of measuring polysorbate levels in a sample. The method comprises applying an aliquot of the sample to a high performance liquid chromatography system having a mixed-mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying an evaporative light scattering detection on the eluent as it exits the column, thereby measuring the polysorbate level. The first mobile phase comprises 0-20% of an acid, 1-70% of acetonitrile, methanol, tetrahydrofuran, tetrahydrofuran, isopropanol or a mixture thereof, and water. The second mobile phase comprises 0-20% of an acid, 80-100% of acetonitrile, methanol, tetrahydrofuran, tetrahydrofuran, isopropanol or a mixture thereof, and optionally water.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 477,904, filed on December 30, 2022, the entire contents of which are incorporated herein by reference.

[0003] public

[0004] The present disclosure relates to methods of measuring polysorbate levels in a sample. In some embodiments, the method provides quantification of polysorbate levels in a sample.

[0005] Polysorbates are used as surfactants in pharmaceutical formulations, particularly in APIs and drug products. Polysorbates are used as surfactants to stabilize biopharmaceutical products. Polysorbates are commonly used to stabilize proteins, particularly antibodies.

[0006] Polysorbates are a class of molecules that include a variety of structures, including but not limited to polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Measuring the level of polysorbates can include measuring the level of one or more polysorbate structures.

[0007] The degradation of polysorbate is a known problem. Polysorbate can be degraded by hydrolysis or oxidation. The degradation of polysorbate reduces the amount of polysorbate in the sample, thereby reducing the ability of other components in the stable sample, such as therapeutic proteins. Therefore, it is necessary to measure the amount of polysorbate in the sample. Especially, measuring the polysorbate level in the bulk drug and the drug product is important for identifying the polysorbate degradation that may cause the instability of other molecules in the preparation.

[0008] Conventional techniques for measuring constituents in a sample do not directly or accurately measure polysorbate levels. Polysorbate molecules lack chemical structures such as strong chromophores, so previous attempts to directly detect polysorbate by UV-Vis or fluorescence have not been successful.

[0009] The method for existing measurement polysorbate level is destructive and / or indirect. A kind of known method for measuring polysorbate focuses on using ultra-high performance liquid chromatography-high resolution mass spectrometry to identify polysorbate subspecies. Another known method digests polysorbate molecule, then characterizes degradation product, usually by liquid chromatography-mass spectrometry, subsequently by liquid chromatography coupled with charged aerosol detection (charged aerosol detection) and with evaporative light scattering detection coupled liquid chromatography. In some embodiments, the degradation product characterized is free fatty acid. Therefore, most of known methods for measuring the polysorbate level in the sample are indirect, and need to destroy polysorbate to characterize gained degradation product or subspecies.

[0010] Furthermore, the previous method identified higher-than-expected levels of polysorbate in samples during robustness studies, including a 30% overestimation compared to expected results. Previous attempts to validate the gradient procedure did not address this issue.

[0011] Previous attempts to create a robust, simple, and cost-effective method for accurately measuring specific polysorbate levels have been unsuccessful. One previous attempt required solid phase extraction to remove the protein before further separation steps for accurate measurement, which is inefficient, time-consuming, and labor-intensive.

[0012] There is a need for an improved method to directly and accurately measure polysorbate levels in a sample.

[0013] The present disclosure addresses this need by providing a robust, simple, cost-effective method to directly and accurately measure polysorbate levels.

[0014] Overview

[0015] The present disclosure relates to methods of measuring polysorbate levels in a sample.The methods of the present disclosure directly and accurately measure polysorbate levels in a sample in a robust, simple and cost-effective manner.

[0016] In some embodiments, the present disclosure relates to a method for measuring the level of polysorbate in a sample. In some embodiments, the method comprises applying an aliquot of the sample to a high performance liquid chromatography system with a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after leaving the column, thereby measuring the level of polysorbate. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or mixtures thereof, and water. In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or mixtures thereof, and optionally water.

[0017] In some embodiments, the acid is a volatile acid.

[0018] In some embodiments, the volatile acid is formic acid.

[0019] In some embodiments, the first mobile phase and / or the second mobile phase comprises 0-2% acid.

[0020] In some embodiments, the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

[0021] In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

[0022] In some embodiments, the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0023] In some embodiments, the polysorbate measured comprises polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80.

[0024] In some embodiments, the polysorbate measured comprises polysorbate 20 and / or polysorbate 80.

[0025] In some embodiments, the polysorbate measured comprises polysorbate 20.

[0026] In some embodiments, the polysorbate measured comprises polysorbate 80.

[0027] In some embodiments, before applying the aliquot to be measured to the column, the method further comprises applying a blank sample to a high performance liquid chromatography system with a mixed mode column, eluting the blank sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the blank sample after it leaves the column. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and water. In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and optionally water.

[0028] In some embodiments, applying evaporative light scattering detection to the blank sample generates a baseline signal.

[0029] In some embodiments, after the first blank sample has been applied to the column and before the aliquot to be measured is applied to the column, the method further comprises applying a second blank sample to a high performance liquid chromatography system having a mixed mode column, eluting the blank sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the blank sample after it leaves the column, wherein applying evaporative light scattering detection to the second blank sample produces a stable baseline signal. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and water. In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and optionally water.

[0030] In some embodiments, the first mobile phase and / or the second mobile phase are prepared within 2 weeks of performing the method.

[0031] In some embodiments, the method further comprises quantifying the measured level of polysorbate in the sample.

[0032] In some embodiments, quantifying the measured level of polysorbate in the sample comprises comparing the measured amount of polysorbate to a calibration curve.

[0033] In some embodiments, comparing the measured amount of polysorbate to a calibration curve determines the amount of polysorbate in the aliquot.

[0034] In some embodiments, the calibration curve is generated as follows: one or more concentration standards containing known amounts of polysorbate are separately applied to a high performance liquid chromatography system with a mixed mode column, the concentration standards are eluted with a gradient from a first mobile phase to a second mobile phase, evaporative light scattering detection is applied to the concentration standards after each concentration standard leaves the column, thereby measuring the polysorbate level, and generating a calibration curve from the measured levels of polysorbate in the concentration standards. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and water. In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof, and optionally water.

[0035] In some embodiments, one or more of the concentration standards produces a larger signal than the aliquot, and one or more of the concentration standards produces a smaller signal than the aliquot.

[0036] In some embodiments, the amount of polysorbate in the one or more concentration standards does not exceed the maximum detectable signal of an evaporative light scattering assay.

[0037] In some embodiments, the amount of polysorbate in the concentration standard comprising the maximum amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0038] In some embodiments, applying evaporative light scattering detection to a blank sample produces a signal that is lower than the signal produced by a concentration standard comprising a minimal amount of polysorbate.

[0039] As used herein, a "blank sample" does not contain polysorbate. In some embodiments, the blank sample is water.

[0040] In some embodiments, the calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0041] In some embodiments, the calibration curve is prepared gravimetrically.

[0042] In some embodiments, the calibration curve has a coefficient of determination (R 2 ).

[0043] In some embodiments, the coefficient of determination (R 2 ) is greater than or equal to 0.998.

[0044] In some embodiments, the calibration curve is prepared using a quadratic fit through zero.

[0045] In some embodiments, the percent relative standard deviation of two or more aliquots of the sample is 10 or less.

[0046] In some embodiments, the gradient comprises 100% first mobile phase and 0% second mobile phase from 0.0 minutes to 2.0 minutes, and / or 85% first mobile phase and 15% second mobile phase from 2.1 minutes to 5.0 minutes, and / or 30% first mobile phase and 70% second mobile phase from 5.1 minutes to 7.6 minutes, and / or 0% first mobile phase and 100% second mobile phase from 7.7 minutes to 9.0 minutes, and / or 100% first mobile phase and 0% second mobile phase from 9.1 minutes to 10.0 minutes.

[0047] In some embodiments, the sample is applied to the mixed mode column at a temperature of 22-28°C.

[0048] In some embodiments, the eluent is eluted from the column at a flow rate of 0.9-1.1 mL / min.

[0049] In some embodiments, the evaporative light scattering detection utilizes an Alltech 3300 detector.

[0050] In some embodiments where the method employs an Alltech 3300 detector, the method is performed using a nebulizer temperature of the detector of 68-72°C.

[0051] In some embodiments where the method employs an Alltech 3300 detector, the method is performed using a gas flow rate to the detector of 2.3-2.7 L / min.

[0052] In some embodiments, the evaporative light scattering detection utilizes an Agilent 1260 evaporative light scattering detector.

[0053] In some embodiments where the method employs an Agilent 1260 Evaporative Light Scattering Detector, the method is performed using a nebulizer temperature of the detector of 43-47°C.

[0054] In some embodiments where the method employs an Agilent 1260 Evaporative Light Scattering Detector, the method is performed using a heated tube temperature of the detector of 70-90°C.

[0055] In some embodiments where the method employs an Agilent 1260 evaporative light scattering detector, the method is performed using a gas flow rate to the detector of 1.3-1.7 L / min.

[0056] In some embodiments, the sample is a drug substance.

[0057] In some embodiments, the sample is a pharmaceutical product.

[0058] In some embodiments, the method comprises: applying a first polysorbate concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a known amount of polysorbate different from the first concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a known amount of polysorbate different from the first concentration standard to the high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column, and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a known amount of polysorbate different from the first concentration standard to the high performance liquid chromatography system having a mixed mode column, The method further comprises: applying a first mobile phase to a high performance liquid chromatography system having a mixed mode column and eluting the sample with a gradient from a first mobile phase to a second mobile phase; applying a second mobile phase to the eluate after it leaves the column and measuring the polysorbate level; and quantifying the amount of polysorbate in the aliquot of the sample by comparison with the calibration curve. In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water, and the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0059] In some embodiments, the method is performed according to any of the features disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The drawings are for illustration purposes only and do not provide any limitation to the present disclosure.

[0062] Figure 1 Illustrated is an exemplary water blank sample chromatogram.

[0063] Figure 2 Illustrated is an example chromatogram of a local reference standard.

[0064] Figure 3 Example chromatograms of concentration standards are shown.

[0065] Figure 4a and Figure 4b Illustrated is an exemplary polysorbate 80 chromatogram. Figure 4a An exemplary chromatogram from a drug substance sample is provided. Figure 4b An exemplary chromatogram from a drug product sample is provided.

[0066] Figure 5a and Figure 5b JMP statistical output for Figure S2. Figure 5a The S2 distribution is depicted. Figure 5b A graph depicting the variability of S2 is shown. Figure 5c The response results of S2 are depicted.

[0067] Figure 6a 、 Figure 6b and Figure 6c JMP statistical output from Figure S3. Figure 6a Depicts the S3 distribution. Figure 6b A graph depicting the variability of S3 is shown. Figure 6c Depicts the response result of S3.

[0068] Figure 7a 、 Figure 7b and Figure 7c Plot JMP statistical output for D1. Figure 7a The D1 distribution is depicted. Figure 7b A variability chart of D1 is depicted. Figure 7c The response results of D1 are depicted.

[0069] Figure 8 The elution peaks for various samples containing polysorbate 80 and a test standard are illustrated.

[0070] Details

[0071] The present disclosure relates to methods of measuring polysorbate levels in a sample.

[0072] The methods of the present disclosure accurately measure polysorbate levels in a sample in a robust, simple, and cost-effective manner.

[0073] In some embodiments, the methods of the present disclosure are non-destructive.

[0074] In some embodiments, the methods of the present disclosure directly measure polysorbate levels.

[0075] High-performance liquid chromatography

[0076] The present disclosure relates to a method of measuring polysorbate levels in a sample.In some embodiments, an aliquot of the sample is applied to a high performance liquid chromatography (HPLC) system with a mixed mode column.

[0077] In some embodiments, the volume of the aliquot is 10 μL.

[0078] High performance liquid chromatography is a known technique for separating components in aliquots applied to a column. In some embodiments, the methods of the present disclosure utilize mixed mode columns. Mixed mode columns are known in the art. In some embodiments, the methods of the present disclosure utilize reversed phase columns. Reverse phase columns are known in the art. Typically, reversed phase columns comprise a non-polar stationary phase.

[0079] In some embodiments, the HPLC system is an Agilent 1200 / 1260 system. In some embodiments, the HPLC system is an Agilent 1260 Infinity II. Those skilled in the art will appreciate that other HPLC systems can be readily used with the methods of the present disclosure, and that these systems are exemplary only.

[0080] In some embodiments, the sample is applied to the mixed mode column at a temperature of 22-28°C.

[0081] In some embodiments, the methods of the present disclosure elute an aliquot of components from a mixed mode column using a gradient from a first mobile phase to a second mobile phase.

[0082] The present disclosure provides mobile phases that result in a robust, simple, and cost-effective method for measuring polysorbate levels.

[0083] In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent, and water.

[0084] In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or a mixture thereof, and water.

[0085] In some embodiments, the acid is a volatile acid. In some embodiments, the volatile acid is formic acid.

[0086] Polysorbate and other components of aliquots may bind to the post, which may cause carry-over problems. In some embodiments, carry-over may occur when components of a previous sample, blank sample, or concentration standard applied to the post bind to the post and interfere with or co-elute with subsequent samples, blank samples, or concentration standard applied to the post. In some embodiments, the presence of an acid (optionally an organic acid) in the first mobile phase and / or the second mobile phase solves the carry-over problem. In some embodiments, this results in a chromatogram with good / improved tailing.

[0087] In some embodiments, the first mobile phase comprises 0-2% acid.

[0088] In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

[0089] In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent, and optionally water.

[0090] In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or mixtures thereof, and optionally water.

[0091] In some embodiments, the acid is a volatile acid. In some embodiments, the volatile acid is formic acid.

[0092] In some embodiments, the second mobile phase comprises 0-2% acid.

[0093] In some embodiments, the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0094] In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water, and the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0095] In some embodiments, a different organic solvent is added to the first mobile phase compared to the second mobile phase. In some embodiments, the organic solvent is selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or a mixture thereof. In some embodiments, selecting two different organic solvents elutes more protein and / or matrix components compared to embodiments in which the same organic solvent is selected for the first and second mobile phases, thereby providing additional resolution strength.

[0096] As used herein, "matrix" refers to a solvent system or formulation system containing an active pharmaceutical ingredient, such as an antibody.

[0097] In some embodiments where a different organic solvent is provided in the first mobile phase compared to the second mobile phase, the method produces more accurate results according to the acceptance criteria in Table 3, thereby improving the robustness of the method.

[0098] In some embodiments, the second mobile phase comprises methanol, tetrahydrofuran, isopropanol, or a mixture thereof. In some embodiments, the second mobile phase does not comprise acetonitrile. In some embodiments, the second mobile phase produces a more efficient elution to obtain a more defined peak, thereby improving tailing and interference.

[0099] In some embodiments, the first mobile phase and / or the second mobile phase are prepared within 2 weeks of performing the method.

[0100] In some embodiments, preparing the first mobile phase and / or the second mobile phase within 2 weeks of performing the method improves the robustness and / or accuracy of the method.

[0101] In some embodiments, the gradient used to elute the polysorbate is 100% first mobile phase and 0% second mobile phase from 0.0 minutes to 2.0 minutes, and / or 85% first mobile phase and 15% second mobile phase from 2.1 minutes to 5.0 minutes, and / or 30% first mobile phase and 70% second mobile phase from 5.1 minutes to 7.6 minutes, and / or 0% first mobile phase and 100% second mobile phase from 7.7 minutes to 9.0 minutes, and / or 100% first mobile phase and 0% second mobile phase from 9.1 minutes to 10.0 minutes.

[0102] In some embodiments, the eluent is eluted from the column at a flow rate of 0.9-1.1 mL / min.

[0103] In some embodiments, the aliquot may contain other components besides polysorbate, including but not limited to proteins, polypeptides, peptides, polysorbate subspecies and / or polysorbate degradants. Typically, the gradient elutes the polysorbate at a different point than the other components of the aliquot.

[0104] Polysorbate subspecies are related molecules or structural variants of polysorbates. Exemplary subspecies include polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, and polyoxyethylene (19) isosorbide monolaurate.

[0105] Polysorbate degradation products are structurally altered polysorbate-related components. Exemplary degradation products include free fatty acids, oxidized polysorbate species, aldehydes, and short-chain ketones. In some cases, polysorbate degradation also increases the amount of polysorbate subspecies.

[0106] In some embodiments, the aliquot comprises more than one polysorbate.In some embodiments, the gradient elutes different polysorbates at different time points.

[0107] Evaporative light scattering detection

[0108] In some embodiments, the eluent is subjected to evaporative light scattering detection (ELSD). Evaporative light scattering detection is a technique known in the art. Briefly, the eluent is passed through a heating chamber to evaporate the mobile phase solvent. The non-volatile components of the aliquot form solid particles after the solvent evaporates. The solid particles scatter UV radiation to produce a detectable signal.

[0109] Evaporative light scattering detection can detect non-UV absorbing components in an aliquot.In some embodiments, the methods of the present disclosure apply evaporative light scattering detection to detect polysorbates.

[0110] Traditionally, evaporative light scattering detection is applied to detect the presence or absence of a component. The present disclosure provides methods for measuring polysorbate levels by evaporative light scattering detection.

[0111] In some embodiments, evaporative light scattering detection identifies one or more signals corresponding to one or more components in the eluate. In some embodiments, the one or more signals correspond to output from evaporative light scattering detection.

[0112] In some embodiments, the method generates a polysorbate signal from which the amount of polysorbate is measured.

[0113] In some embodiments where the aliquot contains more than one polysorbate structure, the method produces a signal for each different polysorbate structure, from which the amount of each different polysorbate is measured.

[0114] In some embodiments, gain is applied during evaporative light scattering detection.

[0115] In some embodiments, evaporative light scattering detection utilizes an Alltech 3300 evaporative light scattering detector. In some embodiments, when using the Alltech 3300 detector, the method is performed using a detector nebulizer temperature of 68-72°C. In some embodiments, when using the Alltech 3300 detector, the method is performed using a detector gas flow rate of 2.3-2.7 L / min. In some embodiments, when using the Alltech 3300 detector, the method produces a tailing result of 1.16 to 1.46.

[0116] In some embodiments, evaporative light scattering detection utilizes an Agilent 1260 evaporative light scattering detector. In some embodiments, when using the Agilent 1260 evaporative light scattering detector, the method is performed using a detector nebulizer temperature of 43-47° C. In some embodiments, when using the Agilent 1260 detector, the method is performed using a detector heater tube temperature of 70-90° C. In some embodiments, when using the Agilent 1260 evaporative light scattering detector, the method is performed using a detector gas flow rate of 1.3-1.7 L / min.

[0117] sample

[0118] The sample in which the polysorbate level is to be measured may be any sample. Typically, the sample comprises or is expected to comprise polysorbate.

[0119] In some embodiments, the sample comprises one or more polysorbates. In some embodiments, the one or more polysorbates can be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture thereof.

[0120] In some embodiments, the sample comprises polysorbate 80.

[0121] In some embodiments, the sample comprises polysorbate 20.

[0122] In some embodiments, the sample is a bulk drug. The term "bulk drug" refers to a composition comprising an active ingredient or active pharmaceutical ingredient. In some embodiments, the bulk drug is intended to provide pharmacological activity or other direct effects in the diagnosis, cure, relief, treatment or prevention of a disease, or to affect the structure or any function of the human body. In some embodiments, the bulk drug does not contain intermediates from synthesis. In some embodiments, the bulk drug does not contain excipients or other ingredients necessary for the final formulation. In some embodiments, the bulk drug also comprises one or more components in addition to the active ingredient or active pharmaceutical ingredient. In some embodiments, the additional component is one or more polysorbates.

[0123] In some embodiments, the drug substance comprises a therapeutic protein or peptide. Optionally, the therapeutic protein is an antibody.

[0124] In some embodiments, the sample is a drug product. The term "drug product" refers to a finished dosage form containing a drug substance. In some embodiments, the drug product is the final formulation administered to a patient. In some embodiments, the drug product comprises a drug substance combined with one or more other ingredients. In some embodiments, the one or more other ingredients comprise one or more excipients. In some embodiments, the one or more other ingredients are not polysorbate.

[0125] The term "polysorbate" is used to define one or more polysorbates. Polysorbates include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0126] In some embodiments, the method measures the level of a polysorbate in a sample. In some embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the polysorbate is polysorbate 20.

[0127] In some embodiments, the method measures the levels of two polysorbates in a sample. In some embodiments, the method measures the levels of two or more polysorbates in a sample. In some embodiments, the two or more polysorbates are selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0128] In some embodiments, the method measures polysorbate 80 levels and polysorbate 20 levels.

[0129] Blank sample

[0130] In some embodiments, before applying the aliquot to be measured to the column, the method includes applying one or more blank samples to a high performance liquid chromatography system having a mixed mode column, eluting the blank samples with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the blank samples after they leave the column. In some embodiments, the first mobile phase and the second mobile phase are as disclosed in the present application.

[0131] In some embodiments, a blank sample generates a baseline signal. In some embodiments, the baseline signal is stable. In some embodiments, a stable baseline signal indicates the absence of polysorbate.

[0132] In some embodiments, after the first blank sample has been applied to the column and before the aliquot to be measured is applied to the column, the method further comprises applying a second blank sample to a high performance liquid chromatography system having a mixed mode column, eluting the blank sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the blank sample after it leaves the column. In some embodiments, the first mobile phase and the second mobile phase are as disclosed herein. In some embodiments, applying evaporative light scattering detection to the second blank sample produces a stable baseline signal.

[0133] Quantifiable methods

[0134] In some embodiments, the method further comprises quantifying the measured level of polysorbate in the sample.

[0135] In some embodiments, quantifying the measured levels of polysorbate provides an accurate, robust measurement of the level of polysorbate in the sample.

[0136] In some embodiments, the method comprises quantifying the measured level of a polysorbate in the sample. Optionally, the polysorbate quantified is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0137] In some embodiments, the method comprises quantifying the measured level of polysorbate 20.

[0138] In some embodiments, the method comprises quantifying the polysorbate 80 measurement level.

[0139] In some embodiments, the method comprises quantifying the measured levels of two or more polysorbates in the sample. Optionally, the two or more polysorbates are selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In some embodiments, the method comprises quantifying the measured levels of polysorbate 80 and polysorbate 20.

[0140] In some embodiments, quantifying the measured level of polysorbate comprises comparing the actual measured level of polysorbate to a calibration curve.

[0141] In some embodiments, the actual measurement of polysorbate is compared to a calibration curve to obtain the amount of polysorbate in the sample.

[0142] In some embodiments, comparing the actual measurement of polysorbate to a calibration curve determines the amount of polysorbate in the aliquot.

[0143] In some embodiments, a calibration curve is generated by applying two or more concentration standards containing known amounts of polysorbate separately to a high performance liquid chromatography system having a mixed mode column, eluting the concentration standards with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the concentration standards as they exit the column, thereby measuring the polysorbate level, and generating a calibration curve from the measured levels of polysorbate in the concentration standards. In some embodiments, the mobile phase is as described herein.

[0144] In some embodiments, each concentration standard comprises the same polysorbate. In some embodiments, each concentration standard comprises polysorbate 80. In some embodiments, each concentration standard comprises polysorbate 20.

[0145] In some embodiments, each concentration standard comprises two or more polysorbates.

[0146] In some embodiments, a calibration curve is generated for two or more different polysorbates, and the method measures the level of each of the two or more polysorbates in the sample.

[0147] In some embodiments, one or more of the concentration standards produces a greater signal than the aliquot, and one or more of the concentration standards produces a smaller signal than the aliquot. In some embodiments, having one or more concentration standards produce a greater signal than the aliquot and one or more concentration standards produce a smaller signal than the aliquot provides a more accurate method compared to methods in which the concentration standards do not produce a greater signal than the aliquot and one or more concentration standards produce a smaller signal than the aliquot.

[0148] In some embodiments, the maximum amount of polysorbate in the one or more concentration standards does not exceed the maximum detectable signal of evaporative light scattering detection. In some embodiments, this produces a more reliable calibration curve.

[0149] In some embodiments, the maximum amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0150] In some embodiments, applying evaporative light scattering detection to a blank sample produces a signal that is lower than the signal produced by a concentration standard comprising a minimal amount of polysorbate.

[0151] In some embodiments, a calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0152] In some embodiments, the calibration curve is prepared gravimetrically. In some embodiments, the calibration curve is prepared gravimetrically by weighing the stock standard and the dilution solvent.

[0153] In some embodiments, the calibration curve has a coefficient of determination (R 2 In some embodiments, the coefficient of determination (R 2 ) is greater than or equal to 0.998.

[0154] In some embodiments, the correlation coefficient (R) is greater than or equal to 0.997. In some embodiments, the correlation coefficient (R) is greater than or equal to 0.999.

[0155] In some embodiments, the calibration curve is prepared using a quadratic fit through zero.

[0156] In some embodiments, the relative standard deviation of the percentages of two or more aliquots of a sample is 10 or less. In some embodiments, the relative standard deviation of the percentages of two or more aliquots is 8 or less, 6 or less, 4 or less, 2 or less, or 1 or less.

[0157] In some embodiments, aliquots of a blank sample, a concentration standard, and one or more samples are applied to the column in one or more of the following orders according to the methods of the present disclosure. In some embodiments, at least two blank samples are applied. Optionally, the blank sample comprises purified water. In some embodiments, for the purpose of equilibrium, a high protein concentration equilibrium standard comprising polysorbate is applied at least twice. In some embodiments, at least three additional blank samples comprising water are applied. In some embodiments, concentration standards are applied sequentially to thereby generate a calibration curve. In some embodiments, additional blank samples comprising water are applied. In some embodiments, a local reference standard comprising a known amount of polysorbate is applied. In some embodiments, additional blank samples comprising water are applied. In some embodiments, a test standard is applied, wherein the test standard comprises a known amount of polysorbate, which is measured against the calibration curve to confirm accuracy before one or more aliquots are applied to the column. In some embodiments, aliquots of one or more samples are applied sequentially. In some embodiments, additional test standards are applied to the column after the one or more aliquots are applied to the column.

[0158] In some embodiments, the method comprises applying a first polysorbate concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a different known amount of polysorbate than the first concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a different known amount of polysorbate than the first concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the eluate after it leaves the column. The method further comprises: applying a sample, applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; generating a calibration curve from the measured polysorbate levels in the polysorbate concentration standard; applying an aliquot of the sample to a high performance liquid chromatography system with a mixed mode column; eluting the sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; and quantifying the amount of polysorbate in the aliquot of the sample by comparison with the calibration curve. In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water, and the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0159] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or variations therefrom will be apparent to those skilled in the art and are included within the spirit of this application and the scope of the appended claims.

[0160] The present disclosure provides an improved method for measuring the level of polysorbate in a sample. The method of the present disclosure provides consistent results that meet all the acceptance criteria listed in Table 3. Without being bound by theory, it is believed that the mobile phase and gradient program disclosed herein contribute to achieving the acceptance criteria listed in Table 3.

[0161] In some embodiments, the mobile phases of the present disclosure are effective in removing trace amounts of proteins. In some embodiments, the addition of acetonitrile to the first mobile phase is effective in removing trace amounts of proteins.

[0162] In some embodiments, the mobile phases of the present disclosure effectively remove trace amounts of matrix components.

[0163] In some embodiments, the gradient program of the present disclosure improves the method to meet the acceptance criteria listed in Table 3.

[0164] In some embodiments, the aliquot volume improves the method to meet the acceptance criteria listed in Table 3.

[0165] Table 1

[0166]

[0167] Listed implementation plans

[0168] Embodiment 1. A method for measuring the level of polysorbate in a sample, the method comprising: applying an aliquot of the sample to a high performance liquid chromatography system having a mixed mode column; eluting the sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the eluate after leaving the column, thereby measuring the level of polysorbate; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or mixtures thereof; and water; and wherein the second mobile phase comprises: 0-20% acid; 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or mixtures thereof; and optionally water.

[0169] Embodiment 2. The method of embodiment 1, wherein the acid is a volatile acid.

[0170] Embodiment 3. The method of embodiment 2, wherein the volatile acid is formic acid.

[0171] Embodiment 4. The method according to any one of the preceding embodiments, wherein the first mobile phase and / or the second mobile phase comprises 0-2% acid.

[0172] Embodiment 5. The method according to any one of the preceding embodiments, wherein the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

[0173] Embodiment 6. The method according to any one of the preceding embodiments, wherein the first mobile phase comprises 2% formic acid, 20% acetonitrile and 78% purified water.

[0174] Embodiment 7. The method according to any one of the preceding embodiments, wherein the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0175] Embodiment 8. The method according to any one of the preceding embodiments, wherein the measured polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60 and / or polysorbate 80.

[0176] Embodiment 9. The method according to any one of the preceding embodiments, wherein the measured polysorbate comprises polysorbate 20 and / or polysorbate 80.

[0177] Embodiment 10. The method according to any one of the preceding embodiments, wherein the measured polysorbate comprises polysorbate 20.

[0178] Embodiment 11. The method according to any one of the preceding embodiments, wherein the measured polysorbate comprises polysorbate 80.

[0179] Embodiment 12. A method according to any of the preceding embodiments, further comprising, before applying the aliquot to be measured to the column: applying a blank sample to a high performance liquid chromatography system having a mixed mode column; eluting the blank sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the blank sample after it leaves the column; wherein the first mobile phase comprises: 0-20% acid; 1-70% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and wherein the second mobile phase comprises: 0-20% acid; 80-100% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and optionally water.

[0180] Embodiment 13. The method of embodiment 12, wherein applying evaporative light scattering detection to the blank sample produces a baseline signal.

[0181] Embodiment 14. A method according to embodiment 12 or embodiment 13, further comprising, after the first blank sample has been applied to the column and before the aliquot to be measured is applied to the column: applying a second blank sample to a high performance liquid chromatography system having a mixed mode column; eluting the blank sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the blank sample after it leaves the column; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and wherein the second mobile phase comprises: 0-20% acid; 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and optionally water; wherein applying evaporative light scattering detection to the second blank sample produces a stable baseline signal.

[0182] Embodiment 15. The method according to any one of the preceding embodiments, wherein the first mobile phase and / or the second mobile phase are prepared within 2 weeks of performing the method.

[0183] Embodiment 16. The method according to any one of the preceding embodiments, further comprising quantifying the measured level of polysorbate in the sample.

[0184] Embodiment 17. The method of embodiment 16, wherein quantifying the measured level of polysorbate in the sample comprises comparing the actual measured amount of polysorbate to a calibration curve.

[0185] Embodiment 18. The method of embodiment 17, wherein the amount of polysorbate in the aliquot is determined by comparing the measured amount of polysorbate to a calibration curve.

[0186] Embodiment 19. A method according to embodiment 17 or embodiment 18, wherein the calibration curve is generated by: applying one or more concentration standards containing known amounts of polysorbate separately to a high performance liquid chromatography system having a mixed mode column; eluting the concentration standards with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the concentration standards after each concentration standard leaves the column, thereby measuring the polysorbate level; and generating a calibration curve from the measured levels of polysorbate in the concentration standards; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or mixtures thereof; and water; and wherein the second mobile phase comprises: 0-20% acid; 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or mixtures thereof; and optionally water.

[0187] Embodiment 20. A method according to embodiment 19, wherein one or more of the concentration standards produces a larger signal than the aliquot, and one or more of the concentration standards produces a smaller signal than the aliquot.

[0188] Embodiment 21. The method of embodiment 19 or embodiment 20, wherein the amount of polysorbate in the two or more concentration standards does not exceed the maximum detectable signal for evaporative light scattering detection.

[0189] Embodiment 22. The method of any one of embodiments 19-21, wherein the amount of polysorbate in the concentration standard comprising the maximum amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0190] Embodiment 23. The method of any one of embodiments 19-22, wherein the signal generated by applying evaporative light scattering detection to a blank sample is lower than the signal generated by a concentration standard comprising a minimal amount of polysorbate.

[0191] Embodiment 24. The method of any one of Embodiments 19-23, wherein the calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0192] Embodiment 25. The method of any one of embodiments 17-24, wherein the calibration curve is prepared gravimetrically.

[0193] Embodiment 26. The method of any one of Embodiments 17-25, wherein the calibration curve has a coefficient of determination (R2) greater than or equal to 0.995.

[0194] Embodiment 27. The method of embodiment 26, wherein the coefficient of determination is greater than or equal to 0.998.

[0195] Embodiment 28. The method according to any one of embodiments 17-27, wherein the calibration curve is prepared using a quadratic fit through zero point.

[0196] Embodiment 29. A method according to any of the preceding embodiments, wherein the percent relative standard deviation of two or more aliquots of the sample is 10 or less.

[0197] Embodiment 30. A method according to any of the preceding embodiments, wherein the gradient comprises: 100% first mobile phase and 0% second mobile phase from 0.0 minutes to 2.0 minutes; 85% first mobile phase and 15% second mobile phase from 2.1 minutes to 5.0 minutes; 30% first mobile phase and 70% second mobile phase from 5.1 minutes to 7.6 minutes; 0% first mobile phase and 100% second mobile phase from 7.7 minutes to 9.0 minutes; and / or 100% first mobile phase and 0% second mobile phase from 9.1 minutes to 10.0 minutes.

[0198] Embodiment 31. The method according to any one of the preceding embodiments, wherein the sample is applied to the mixed mode column at a temperature of 22-28°C.

[0199] Embodiment 32. A method according to any one of the preceding embodiments, wherein the eluent is eluted from the column at a flow rate of 0.9-1.1 mL / min.

[0200] Embodiment 33. A method according to any of the preceding embodiments, wherein the evaporative light scattering detection utilizes an Alltech 3300 detector.

[0201] Embodiment 34. The method of embodiment 33, wherein the method is performed using a nebulizer temperature of the detector of 68-72°C.

[0202] Embodiment 35. The method of embodiment 33 or embodiment 34, wherein the method is performed using a gas flow rate to the detector of 2.3-2.7 L / min.

[0203] Embodiment 36. A method according to any one of embodiments 1-32, wherein the evaporative light scattering detection uses an Agilent 1260 evaporative light scattering detector.

[0204] Embodiment 37. The method of embodiment 36, wherein the method is performed using a nebulizer temperature of the detector of 43-47°C.

[0205] Embodiment 38. The method of embodiment 36 or embodiment 37, wherein the method is performed using a heating tube temperature of the detector of 70-90°C.

[0206] Embodiment 39. A method according to any one of Embodiments 36-38, wherein the method is performed using a gas flow rate to the detector of 1.3-1.7 L / min.

[0207] Embodiment 40. A method according to any one of the preceding embodiments, wherein the sample is a drug substance.

[0208] Embodiment 41. The method of any one of embodiments 1-39, wherein the sample is a pharmaceutical product.

[0209] Embodiment 42. A method of quantifying polysorbate levels in a sample, the method comprising: applying a first polysorbate concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after leaving the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a different known amount of polysorbate than the first concentration standard to a high performance liquid chromatography system having a reversed direction mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after leaving the column; applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; generating a calibration curve from the measured polysorbate levels in the polysorbate concentration standards; applying an aliquot of the sample to a high performance liquid chromatography system having a mixed mode column and eluting the sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the eluate after it leaves the column, thereby measuring the polysorbate level; and quantifying the amount of polysorbate in the aliquot of the sample by comparison with the calibration curve; wherein the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water, and the second mobile phase comprises 2% formic acid and 98% isopropanol.

[0210] Embodiment 43. The method of Embodiment 40, wherein the method is implemented according to any one of Embodiments 8-15 or 20-41. Example

[0211] The following examples are only intended to illustrate the methods of the present disclosure and are not intended to limit the scope of the present disclosure as set forth in the claims.

[0212] Example 1

[0213] Example 1 provides an exemplary application of the method of the present disclosure.

[0214] The methods of the present disclosure are illustrated using an exemplary sample comprising an antibody in a matrix comprising polysorbate 80.

[0215] A calibration curve was generated using standards containing known amounts of polysorbate 80.

[0216] This sample is a high protein concentration sample containing a polysorbate 80 equilibrium sample.

[0217] A mixed mode column was selected. Specifically, an exemplary column is a Waters Oasis Max Online Column, 3 mm x 20 mm, 30 μm, part number: 186002053.

[0218] The primary mobile phase for HPLC consisted of 2% formic acid, 20% acetonitrile, and 78% purified water. The reagents were added and mixed thoroughly and maintained under ambient conditions for up to 2 weeks.

[0219] The second mobile phase consisted of 2% formic acid in isopropanol. Add 20 mL of formic acid to 980 mL of isopropanol and mix thoroughly. Maintain the second mobile phase at ambient conditions for 2 weeks.

[0220] Use 95:5 H2O:isopropanol for syringe wash / needle dip.

[0221] An Agilent 1200 / 1260 series HPLC system with an evaporative light scattering detector (ELSD) was used for the measurement of polysorbates. An Agilent 1260 Infinity II was used as the HPLC system.

[0222] A standard curve was prepared gravimetrically using the concentration standards described in Table 4. The skilled artisan will appreciate that calibration curves can be readily generated using polysorbate concentration standards having varying concentrations and / or using varying quantities of concentration standards, and that the following standards are exemplary only.

[0223] Table 2

[0224]

[0225] Concentration standards are stable for up to 3 days at ambient conditions or when refrigerated (2-8°C).

[0226] All samples, blanks, and concentration standards were allowed to equilibrate to ambient temperature and mixed thoroughly by inversion.

[0227] Transfer aliquots into HPLC vials.

[0228] Select evaporative light scattering detection settings so that the highest concentration standard will not exceed full scale.

[0229] The peak height of the highest concentration standard is -80% of full scale. This represents an optimal position between sensitivity and range.

[0230] Adjust the detector gain to achieve the desired sensitivity.

[0231] The evaporative light scattering detector was steam washed with 100% purified water at a flow rate of 1 mL / min for 1 hour before the analytical run. The detector settings for the steam wash were: gas flow rate of 1.3 SLM, evaporation temperature of 100°C, and nebulizer temperature of 50°C.

[0232] The column temperature was 25°C and the autosampler temperature was 5°C.

[0233] A 20 μL aliquot was applied to the column.

[0234] The evaporative light scattering detector had a gas flow of 1.5 SLM, a nebulizer temperature of 45°C, an evaporation temperature of 80°C, a time constant of 30, a data output rate of 10 Hz, and a detector gain of 1.

[0235] Use flush port. If running this method with a flush port, such as on an Agilent 1260 Infinity II, select Use flush port instead of Wash vial. A syringe wash is used with the flush port, and the syringe wash time is increased from the default of 3 seconds as needed.

[0236] The run time was 10 minutes, using the gradient program in Table 5.

[0237] Table 5

[0238] Time (minutes) % first mobile phase % Second mobile phase 0.0 100 0 2.0 100 0 2.1 85 15 5.0 85 15 5.1 30 70 7.6 30 70 7.7 0 100 9.0 0 100 9.1 100 0 10.0 100 0

[0239] A blank sample, concentration standards, and one or more samples were applied in the order listed in Table 6 according to the methods of the present disclosure.

[0240] Table 3

[0241]

[0242]

[0243] Concentration standards and samples were integrated with a linear baseline.

[0244] Alternative exemplary sequences will be apparent to those skilled in the art.One skilled in the art will understand that the exemplary injection sequence is an example and should not be taken as limiting the scope of the claims in any way.

[0245] The skilled artisan will appreciate that this method is applicable to other polysorbates, including but not limited to polysorbate 20, polysorbate 40, and polysorbate 60.

[0246] Acceptance criteria for evaluating this method are provided. The acceptance criteria are listed in Table 3.

[0247] A blank sample was applied immediately prior to evaluating the standard. To meet the acceptance criteria, a stable baseline must be observed. A stable baseline was observed for both injections prior to the first standard.

[0248] Blank sample chromatogram and Figure 1 The exemplary chromatograms in are comparable.

[0249] It is normal to have a peak in the water blank sample at the retention time of polysorbate 80. If a peak is present in the blank sample, it should be smaller than the peak resulting from the standard containing the lowest concentration of polysorbate 80.

[0250] A control sample was also evaluated that did not contain polysorbate 80. The control sample did not have interfering peaks corresponding to equivalent levels of greater than 0.005% polysorbate 80, and therefore met the acceptance criteria.

[0251] The local reference standard was also evaluated for its chromatographic interference-free nature. The acceptance criterion for interference with the local reference standard was that any interfering peak in the local reference standard was smaller than that in the standard containing the lowest amount of polysorbate.

[0252] Chromatograms of local reference standards are provided in Figure 2 middle.

[0253] Evaluate the calibration curve generated from the concentration standards. Evaluate the correlation coefficient between the chromatographic profile of each standard and the calibration curve.

[0254] Chromatogram and Figure 3 Quite, and therefore acceptable.

[0255] R 2 ≥0.995 was considered acceptable (quadratic fit through zero).

[0256] The test STD was evaluated against the calibration curve. The acceptance criterion was that the measured amount of polysorbate 80% (w / v) was ±20% of the theoretical amount of polysorbate calculated by the percentage difference from the following theoretical equation:

[0257]

[0258] Calibration curves were generated as a quadratic fit through the zero point using validation software for concentration vs. peak area.

[0259] The concentration of polysorbate 80 in the samples was determined from a secondary calibration curve.

[0260] Figure 4a -b provides a chromatogram of polysorbate 80. Figure 4a A chromatogram from a drug substance sample is provided. Figure 4b A chromatogram from a drug product sample is provided.

[0261] The skilled artisan will appreciate that the exemplary method of Example 1 can be readily applied to other polysorbates, including but not limited to polysorbate 20, polysorbate 40, and polysorbate 60.

[0262] Example 2

[0263] The recovery of polysorbate 80 was measured to evaluate the robustness of the method of Example 1.

[0264] Initial testing provided percent recoveries within 20% (Table 7).

[0265]

[0266] Table 7

[0267] Further upgrades to the method further improved the recoveries to within 10% (Table 7).

[0268] The recoveries of the spiked samples also demonstrated robustness according to the acceptance criteria (Table 8). For the upgraded runs, the spiked samples showed a percent recovery of 105-109%.

[0269] Table 8

[0270]

[0271] Example 3

[0272] The robustness of the method of Example 1 was evaluated to investigate the effects of different parameters and ranges used for the method, as well as the effect of using evaporative light scattering detectors of different models and manufacturers.

[0273] Robustness studies were performed to investigate the parameters and ranges of the method used in Example 1 using an Alltech 3300 and an Agilent 1260 evaporative light scattering detector.

[0274] For the Alltech 3300 detector, different nebulizer temperatures were evaluated for evaporative light scattering detection, different column batches and column temperatures were evaluated for the column, and different flow rates were evaluated for HPLC. The parameters evaluated are listed in Table 9.

[0275] Two different Water Oasis Max Mixed-mode 3x20 mm, 30 μm column batches were evaluated. One column batch with a previous injection and a second column batch without a previous injection were selected.

[0276] Table 9

[0277]

[0278] For the Agilent 1260 Evaporative Light Scattering Detector, different nebulizer temperatures, heater tube temperatures, and gas flows were evaluated for the evaporative light scattering detector. The parameters evaluated are listed in Table 10.

[0279] Table 10

[0280]

[0281] The fractional factorial design of experiments was selected for the robustness study. The experimental design for the Alltech 3300 is shown in Table 11. The experimental design for the Agilent 1260 evaporative light scattering detector is listed in Table 12.

[0282] Table 11

[0283] run model Atomizer temperature Gas flow Column batch Column temperature flow 1 00-00 70 2.5 Batch A 25 1 2 ++--- 72 2.7 Batch A 22 0.9 3 --+-- 68 2.3 Batch B 22 0.9 4 -+-+- 68 2.7 Batch A 28 0.9 5 00-00 70 2.5 Batch A 25 1 6 ---++ 68 2.3 Batch A 28 1.1 7 +-++- 72 2.3 Batch B 28 0.9 8 00+00 70 2.5 Batch B 25 1 9 -++-+ 68 2.7 Batch B 22 1.1 10 +---+ 72 2.3 Batch A 22 1.1 11 +++++ 72 2.7 Batch B 28 1.1 12 00+00 70 2.5 Batch B 25 1

[0284] Table 12

[0285] test temperature Gas flow 1 Atomizer 47℃, heating tube 90℃ 1.7 2 Atomizer 45℃, heating tube 80℃ 1.5 3 Atomizer 43℃, heating tube 70℃ 1.3

[0286] The data confirm that different evaporative light scattering detector models and manufacturers have no real impact on the test results. Therefore, the method of the present disclosure is effective regardless of the model and / or manufacturer of the evaporative light scattering detector.

[0287] The study was conducted according to the robustness scheme.

[0288] System suitability acceptance criteria were defined for this method. The acceptance criteria are defined in Table 3.

[0289] All robustness runs met the method suitability criteria.

[0290] USP tailing data were also collected for analysis.

[0291] The system suitability results for the Alltech 3300 are listed in Tables 13A and 13B. For the Alltech 3300, the tailing results for the start and end check standards ranged from 1.16 to 1.46.

[0292] For all runs, R 2 >0.999, meeting the calibration curve acceptance criteria.

[0293] The check standard results for all samples showed that the difference from the theoretical polysorbate amount was approximately 0.03% for all samples, which fell within the acceptance criteria.

[0294] Table 13A

[0295] run Atomizer temperature Gas flow batch temperature flow 1 70 2.5 Batch A 25 1 2 72 2.7 Batch A 22 0.9 3 68 2.3 Batch B 22 0.9 4 68 2.7 Batch A 28 0.9 5 70 2.5 Batch A 25 1 6 68 2.3 Batch A 28 1.1 7 72 2.3 Batch B 28 0.9 8 70 2.5 Batch B 25 1 9 68 2.7 Batch B 22 1.1 10 72 2.3 Batch A 22 1.1 11 72 2.7 Batch B 28 1.1 12 70 2.5 Batch B 25 1

[0296] Table 13B

[0297]

[0298] The 12 runs listed in Table 14 were performed with three different samples, S2, S3, and D1, and statistical analysis was performed as described in Table 10. For all runs, the % PS80 was within 20%.

[0299] Table 14

[0300]

[0301] The corresponding JMP software analysis tables are listed in Tables 15A and 15B, and the JMP statistical output is listed in Figures 5-7.

[0302] Table 15A

[0303]

[0304] Table 15B

[0305]

[0306] The data for the S3 sample showed that column temperature had a statistically significant effect on the method at the 0.05 level. No other method parameters showed statistical significance at the 0.05 level.

[0307] To assess whether column temperature exhibited a real impact on the method, the total standard deviation of the 12 runs of the S3 sample was compared to the validation acceptance criteria for intermediate precision standard deviation from other studies. The standard deviation of the 12 runs of the S3 sample was 0.0008, and the validation acceptance criteria for intermediate precision standard deviation was SD ≤ 0.007. Therefore, this analysis indicated that the total standard deviation of the S3 sample was less than the validation acceptance criteria. Therefore, column temperature exhibited no real impact on the method.

[0308] Example 4

[0309] In order to demonstrate that the method is applicable to different detectors, the disclosed method was applied using two different models from two different manufacturers.

[0310] A fractional factorial design with 12 runs was performed using an Alltech 3300 evaporative light scattering detector.A second study with three runs was performed using an Agilent 1260 evaporative light scattering detector.

[0311] Comparable results were determined using both ELSD models. Table 16 shows comparable results for S1, S2, and S3. The calculated results for S1 and S2 were higher than expected but within the acceptance criteria.

[0312] Table 16

[0313]

[0314] Therefore, the method of the present disclosure is robust across different manufacturers and models of evaporative light scattering detectors.

[0315] The method of the present disclosure is robust with respect to nebulizer temperature, gas flow rate, column batch, column temperature and flow rate.

[0316] All robustness runs met the method system suitability criteria and generated acceptable data for each run.

[0317] The tailing results for polysorbate 80 on the Alltech 3300 ranged from 1.16 to 1.46.

[0318] Furthermore, the disclosed methods exhibit consistent performance in determining polysorbate concentrations across a variety of antibody molecules.

[0319] No real problems were found from the analysis of the evaluated method parameters, indicating that the method is robust for this operating range. The evaluated operating ranges are described in Tables 17 and 18.

[0320] Table 17

[0321]

[0322] Table 18

[0323]

[0324] This example demonstrates that the method of Example 1 is applicable to different evaporative light scattering detectors from different manufacturers under different conditions within the acceptable ranges of Tables 23 and 24.

[0325] Example 5

[0326] To evaluate the robustness of a method for measuring polysorbate levels in drug substances and drug products to demonstrate its applicability to samples from different sources.

[0327] This method was further evaluated with three antibody samples as listed in Table 19. Two samples, DS (drug substance) and DP (drug product), contained polysorbate 80, while the third sample, TFF, did not contain polysorbate 80.

[0328] Table 19

[0329]

[0330]

[0331] The suitability of this method was assessed for each sample by applying multiple aliquots from each sample according to the method of Example 1 .

[0332] At least one blank sample was applied after the standards to assess carryover.

[0333] All results met the method system acceptance criteria in Table 3. A stable baseline was observed on the blank sample before the first standard injection, and the blank chromatogram was consistent with the Figure 1 Comparable. No peaks above the lowest standard level were observed in the blank samples. Chromatographic non-interference was demonstrated for each sample. No carryover was observed.

[0334] The results are listed in Table 20.

[0335] Table 20

[0336] Sample ID <![CDATA[Coefficient of correlation, R 2 > Test solution, % difference from theory 1 0.998 -3、-4、-4、-2、-2 2 0.999 -2、-3、-2、-3 3 0.998 -2、-4、-3、-3 4 0.999 -2、-2、-2、-3 Method Standards ≥0.995 ±20％

[0337] On day 0, the criterion for the percent difference between the test standard and theory was ±10%.

[0338] Specificity was assessed by the absence of chromatographic interferences, which was demonstrated by analyzing blank samples, local reference standards, and STD 1 according to the method of Example 1.

[0339] The blank sample produced a peak at the retention time of polysorbate 80. However, the calibration curve compensated for this interference. The baseline was consistent.

[0340] Any interfering peaks above the baseline of the local reference standard should be no larger than the corresponding peaks in STD 1.

[0341] The acceptance criteria are met. Figure 1-3 As shown in . Figure 1 A blank sample is depicted, Figure 2 A local reference standard is depicted, and Figure 3 STD1 is depicted.

[0342] Example 6

[0343] The precision and reproducibility of the method of Example 1 were determined by preparing six replicate samples of drug substance and six replicate samples of drug product.

[0344] The acceptance criterion required that the percentage relative standard deviation of the six replicates was no greater than 10.

[0345] As shown in Table 21, the average percent relative standard deviation of the drug substance was 0.

[0346] Table 21

[0347] preparation Polysorbate 80 (%) 1 0.041737 2 0.042123 3 0.042082 4 0.042215 5 0.042223 6 0.041821 Average (n=6) 0.0420335 Percentage relative standard deviation (n=6) 0

[0348] As shown in Table 22, the mean percent relative standard deviation of drug product was 1.

[0349] Table 22

[0350] preparation Polysorbate 80 (%) 1 0.041847 2 0.041181 3 0.041416 4 0.041415 5 0.041116 6 0.041187 Average (n=6) 0.041367667 Percentage relative standard deviation (n=6) 1

[0351] This example demonstrates that the method of Example 1 is reproducible, with the percentage relative standard deviations for both the API and the drug product being no greater than 10. Representative chromatograms of the API and drug product are shown in Figures 1 and 2. Figure 4a and 4b Described in.

[0352] Example 7

[0353] Evaluate the stability of standard solutions to determine the period after which new concentration standards should be prepared.

[0354] Calibration curves were prepared in duplicate on day 0. One set of standards was stored at ambient conditions, while the other set of standards was stored under refrigerated conditions (5° C.±3° C.). “Ambient conditions” constituted room temperature.

[0355] On days 1, 2, and 3, drug product samples were analyzed using calibration curves generated from both sets of standards and using freshly prepared standards.

[0356] The acceptance criterion was that the difference in sample results calculated from the fresh and aged standards be no greater than 20% to designate the aged standards as stable.

[0357] All standards remained stable over the 3-day test. The results are shown in Table 23.

[0358] Table 23

[0359]

[0360] The percent difference is calculated according to the following equation:

[0361]

[0362] Where A = sample results from the aged curve, and B = sample results from the fresh curve.

[0363] Example 8

[0364] To assess the specificity of the disclosed method and to confirm that the robustness of the method is consistent across multiple molecules, different antibody molecules were evaluated.

[0365] Different antibody molecules are present in different matrices. The matrix compositions from the antibody molecules are described in Table 24. The polysorbate in each is polysorbate 80.

[0366] Table 24

[0367] This method was performed to demonstrate that specificity for polysorbate 80 was achieved in all antibody molecules tested. For all samples, evaporative light scattering detection identified a single peak signal at approximately 6.5 minutes ( Figure 8 No significant matrix effect was observed. Therefore, the method disclosed herein is suitable for determining the concentration of polysorbate 80 in different samples.

[0368] Example 9

[0369] To demonstrate the robustness of the method of Example 1 to different polysorbates and different laboratories, further studies were performed.

[0370] The method of this example was applied by two different laboratories on samples containing either polysorbate 20 (Table 25) or polysorbate 80 (Table 26).

[0371] Table 25

[0372]

[0373] Table 26

[0374]

[0375] This study demonstrates that the disclosed method is robust to different polysorbates and that robust results were achieved by this method in different laboratories.

[0376] Further robustness studies of polysorbate 20 determined that the percent relative standard deviation for both samples was less than 10 (Table 27).

[0377] Table 27

[0378]

[0379] Two further studies confirmed acceptable robustness by percent relative standard deviation of three samples containing polysorbate 80 (Tables 28 and 29).

[0380] Table 28

[0381]

[0382]

[0383] Sample S1 in Table 28 is a stressed sample. The method of the present disclosure is also robust on stressed samples.

[0384] Table 29

[0385]

[0386] D1 is an overstressed sample maintained at 40° C. for 5 months. This sample exhibits high viscosity.

[0387] Therefore, the method of the present disclosure is robust to different polysorbates.

[0388] equivalent

[0389] Those skilled in the art will recognize many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited by the above description, but is instead set forth in the claims.

Claims

1. A method for measuring the level of polysorbate in a sample, the method comprising: applying an aliquot of the sample to a high performance liquid chromatography system having a mixed mode column; eluting the sample with a gradient from a first mobile phase to a second mobile phase; and Polysorbate levels were measured by applying evaporative light scattering detection to the eluate after it exited the column; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and The second mobile phase comprises: 0-20% of an acid; 80-100% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and optionally water. 2 . The method according to claim 1 , wherein the acid in the first mobile phase and / or the second mobile phase is a volatile acid.

3. The method of claim 2, wherein the volatile acid is formic acid.

4. The method according to any one of the preceding claims, wherein the first mobile phase and / or the second mobile phase comprises 0-2% acid.

5. The method according to any one of the preceding claims, wherein the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

6. The method according to any one of the preceding claims, wherein the first mobile phase comprises 2% formic acid, 20% acetonitrile and 78% purified water.

7. The method according to any one of the preceding claims, wherein the second mobile phase comprises 2% formic acid and 98% isopropanol.

8. The method according to any one of the preceding claims, wherein the measured polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60 and / or polysorbate 80.

9. The method according to any one of the preceding claims, wherein the measured polysorbate comprises polysorbate 20 and / or polysorbate 80.

10. The method according to any one of the preceding claims, wherein the measured polysorbate comprises polysorbate 20.

11. The method according to any one of the preceding claims, wherein the measured polysorbate comprises polysorbate 80.

12. The method according to any one of the preceding claims, further comprising, before applying the aliquot to be measured to the column: A blank sample was applied to a HPLC system with a mixed-mode column; eluting the blank sample with a gradient from the first mobile phase to the second mobile phase; and applying evaporative light scattering detection to the blank sample after it leaves the column; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and The second mobile phase comprises: 0-20% of an acid; 80-100% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and optionally water.

13. The method of claim 12, wherein applying evaporative light scattering detection to the blank sample generates a baseline signal.

14. The method according to claim 12 or claim 13, further comprising, after the first blank sample has been applied to the column and before the aliquot to be measured is applied to the column: applying a second blank sample to a high performance liquid chromatography system with a mixed mode column; eluting the blank sample with a gradient from the first mobile phase to the second mobile phase; and applying evaporative light scattering detection to the blank sample after it leaves the column; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and wherein the second mobile phase comprises: 0-20% of an acid; 80-100% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol, or a mixture thereof; and optionally water; The second blank sample is subjected to evaporative light scattering detection to generate a stable baseline signal.

15. The method according to any one of the preceding claims, wherein the first mobile phase and / or the second mobile phase are prepared within 2 weeks of performing the method.

16. The method of any preceding claim, further comprising quantifying the measured level of polysorbate in the sample.

17. The method of claim 16, wherein quantifying the measured level of polysorbate in the sample comprises comparing the actual measured amount of polysorbate to a calibration curve.

18. The method of claim 17, wherein comparing the measured amount of polysorbate to a calibration curve determines the amount of polysorbate in the aliquot.

19. The method of claim 17 or claim 18, wherein the calibration curve is generated as follows: separately applying one or more concentration standards containing known amounts of polysorbate to a high performance liquid chromatography system having a mixed mode column; eluting the concentration standard with a gradient from a first mobile phase to a second mobile phase; and measuring the polysorbate level by subjecting each concentration standard to evaporative light scattering detection after it leaves the column; and generating a calibration curve from the measured levels of polysorbate in the concentration standards; wherein the first mobile phase comprises: 0-20% acid; 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and water; and The second mobile phase comprises: 0-20% of an acid; 80-100% of an organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropanol or a mixture thereof; and optionally water.

20. The method of claim 19, wherein one or more of the concentration standards produces a greater signal than the aliquot and one or more of the concentration standards produces a smaller signal than the aliquot.

21. The method of claim 19 or claim 20, wherein the amount of polysorbate in the two or more concentration standards does not exceed the maximum detectable signal of the evaporative light scattering assay.

22. The method of any one of claims 19-21, wherein the amount of polysorbate in the concentration standard comprising the maximum amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

23. The method of any one of claims 19 to 22, wherein the signal generated by evaporative light scattering detection applied to a blank sample is lower than the signal generated by a concentration standard comprising a minimal amount of polysorbate.

24. The method of any one of claims 19-23, wherein the calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

25. The method according to any one of claims 17 to 24, wherein the calibration curve is prepared gravimetrically.

26. The method of any one of claims 17 to 25, wherein the calibration curve has a coefficient of determination (R) greater than or equal to 0.

995. 2 ).

27. The method of claim 26, wherein the coefficient of determination is greater than or equal to 0.

998.

28. The method of any one of claims 17 to 27, wherein the calibration curve is prepared using a quadratic fit through zero point.

29. The method of any preceding claim, wherein the percent relative standard deviation of two or more aliquots of the sample is 10 or less.

30. The method of any preceding claim, wherein the gradient comprises: 100% first mobile phase and 0% second mobile phase from 0.0 min to 2.0 min; 85% first mobile phase and 15% second mobile phase from 2.1 min to 5.0 min; 30% first mobile phase and 70% second mobile phase from 5.1 to 7.6 minutes; 0% first mobile phase and 100% second mobile phase from 7.7 minutes to 9.0 minutes; and / or 100% first mobile phase and 0% second mobile phase from 9.1 min to 10.0 min.

31. The method according to any one of the preceding claims, wherein the sample is applied to a mixed mode column at a temperature of 22-28°C.

32. A method according to any preceding claim, wherein the eluent is eluted from the column at a flow rate of 0.9-1.1 mL / min.

33. The method according to any one of the preceding claims, wherein the evaporative light scattering detection uses an Alltech 3300 detector.

34. The method of claim 33, wherein the method is performed using a nebulizer temperature of the detector of 68-72°C.

35. A method according to claim 33 or claim 34, wherein the method is performed using a gas flow rate to the detector of 2.3-2.7 L / min.

36. The method according to any one of claims 1 to 32, wherein the evaporative light scattering detection employs an Agilent 1260 evaporative light scattering detector.

37. The method of claim 36, wherein the method is performed using a nebulizer temperature of the detector of 43-47°C.

38. A method according to claim 36 or claim 37, wherein the method is performed using a heater tube temperature of the detector of 70-90°C.

39. The method according to any one of claims 36 to 38, wherein the method is performed using a gas flow rate to the detector of 1.3 to 1.7 L / min.

40. The method of any preceding claim, wherein the sample is a drug substance.

41. The method of any one of claims 1-39, wherein the sample is a pharmaceutical product.

42. A method of quantifying the level of polysorbate in a sample, the method comprising: applying a first polysorbate concentration standard to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after exiting the column, thereby measuring the polysorbate level; applying one or more additional polysorbate concentration standards comprising a different known amount of polysorbate than the first concentration standard to a high performance liquid chromatography system having a reversed mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluate after exiting the column, thereby measuring the polysorbate level; generating a calibration curve from the measured polysorbate levels in the polysorbate concentration standards; applying an aliquot of the sample to a high performance liquid chromatography system having a mixed mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the eluate after leaving the column, thereby measuring the polysorbate level; and quantifying the amount of polysorbate in an aliquot of the sample by comparison to a calibration curve; The first mobile phase comprises 2% formic acid, 20% acetonitrile and 78% purified water, and the second mobile phase comprises 2% formic acid and 98% isopropanol.

43. The method of claim 40, wherein the method is performed according to any one of claims 8-5 or 20-41.

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