Methods for measuring polysorbate levels

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 477,904, filed December 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to methods for measuring the level of polysorbate in a sample. In some embodiments, the methods provide for quantification of the level of polysorbate in a sample. [Background technology]

[0003] Polysorbates are used as surfactants in pharmaceutical formulations, especially in drug substances and formulations. Polysorbates are used as surfactants to stabilize biopharmaceuticals. Polysorbates are commonly used to stabilize proteins, especially antibodies.

[0004] Polysorbates are a class of molecules that contain multiple structures, including, but not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Measuring the level of polysorbate can include measuring the level of one or more polysorbate structures.

[0005] Degradation of polysorbates is a known problem. Polysorbates can be degraded by hydrolysis or oxidation. Degradation of polysorbates reduces the amount of polysorbates in a sample, and therefore reduces their ability to stabilize other components, such as therapeutic proteins, in the sample. Therefore, it is necessary to measure the amount of polysorbates in a sample. In particular, measuring the level of polysorbates in drug substances and drug products is important for identifying polysorbate degradation, which may lead to instability of other molecules in the formulation.

[0006] Conventional techniques for measuring constituents in a sample do not directly or accurately measure polysorbate levels, and previous attempts to directly detect polysorbate by UV-Vis or fluorescence have been unsuccessful because the polysorbate molecule lacks a chemical structure such as a strong chromophore.

[0007] Existing methods for measuring polysorbate levels are destructive and / or indirect. One known method for measuring polysorbate focuses on identifying polysorbate subspecies using ultra-high performance liquid chromatography combined with high-resolution mass spectrometry. Another known method digests polysorbate molecules and then characterizes the degradation products, typically by liquid chromatography coupled to mass spectrometry, followed by liquid chromatography coupled to charged aerosol detection and liquid chromatography coupled to evaporative light scattering detection. In some embodiments, the characterized degradation products do not contain fatty acids. Therefore, most known methods for measuring polysorbate levels in a sample are indirect and require the destruction of polysorbate to characterize the resulting degradation products or subspecies.

[0008] Furthermore, during robustness testing, the conventional method identified higher than expected polysorbate levels in samples, including a 30% overestimation compared to the expected results. Previous attempts to identify gradient programs did not resolve this issue.

[0009] Previous attempts to create a robust, simple, and cost-effective method for accurately measuring levels of specific polysorbates have been unsuccessful. One previous attempt required solid phase extraction to remove proteins prior to further separation steps for accurate measurement, which is inefficient, time-consuming, and labor-intensive. Summary of the Invention [Problem to be solved by the invention]

[0010] There is a need for improved methods for directly and accurately measuring polysorbate levels in a sample.

[0011] The present disclosure meets this need by providing a robust, simple, and cost-effective method for directly and accurately measuring polysorbate levels. [Means for solving the problem]

[0012] The present disclosure is directed to a method for measuring the level of polysorbate in a sample. The disclosed method directly and accurately measures polysorbate levels in a sample in a robust, simple, and cost-effective manner.

[0013] In some embodiments, the present disclosure is directed to a method for measuring polysorbate levels in a sample. In some embodiments, the method includes applying an aliquot of the sample to a high-performance liquid chromatography system equipped with a mixed-mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby measuring the polysorbate level. In some embodiments, the first mobile phase includes 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and water. In some embodiments, the second mobile phase includes 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

[0014] In some embodiments, the acid is a volatile acid.

[0015] In some embodiments, the volatile acid is formic acid.

[0016] In some embodiments, the first mobile phase and / or the second mobile phase comprises 0-2% acid.

[0017] In some embodiments, the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

[0018] In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

[0019] In some embodiments, the second mobile phase comprises 2% formic acid and 98% isopropyl alcohol.

[0020] In some embodiments, the polysorbates measured include polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80.

[0021] In some embodiments, the polysorbates measured include polysorbate 20 and / or polysorbate 80.

[0022] In some embodiments, the polysorbate measured includes polysorbate 20.

[0023] In some embodiments, the polysorbate measured includes polysorbate 80.

[0024] In some embodiments, prior to applying the aliquot to be measured to the column, the method further includes applying a blank to a high performance liquid chromatography system equipped with a mixed-mode column, eluting the blank using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the blank after it exits the column. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, 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, isopropyl alcohol, or mixtures thereof, and optionally water.

[0025] In some embodiments, a baseline signal is generated by applying evaporative light scattering detection to a blank.

[0026] In some embodiments, after applying the first blank to the column and before applying the aliquot to be measured to the column, the method further includes applying a second blank to the high-performance liquid chromatography system equipped with a mixed-mode column, eluting the blank using a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the blank after it exits the column, wherein applying evaporative light scattering detection to the second blank generates a stable baseline signal. In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, 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, isopropyl alcohol, or mixtures thereof, and optionally water.

[0027] In some embodiments, the first mobile phase and / or the second mobile phase were prepared within two weeks of performing the method.

[0028] In some embodiments, the method further comprises quantifying the measured level of polysorbate in the sample.

[0029] In some embodiments, quantifying the measured level of polysorbate in the sample comprises comparing the measured amount of polysorbate to a standard curve.

[0030] In some embodiments, the amount of polysorbate in an aliquot is determined by comparing the measured amount of polysorbate to a standard curve.

[0031] In some embodiments, a calibration curve is generated by separately applying one or more concentration standards containing known amounts of polysorbate to a high-performance liquid chromatography system equipped with a mixed-mode column, eluting the concentration standards using a gradient from a first mobile phase to a second mobile phase, applying evaporative light scattering detection to the concentration standards after each exits the column, thereby measuring the polysorbate levels, 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, isopropyl alcohol, 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, isopropyl alcohol, or mixtures thereof, and optionally water.

[0032] In some embodiments, one or more of the concentration standards produces a signal that is greater than the aliquot, and one or more of the concentration standards produces a signal that is less than the aliquot.

[0033] In some embodiments, the amount of polysorbate in the one or more concentration standards does not exceed the maximum detectable signal for evaporative light scattering detection.

[0034] In some embodiments, the amount of polysorbate in the concentration standard containing the greatest amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0035] In some embodiments, applying evaporative light scattering detection to a blank produces a signal that is lower than the signal produced by a concentration standard containing minimal amounts of polysorbate.

[0036] As used herein, a "blank" does not contain polysorbate. In some embodiments, the blank is water.

[0037] In some embodiments, a calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0038] In some embodiments, the calibration curve is generated by a gravimetric method.

[0039] In some embodiments, the calibration curve has a coefficient of determination (R) of 0.995 or greater. 2 )

[0040] In some embodiments, the coefficient of determination (R 2 ) is 0.998 or greater.

[0041] In some embodiments, the calibration curve is generated using a quadratic fit through zero.

[0042] In some embodiments, the percentage relative standard deviation of two or more aliquots of a sample is 10 or less.

[0043] In some embodiments, the gradient comprises 100% of the first mobile phase and 0% of the second mobile phase from 0.0 to 2.0 minutes, and / or 85% of the first mobile phase and 15% of the second mobile phase from 2.1 to 5.0 minutes, and / or 30% of the first mobile phase and 70% of the second mobile phase from 5.1 to 7.6 minutes, and / or 0% of the first mobile phase and 100% of the second mobile phase from 7.7 to 9.0 minutes, and / or 100% of the first mobile phase and 0% of the second mobile phase from 9.1 to 10.0 minutes.

[0044] In some embodiments, the sample is applied to the mixed-mode column at a temperature of 22-28°C.

[0045] In some embodiments, the eluent is eluted from the column at a flow rate of 0.9 to 1.1 mL / min.

[0046] In some embodiments, evaporative light scattering detection utilizes an Alltech 3300 detector.

[0047] In some embodiments where the method utilizes an Alltech 3300 detector, the method is carried out using a detector nebulizer temperature of 68-72°C.

[0048] In some embodiments where the method utilizes an Alltech 3300 detector, the method is carried out using a detector gas flow rate of 2.3 to 2.7 L / min.

[0049] In some embodiments, the evaporative light scattering detection utilizes an Agilent 1260 evaporative light scattering detector.

[0050] In some embodiments where the method utilizes an Agilent 1260 evaporative light scattering detector, the method is carried out using a detector nebulizer temperature of 43-47°C.

[0051] In some embodiments where the method utilizes an Agilent 1260 evaporative light scattering detector, the method is carried out using a detector heater tube temperature of 70-90°C.

[0052] In some embodiments where the method utilizes an Agilent 1260 evaporative light scattering detector, the method is carried out using a detector gas flow rate of 1.3-1.7 L / min.

[0053] In some embodiments, the sample is a drug substance.

[0054] In some embodiments, the sample is a formulation.

[0055] In some embodiments, the method includes applying a first polysorbate concentration standard to a high performance liquid chromatography system equipped with a mixed mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby measuring the polysorbate level; and applying one or more additional polysorbate concentration standards, each having a different known amount of polysorbate compared to the first concentration standard, to a high performance liquid chromatography system equipped with a mixed mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby measuring the polysorbate level. applying evaporative light scattering detection to the eluent after it exits a column to thereby measure 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 equipped with a mixed-mode column and eluting the sample with a gradient from a first mobile phase to a second mobile phase, applying evaporative light scattering detection to the eluent after it exits the column to thereby measure the polysorbate level, and quantifying the amount of polysorbate in the sample aliquot 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% isopropyl alcohol.

[0056] In some embodiments, the method is performed according to any of the disclosed features. [Brief explanation of the drawings]

[0057] The drawings are for illustrative purposes only and are not intended to impose any limitations on the disclosure. [Figure 1] An exemplary water blank chromatogram is shown. [Figure 2] 1 shows an exemplary chromatogram of a local reference standard. [Figure 3] 1 shows an exemplary chromatogram of a concentration standard. [Figure 4a] 1 shows an exemplary chromatogram of Polysorbate 80. 2 provides an exemplary chromatogram from a drug substance sample. [Figure 4b] 1 shows an exemplary chromatogram of Polysorbate 80. 2 provides an exemplary chromatogram from a formulation sample. [Figure 5a] Figure 5 shows the JMP statistics output for S2. Figure 5a shows the S2 distribution. Figure 5b shows the variability chart for S2. Figures 5c and 5d show the response results for S2. [Figure 5b] Figure 5 shows the JMP statistics output for S2. Figure 5a shows the S2 distribution. Figure 5b shows the variability chart for S2. Figures 5c and 5d show the response results for S2. [Figure 5c] Figure 5 shows the JMP statistics output for S2. Figure 5a shows the S2 distribution. Figure 5b shows the variability chart for S2. Figures 5c and 5d show the response results for S2. [Figure 5d] Figure 5 shows the JMP statistics output for S2. Figure 5a shows the S2 distribution. Figure 5b shows the variability chart for S2. Figures 5c and 5d show the response results for S2. [Figure 6a] Figure 6 shows the JMP statistics output for S3. Figure 6a shows the S3 distribution. Figure 6b shows the variability chart for S3. Figures 6c and 6d show the response results for S3. [Figure 6b] Figure 6 shows the JMP statistics output for S3. Figure 6a shows the S3 distribution. Figure 6b shows the variability chart for S3. Figures 6c and 6d show the response results for S3. [Figure 6c]Figure 6 shows the JMP statistics output for S3. Figure 6a shows the S3 distribution. Figure 6b shows the variability chart for S3. Figures 6c and 6d show the response results for S3. [Figure 6d] Figure 6 shows the JMP statistics output for S3. Figure 6a shows the S3 distribution. Figure 6b shows the variability chart for S3. Figures 6c and 6d show the response results for S3. [Figure 7a] Figure 7 shows the JMP statistics output for D1. Figure 7a shows the D1 distribution. Figure 7b shows the variability chart for D1. Figures 7c and 7d show the response results for D1. [Figure 7b] Figure 7 shows the JMP statistics output for D1. Figure 7a shows the D1 distribution. Figure 7b shows the variability chart for D1. Figures 7c and 7d show the response results for D1. [Figure 7c] Figure 7 shows the JMP statistics output for D1. Figure 7a shows the D1 distribution. Figure 7b shows the variability chart for D1. Figures 7c and 7d show the response results for D1. [Figure 7d] Figure 7 shows the JMP statistics output for D1. Figure 7a shows the D1 distribution. Figure 7b shows the variability chart for D1. Figures 7c and 7d show the response results for D1. [Figure 8] The eluent peaks of several samples containing polysorbate 80 and check standards are shown. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present disclosure is directed to methods for measuring the level of polysorbate in a sample.

[0059] The disclosed methods accurately measure polysorbate levels in a sample in a robust, simple, and cost-effective manner.

[0060] In some embodiments, the methods of the present disclosure are non-destructive.

[0061] In some embodiments, the methods of the present disclosure measure polysorbate levels directly.

[0062] High-performance liquid chromatography The present disclosure is directed to a method for measuring polysorbate levels in a sample. In some embodiments, an aliquot of the sample is applied to a high performance liquid chromatography (HPLC) system equipped with a mixed-mode column.

[0063] In some embodiments, the aliquot has a volume of 10 μL.

[0064] High performance liquid chromatography is a known technique for separating components in an aliquot applied to a column. In some embodiments, the disclosed methods utilize a mixed-mode column. Mixed-mode columns are known in the art. In some embodiments, the disclosed methods utilize a reversed-phase column. Reversed-phase columns are known in the art. Typically, reversed-phase columns include a non-polar stationary phase.

[0065] In some embodiments, the high performance liquid chromatography system is an Agilent 1200 / 1260 system. In some embodiments, the high performance liquid chromatography system is an Agilent 1260 Infinity II. Those skilled in the art will appreciate that other high performance liquid chromatography systems can readily be utilized in the methods of the present disclosure and that these systems are purely exemplary.

[0066] In some embodiments, the sample is applied to the mixed-mode column at a temperature of 22-28°C.

[0067] In some embodiments, the methods of the present disclosure elute the components of the aliquot from the mixed-mode column using a gradient from a first mobile phase to a second mobile phase.

[0068] The present disclosure provides a mobile phase that provides a robust, simple, and cost-effective method for measuring polysorbate levels.

[0069] In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent, and water.

[0070] In some embodiments, the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and water.

[0071] In some embodiments, the acid is a volatile acid, hi some embodiments, the volatile acid is formic acid.

[0072] Polysorbates and other components of the aliquots can bind to the column, potentially creating carryover problems. In some embodiments, carryover can occur when components of a previous sample, blank, or concentration standard applied to the column bind to the column and interfere with or co-elute with the next sample, blank, or concentration standard applied to the column. 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 carryover problem. In some embodiments, this results in a chromatogram with better / improved tailing.

[0073] In some embodiments, the first mobile phase comprises 0-2% acid.

[0074] In some embodiments, the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

[0075] In some embodiments, the second mobile phase comprises 0-20% acid, 80-100% organic solvent, and optionally water.

[0076] In some embodiments, the second mobile phase comprises 0-20% acid and 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

[0077] In some embodiments, the acid is a volatile acid, hi some embodiments, the volatile acid is formic acid.

[0078] In some embodiments, the second mobile phase comprises 0-2% acid.

[0079] In some embodiments, the second mobile phase comprises 2% formic acid and 98% isopropyl alcohol.

[0080] 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% isopropyl alcohol.

[0081] 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, isopropyl alcohol, or a mixture thereof. In some embodiments, by selecting two different organic solvents, more proteins and / or matrix components are eluted compared to embodiments in which the same organic solvent is selected for the first and second mobile phases, thereby providing additional resolution.

[0082] As used herein, "matrix" refers to a solvent or formulation system that contains an active pharmaceutical ingredient, such as an antibody.

[0083] In some embodiments in which 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 criteria in Table 3, thereby improving the robustness of the method.

[0084] In some embodiments, the second mobile phase comprises methanol, tetrahydrofuran, isopropyl alcohol, or a mixture thereof. In some embodiments, the second mobile phase does not comprise acetonitrile. In some embodiments, the second mobile phase provides more efficient elution, resulting in sharper peaks, thereby improving tailing and interference.

[0085] In some embodiments, the first mobile phase and / or the second mobile phase were prepared within two weeks of performing the method.

[0086] In some embodiments, preparing the first mobile phase and / or the second mobile phase within two weeks of performing the method improves the robustness and / or accuracy of the method.

[0087] In some embodiments, the gradient used to elute the polysorbate comprises 100% of the first mobile phase and 0% of the second mobile phase from 0.0 to 2.0 minutes, and / or 85% of the first mobile phase and 15% of the second mobile phase from 2.1 to 5.0 minutes, and / or 30% of the first mobile phase and 70% of the second mobile phase from 5.1 to 7.6 minutes, and / or 0% of the first mobile phase and 100% of the second mobile phase from 7.7 to 9.0 minutes, and / or 100% of the first mobile phase and 0% of the second mobile phase from 9.1 to 10.0 minutes.

[0088] In some embodiments, the eluent is eluted from the column at a flow rate of 0.9 to 1.1 mL / min.

[0089] In some embodiments, the aliquot may contain other components in addition to polysorbate, including, but not limited to, proteins, polypeptides, peptides, polysorbate subspecies, and / or polysorbate degradation products. Typically, the gradient elutes the polysorbate at a different time point compared to the other components of the aliquot.

[0090] Polysorbate subspecies are polysorbate-related molecular or structural variants. Exemplary subspecies include polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, and polyoxyethylene (19) isosorbitan monolaurate.

[0091] 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 situations, polysorbate degradation also increases the amount of polysorbate subspecies.

[0092] In some embodiments, the aliquot comprises two or more polysorbates. In some embodiments, the gradient elutes different polysorbates at different time points.

[0093] Evaporative light scattering detection In some embodiments, evaporative light scattering detection (ELSD) is applied to the eluent. Evaporative light scattering detection is a technique known in the art. Briefly, the eluent is passed through a heated chamber to evaporate the mobile phase solvent. The non-volatile components of the aliquot form solid particles upon solvent evaporation. The solid particles scatter UV radiation, resulting in a detectable signal.

[0094] Evaporative light scattering detection can detect non-UV absorbing components in the aliquot. In some embodiments, the methods of the present disclosure apply evaporative light scattering detection to detect polysorbates.

[0095] Conventionally, evaporative light scattering detection is applied to detect the presence or absence of a component. The present disclosure provides a method for measuring the level of polysorbates by evaporative light scattering detection.

[0096] In some embodiments, the evaporative light scattering detection identifies one or more signals corresponding to one or more components in the eluent, hi some embodiments, the one or more signals correspond to output from the evaporative light scattering detection.

[0097] In some embodiments, the method generates a signal for the polysorbate, and the amount of polysorbate is determined from the generated signal.

[0098] In some embodiments, when the aliquot contains multiple polysorbate structures, the method generates a signal for each different polysorbate structure, and the amount of each different polysorbate is determined from the generated signals.

[0099] In some embodiments, gain is applied during evaporative light scattering detection.

[0100] In some embodiments, the evaporative light scattering detection utilizes an Alltech 3300 evaporative light scattering detector. In some embodiments, when using an Alltech 3300 detector, the method is performed using a detector nebulizer temperature of 68-72°C. In some embodiments, when using an 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 an Alltech 3300 detector, the method results in a tailing result of 1.16-1.46.

[0101] In some embodiments, the evaporative light scattering detection utilizes an Agilent 1260 evaporative light scattering detector. In some embodiments, when using an Agilent 1260 evaporative light scattering detector, the method is performed using a detector nebulizer temperature of 43-47° C. In some embodiments, when using an Agilent 1260 detector, the method is performed using a detector heater tube temperature of 70-90° C. In some embodiments, when using an Agilent 1260 evaporative light scattering detector, the method is performed using a detector gas flow rate of 1.3-1.7 L / min.

[0102] sample The sample in which the polysorbate level is measured can be any sample. Typically, the sample contains polysorbate or is suspected to contain polysorbate.

[0103] In some embodiments, the sample comprises one or more polysorbates, which may be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or mixtures thereof.

[0104] In some embodiments, the sample contains polysorbate 80.

[0105] In some embodiments, the sample contains polysorbate 20.

[0106] In some embodiments, the sample is a drug substance. The term "drug substance" refers to an active ingredient or a composition containing an effective active ingredient. In some embodiments, a drug substance is intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or function of the human body. In some embodiments, a drug substance does not include intermediates from synthesis. In some embodiments, a drug substance does not include excipients or other components required for the final formulation. In some embodiments, a drug substance includes one or more components in addition to the active ingredient or effective active ingredient. In some embodiments, the additional component is one or more polysorbates.

[0107] In some embodiments, the drug substance comprises a therapeutic protein or peptide. Optionally, the therapeutic protein is an antibody.

[0108] In some embodiments, the sample is a formulation. The term "formulation" refers to the final dosage form containing the drug substance. In some embodiments, the formulation is the final formulation approved for patients. In some embodiments, the formulation includes the drug substance in association with one or more additional ingredients. In some embodiments, the one or more additional ingredients include one or more excipients. In some embodiments, the one or more additional ingredients are not polysorbates.

[0109] The term "polysorbate" is used to define one or more polysorbates, including, but not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0110] In some embodiments, the method measures the level of one 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.

[0111] 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.

[0112] In some embodiments, the method measures the level of polysorbate 80 and the level of polysorbate 20.

[0113] blank In some embodiments, prior to applying the aliquot to be measured to the column, the method includes applying one or more blanks to a high performance liquid chromatography system equipped with a mixed-mode column, eluting the blanks with a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the blanks after they exit the column. In some embodiments, the first mobile phase and the second mobile phase are as disclosed herein.

[0114] In some embodiments, the blank generates a baseline signal. In some embodiments, the baseline signal is stable. In some embodiments, a stable baseline signal indicates the absence of polysorbate.

[0115] In some embodiments, after applying the first blank to the column and before applying the aliquot to be measured to the column, the method further includes applying a second blank to the high performance liquid chromatography system equipped with a mixed-mode column, eluting the blank using a gradient from the first mobile phase to the second mobile phase, and applying evaporative light scattering detection to the blank after it exits 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 generates a stable baseline signal.

[0116] Quantifiable Methods In some embodiments, the method further comprises quantifying the measured level of polysorbate in the sample.

[0117] In some embodiments, quantifying the measured levels of polysorbate provides an accurate and robust measurement of the levels of polysorbate in a sample.

[0118] In some embodiments, the method further comprises quantifying the measured level of one polysorbate in the sample. Optionally, the quantified polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0119] In some embodiments, the methods include quantifying the measured level of polysorbate 20.

[0120] In some embodiments, the methods include quantifying the measured level of polysorbate 80.

[0121] In some embodiments, the method comprises quantifying 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 measured levels of polysorbate 80 and polysorbate 20.

[0122] In some embodiments, quantifying the measured level of polysorbate comprises comparing the measured amount of polysorbate to a standard curve.

[0123] In some embodiments, the amount of polysorbate in the sample is obtained by comparing the measured amount of polysorbate to a standard curve.

[0124] In some embodiments, the amount of polysorbate in an aliquot is determined by comparing the measured amount of polysorbate to a standard curve.

[0125] In some embodiments, the calibration curve is generated by separately applying two or more concentration standards containing known amounts of polysorbate to a high-performance liquid chromatography system equipped with a mixed-mode column, eluting the concentration standards with a gradient from a first mobile phase to a second mobile phase, applying evaporative light scattering detection to the concentration standards as each exits the column, thereby measuring the polysorbate levels, 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.

[0126] 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.

[0127] In some embodiments, each concentration standard contains two or more polysorbates.

[0128] 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.

[0129] In some embodiments, one or more of the concentration standards produce a larger signal than the aliquot and one or more of the concentration standards produce a smaller signal than the aliquot. In some embodiments, having one or more concentration standards produce a larger 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 larger or smaller signal than the aliquot.

[0130] In some embodiments, the maximum amount of polysorbate in one or more concentration standards does not exceed the maximum detectable signal for evaporative light scattering detection, which in some embodiments generates a more reliable calibration curve.

[0131] In some embodiments, the maximum amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0132] In some embodiments, applying evaporative light scattering detection to a blank produces a signal that is lower than the signal produced by a concentration standard containing minimal amounts of polysorbate.

[0133] In some embodiments, a calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0134] In some embodiments, the calibration curve is prepared gravimetrically. In some embodiments, the calibration curve is prepared gravimetrically by weighing the stock standards and dilution solvent.

[0135] In some embodiments, the calibration curve has a coefficient of determination (R) of 0.995 or greater. 2 In some embodiments, the coefficient of determination (R 2 ) is 0.998 or greater.

[0136] In some embodiments, the correlation coefficient (R) is 0.997 or greater. In some embodiments, the correlation coefficient (R) is 0.999 or greater.

[0137] In some embodiments, the calibration curve is generated using a quadratic fit through zero.

[0138] In some embodiments, the percentage relative standard deviation of two or more aliquots of a sample is less than or equal to 10. In some embodiments, the percentage relative standard deviation of two or more aliquots is less than or equal to 8, less than or equal to 6, less than or equal to 4, less than or equal to 2, or less than or equal to 1.

[0139] In some embodiments, blanks, concentration standards, and one or more sample aliquots are applied to a column according to the disclosed methods in one or more of the following orders: In some embodiments, at least two blanks are applied. Optionally, the blank comprises purified water. In some embodiments, a high protein concentration equilibration standard comprising polysorbate is applied at least twice for equilibration purposes. In some embodiments, at least three additional blanks comprising water are applied. In some embodiments, concentration standards are applied sequentially from which a calibration curve is generated. In some embodiments, an additional blank comprising water is applied. In some embodiments, a local reference standard comprising a known amount of polysorbate is applied. In some embodiments, an additional blank comprising water is applied. In some embodiments, a check standard is applied, the check standard comprising a known amount of polysorbate that is measured against the calibration curve to confirm accuracy before applying one or more aliquots to the column. In some embodiments, one or more sample aliquots are applied sequentially. In some embodiments, an additional check standard is applied to the column after one or more aliquots have been applied to the column.

[0140] In some embodiments, the method includes applying a first polysorbate concentration standard to a high performance liquid chromatography system equipped 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 eluent after it exits the column, thereby measuring the polysorbate level; and applying one or more additional polysorbate concentration standards, each having a different known amount of polysorbate compared to the first concentration standard, to a high performance liquid chromatography system equipped 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 eluent after it exits the column, thereby measuring the polysorbate level. applying evaporative light scattering detection to the eluent after it exits the column to thereby measure 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 equipped with a mixed-mode column, eluting the sample with a gradient from a first mobile phase to a second mobile phase, applying evaporative light scattering detection to the eluent after it exits the column to thereby measure 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% isopropyl alcohol.

[0141] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations therein will be known to those skilled in the art and are within the spirit of this application and the scope of the appended claims.

[0142] The present disclosure provides improved methods for measuring polysorbate levels in a sample. The disclosed methods provide consistent results that meet all of the criteria set forth in Table 3. Without being bound by theory, it is believed that the mobile phase and gradient program disclosed herein contribute to achieving the criteria set forth in Table 3.

[0143] In some embodiments, the mobile phases of the present disclosure effectively remove trace amounts of proteins. In some embodiments, the addition of acetonitrile to the first mobile phase effectively removes trace amounts of proteins.

[0144] In some embodiments, the mobile phases of the present disclosure effectively remove trace amounts of matrix components.

[0145] In some embodiments, the gradient program of the present disclosure improves the method to meet the criteria set forth in Table 3.

[0146] In some embodiments, the volume of the aliquot improves the method to meet the criteria set forth in Table 3.

[0147] [Table 1]

[0148] Enumerated Embodiments Embodiment 1. A method for measuring polysorbate levels in a sample, the method comprising: applying an aliquot of the sample to a high performance liquid chromatography system equipped 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 eluent after exiting the column, thereby measuring the polysorbate level, wherein the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and water; and the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

[0149] Embodiment 2. The method of embodiment 1, wherein the acid is a volatile acid.

[0150] Embodiment 3. The method of embodiment 2, wherein the volatile acid is formic acid.

[0151] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the first mobile phase and / or the second mobile phase comprises 0-2% acid.

[0152] Embodiment 5. The method of any one of embodiments 1-4, wherein the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

[0153] Embodiment 6. The method of any one of embodiments 1-5, wherein the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

[0154] Embodiment 7. The method of any one of embodiments 1-6, wherein the second mobile phase comprises 2% formic acid and 98% isopropyl alcohol.

[0155] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the measured polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80.

[0156] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the measured polysorbate comprises polysorbate 20 and / or polysorbate 80.

[0157] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the measured polysorbate comprises polysorbate 20.

[0158] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the measured polysorbate comprises polysorbate 80.

[0159] Embodiment 12. The method of any one of embodiments 1 to 11, further comprising applying a blank to a high performance liquid chromatography system equipped with a mixed-mode column before applying the aliquot to be measured to the column; eluting the blank using a gradient from a first mobile phase to a second mobile phase; and applying evaporative light scattering detection to the blank after exiting the column, wherein the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and water; and wherein the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

[0160] Embodiment 13. The method of embodiment 12, wherein the baseline signal is generated by applying evaporative light scattering detection to a blank.

[0161] Embodiment 14. The method of embodiment 12 or embodiment 13, further comprising applying a second blank to a high performance liquid chromatography system equipped with a mixed-mode column after applying the first blank to the column and before applying the aliquot to be measured to the column; eluting the blank using a gradient from the first mobile phase to the second mobile phase; and applying evaporative light scattering detection to the blank after it exits the column, wherein the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and water; and the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water; and wherein applying evaporative light scattering detection to the second blank generates a stable baseline signal.

[0162] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the first mobile phase and / or the second mobile phase have been prepared within two weeks of performing the method.

[0163] Embodiment 16. The method of any one of embodiments 1 to 15, further comprising quantifying the measured level of polysorbate in the sample.

[0164] Embodiment 17. The method of embodiment 16, wherein quantifying the measured level of polysorbate in the sample comprises comparing the measured amount of polysorbate to a standard curve.

[0165] 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 standard curve.

[0166] Embodiment 19. The method of embodiment 17 or embodiment 18, wherein the calibration curve is generated by separately applying one or more concentration standards containing known amounts of polysorbate to a high performance liquid chromatography system equipped with a mixed-mode column, eluting the concentration standards using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the concentration standards after each exits the column, thereby measuring the polysorbate levels, 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, isopropyl alcohol, or mixtures thereof, and water, and wherein the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

[0167] Embodiment 20. The method of embodiment 19, wherein one or more of the concentration standards produces a signal that is greater than the aliquot, and one or more of the concentration standards produces a signal that is less than the aliquot.

[0168]

[0023] 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.

[0169] Embodiment 22. The method of any one of embodiments 19-21, wherein the amount of polysorbate in the concentration standard containing the greatest amount of polysorbate has a peak height of about 80% of the maximum detectable signal.

[0170] Embodiment 23. The method of any one of embodiments 19 to 22, wherein applying evaporative light scattering detection to the blank produces a signal that is lower than the signal produced by a concentration standard containing the least amount of polysorbate.

[0171] Embodiment 24. The method of any one of embodiments 19 to 23, wherein the calibration curve is generated using three or more, four or more, five or more, or six or more concentration standards.

[0172] Embodiment 25. The method of any one of embodiments 17 to 24, wherein the calibration curve is generated by a gravimetric method.

[0173] Embodiment 26. The method of any one of embodiments 17 to 25, wherein the calibration curve has a coefficient of determination (R2) of 0.995 or greater.

[0174] Embodiment 27. The method of embodiment 26, wherein the coefficient of determination is 0.998 or greater.

[0175] Embodiment 28. The method of any one of embodiments 17 to 27, wherein the calibration curve is generated using a quadratic fit through zero.

[0176] Embodiment 29. The method of any one of embodiments 1 to 28, wherein the percentage relative standard deviation of two or more aliquots of the sample is 10 or less.

[0177] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the gradient comprises 100% of the first mobile phase and 0% of the second mobile phase from 0.0 to 2.0 minutes, 85% of the first mobile phase and 15% of the second mobile phase from 2.1 to 5.0 minutes, 30% of the first mobile phase and 70% of the second mobile phase from 5.1 to 7.6 minutes, 0% of the first mobile phase and 100% of the second mobile phase from 7.7 to 9.0 minutes, and / or 100% of the first mobile phase and 0% of the second mobile phase from 9.1 to 10.0 minutes.

[0178] Embodiment 31. The method of any one of embodiments 1 to 30, wherein the sample is applied to the mixed-mode column at a temperature of 22 to 28°C.

[0179] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the eluent is eluted from the column at a flow rate of 0.9 to 1.1 mL / min.

[0180] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the evaporative light scattering detection utilizes an Alltech 3300 detector.

[0181] Embodiment 34. The method of embodiment 33, wherein the method is carried out using a detector nebulizer temperature of 68 to 72°C.

[0182] Embodiment 35. The method of embodiment 33 or embodiment 34, wherein the method is performed using a detector gas flow rate of 2.3 to 2.7 L / min.

[0183] Embodiment 36. The method of any one of embodiments 1 to 32, wherein the evaporative light scattering detection utilizes an Agilent 1260 evaporative light scattering detector.

[0184] Embodiment 37. The method of embodiment 36, wherein the method is carried out using a detector nebulizer temperature of 43 to 47°C.

[0185] Embodiment 38. The method of embodiment 36 or claim 37, wherein the method is carried out using a detector heater tube temperature of 70 to 90°C.

[0186] Embodiment 39. The method of any one of embodiments 36 to 38, wherein the method is carried out using a detector gas flow rate of 1.3 to 1.7 L / min.

[0187] Embodiment 40. The method of any one of embodiments 1 to 39, wherein the sample is a drug substance.

[0188] Embodiment 41. The method of any one of embodiments 1 to 39, wherein the sample is a formulation.

[0189] Embodiment 42. A method for quantifying polysorbate levels in a sample, the method comprising: applying a first polysorbate concentration standard to a high performance liquid chromatography system equipped 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 eluent after it exits the column, thereby measuring the polysorbate level; and applying one or more additional polysorbate concentration standards, each having a different known amount of polysorbate compared to the first concentration standard, to a high performance liquid chromatography system equipped with a reversed-phase mixed-mode column, eluting the sample with a gradient from the first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby measuring the polysorbate level. measuring polysorbate levels in polysorbate concentration standards; generating a calibration curve from the measured polysorbate levels in polysorbate concentration standards; applying an aliquot of the sample to a high performance liquid chromatography system equipped with a mixed-mode column and eluting the sample with a gradient from a first mobile phase to a second mobile phase; applying evaporative light scattering detection to the eluent after exiting the column, thereby measuring the polysorbate levels; 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% isopropyl alcohol.

[0190] Embodiment 43. The method according to embodiment 40, wherein the method is carried out according to any one of embodiments 8 to 15 or embodiments 20 to 41. [Example]

[0191] The following examples are 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.

[0192] Example 1 Example 1 provides an exemplary application of the methods of the present disclosure.

[0193] The methods of the present disclosure were illustrated using an exemplary sample comprising antibodies in a matrix comprising polysorbate 80.

[0194] A calibration curve was generated using standards containing known amounts of polysorbate 80.

[0195] The samples were high protein concentration samples including polysorbate 80 equilibrated samples.

[0196] A mixed-mode column was selected, specifically, the exemplary column was a Waters Oasis Max Online Column (3 mm x 20 mm, 30 μm, part number: 186002053).

[0197] The first mobile phase for high performance liquid chromatography contained 2% formic acid, 20% acetonitrile, and 78% purified water. The reagents were added, mixed thoroughly, and kept at ambient conditions for up to 2 weeks.

[0198] The second mobile phase contained 2% formic acid in isopropyl alcohol. 20 mL of formic acid was added to 980 mL of isopropyl alcohol and mixed thoroughly. The second mobile phase was maintained at ambient conditions for 2 weeks.

[0199] An injector wash / needle soak was applied using 95:5 H2O:isopropyl alcohol.

[0200] To measure polysorbates, an Agilent 1200 / 1260 series high-performance liquid chromatography system equipped with an evaporative light scattering detector (ELSD) was used. The high-performance liquid chromatography system used was an Agilent 1260 Infinity II.

[0201] A calibration curve was generated gravimetrically using the concentration standards listed in Table 4. One of skill in the art will appreciate that calibration curves can readily be generated utilizing different concentrations of polysorbate concentration standards and / or using a different number of concentration standards, and that the following standards are exemplary only.

[0202] [Table 2]

[0203] The concentration standards were stable for up to 3 days under ambient or refrigerated (2-8°C) conditions.

[0204] All samples, blanks, and concentration standards were equilibrated to ambient temperature and mixed thoroughly by inversion.

[0205] Aliquots were transferred to HPLC vials.

[0206] Evaporative light scattering detection settings were selected such that the highest concentration standard did not exceed full scale.

[0207] The highest concentration standard had a peak height of approximately 80% of full scale, which represented a good compromise between sensitivity and range.

[0208] The detector gain was adjusted to achieve the desired sensitivity.

[0209] Prior to analysis, the evaporative light scattering detector was steam cleaned with 100% purified water for 1 hour at a flow rate of 1 mL / min. The detector settings for the steam clean were a gas flow rate of 1.3 SLM, an evaporation temperature of 100°C, and a nebulizer temperature of 50°C.

[0210] The column temperature was 25°C and the autosampler temperature was 5°C.

[0211] A 20 μL aliquot was applied to the column.

[0212] The evaporative light scattering detector has a gas flow rate 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.

[0213] A flush port was applied. For example, when running the method with a flush port on an Agilent 1260 Infinity II, we chose to use the flush port instead of a wash vial. We used an injector wash with the flush port and increased the injector wash time from the default of 3 seconds, if necessary.

[0214] The run time was 10 minutes and the gradient program in Table 5 was used.

[0215] [Table 3]

[0216] A blank, concentration standards, and one or more samples were applied according to the disclosed method in the order listed in Table 6.

[0217] [Table 4]

[0218] Concentration standards and samples were integrated with a linear baseline.

[0219] Alternative exemplary orders will be apparent to those skilled in the art. Those skilled in the art will understand that the illustrated injection orders are examples and should not be construed as limiting the scope of the claims in any way.

[0220] Those skilled in the art will understand that the present method is applicable to other polysorbates, including but not limited to polysorbate 20, polysorbate 40, and polysorbate 60.

[0221] Criteria were provided for comparing and evaluating the methods and are listed in Table 3.

[0222] A blank applied immediately before the standard was evaluated. To meet the acceptance criteria, a stable baseline must be observed. A stable baseline was observed for the two injections prior to the first standard.

[0223] The blank chromatogram was equivalent to the chromatogram illustrated in FIG.

[0224] It is normal to have a peak in the water blank at the retention time of polysorbate 80. If a peak is present in the blank, it should be smaller than the peak produced by the standard containing the lowest concentration of polysorbate 80.

[0225] A control sample was also evaluated that did not contain polysorbate 80. The control sample did not have any interfering peaks corresponding to equivalent levels of polysorbate 80 above 0.005% and therefore met the acceptance criteria.

[0226] The chromatographic non-interference of the local reference standard was also assessed. The criterion for interference of the local reference standard is that any interfering peaks in the local reference standard are smaller than the standard containing the lowest amount of polysorbate.

[0227] The chromatogram of the local reference standard is shown in FIG.

[0228] The calibration curves prepared from the concentration standards were evaluated, and the correlation coefficients between the chromatographic profiles of each standard and the calibration curves were evaluated.

[0229] The chromatographic profile was comparable to Figure 3 and therefore acceptable.

[0230] R 2 A value of ≧0.995 was considered acceptable (quadratic fit through zero).

[0231] The check STD was evaluated against the calibration curve, and the acceptance criteria was a measured % (w / v) of polysorbate 80 of ±20% of the theoretical amount of polysorbate calculated from the percentage difference obtained from the theoretical formula described below.

[0232]

number

[0233] Calibration curves were generated using validated software of concentration versus peak area as a quadratic fit through zero.

[0234] The concentration of polysorbate 80 in the samples was determined from a secondary calibration curve.

[0235] Figures 4a-b provide chromatograms of polysorbate 80. Figure 4a provides a chromatogram from a drug substance sample. Figure 4b provides a chromatogram from a formulation sample.

[0236] Those skilled in the art will understand 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.

[0237] Example 2 The recovery of polysorbate 80 was measured to assess the robustness of the method of Example 1.

[0238] Initial testing provided recoveries within 20% (Table 7).

[0239] [Table 5]

[0240] Further updates to this method further improved recovery to within 10% (Table 7).

[0241] The recoveries of the spiked samples also demonstrated robustness according to the criteria (Table 8). The spiked samples showed recoveries of 105-109% for the renewal tests.

[0242] [Table 6]

[0243] Example 3 The robustness of the method of Example 1 was evaluated to investigate the influence of different parameters and ranges of the method, as well as the effect of using different models and manufacturers of evaporative light scattering detectors.

[0244] To explore the parameters and scope of the method of Example 1, robustness tests were performed using an Alltech 3300 evaporative light scattering detector and an Agilent 1260 evaporative light scattering detector.

[0245] For the Alltech 3300 detector, various nebulizer temperatures were evaluated for evaporative light scattering detection, various column lots and column temperatures were evaluated for the column, and various flow rates were evaluated for high performance liquid chromatography. The parameters evaluated were as listed in Table 9.

[0246] Two different Water Oasis Max Mixed-mode 3 x 20 mm, 30 μm column lots were evaluated: one column lot was pre-injected and a second column lot was not pre-injected.

[0247] [Table 7]

[0248] For the Agilent 1260 evaporative light scattering detector, various nebulizer temperatures, heater tube temperatures, and gas flow rates were evaluated for the evaporative light scattering detector. The parameters evaluated were as listed in Table 10.

[0249] [Table 8]

[0250] A fractional factorial design of experiments was chosen for robustness testing. The experimental design for the Alltech 3300 was as described in Table 11. The experimental design for the Agilent 1260 evaporative light scattering detector was as described in Table 12.

[0251] [Table 9]

[0252] [Table 10]

[0253] The data confirmed that different evaporative light scattering detector models and manufacturers did not have any real effect on the test results, and therefore the disclosed method is valid regardless of the model and / or manufacturer of the evaporative light scattering detector.

[0254] The study was carried out according to a robustness protocol.

[0255] System suitability criteria were defined for this method, as defined in Table 3.

[0256] All robustness tests met the suitability criteria for the method.

[0257] USP tailing data was also collected for analysis.

[0258] System suitability results for the Alltech 3300 are shown in Tables 13 and 13B. Tailing results for the start and end check standards ranged from 1.16 to 1.46 for the Alltech 3300.

[0259] R 2 was >0.999 in all tests, meeting the standard curve acceptance criteria.

[0260] The results of the check standards for all samples showed that the difference from the theoretical amount of polysorbate was approximately 0.03%, which was within the acceptance criteria for all samples.

[0261] [Table 11]

[0262] [Table 12]

[0263] Twelve tests, listed in Table 14, were performed using three different samples, S2, S3, and D1, and statistical analysis was performed as listed in Table 10. The %PS80 was within 20% for all tests.

[0264] [Table 13]

[0265] The corresponding JMP software analysis tables are shown in Tables 15A and 15B, and the JMP statistical outputs are shown in Figures 5-7.

[0266] [Table 14]

[0267] [Table 15]

[0268] The data for sample S3 indicated that column temperature may have a statistically significant effect on the method at the 0.05 level. No other method parameters showed statistical significance at the 0.05 level.

[0269] To assess whether the column temperature had a practical effect on the method, the overall standard deviation of the 12 tests for the S3 sample was compared with the validation criterion of the intermediate precision standard deviation of the other tests. The standard deviation of the 12 tests for sample S3 was 0.0008, and the validation criterion of the intermediate precision standard deviation was SD≦0.007. Therefore, the analysis showed that the overall standard deviation of the S3 sample was smaller than the validation criterion. Therefore, the column temperature did not have a practical effect on the method.

[0270] Example 4 To confirm that the method is applicable to different detectors, the disclosed method was applied using two different models obtained from two different manufacturers.

[0271] A fractional factorial design of 12 trials was applied using an Alltech 3300 evaporative light scattering detector. A second trial of three trials was applied using an Agilent 1260 evaporative light scattering detector.

[0272] Similar results were observed for both models of evaporative light scattering detector. Table 16 shows similar results for S1, S2, and S3. The calculated results for S1 and S2 were higher than expected, but within the acceptance criteria.

[0273] [Table 16]

[0274] Thus, the disclosed method is robust across different makes and models of evaporative light scattering detectors.

[0275] The disclosed method is robust with respect to nebulizer temperature, gas flow rate, column lot, column temperature, and flow rate.

[0276] All robustness experiments met the method-system suitability criteria and produced acceptable data for each test.

[0277] Tailing results for polysorbate 80 on the Alltech 3300 ranged from 1.16 to 1.46.

[0278] Furthermore, the disclosed method demonstrated consistent performance in determining polysorbate concentrations across multiple antibody molecules.

[0279] Analysis of the evaluated method parameters identified no practical concerns and demonstrated that the method is robust over a range of operating conditions, as shown in Tables 17 and 18.

[0280] [Table 17]

[0281] [Table 18]

[0282] This example demonstrates that the method of Example 1 works with a variety of evaporative light scattering detectors from a variety of manufacturers under a variety of conditions within the tolerances of Tables 23 and 24.

[0283] Example 5 The robustness of the method for measuring polysorbate levels in drug substances and drug products was evaluated to confirm its applicability to samples from different origins.

[0284] The method was further evaluated using three antibody samples, as listed in Table 19. Two samples (DS (drug substance) and DP (formulation)) contained polysorbate 80, while the third sample (TFF) did not contain polysorbate 80.

[0285] [Table 19]

[0286] The suitability of the method was assessed for each sample by applying multiple aliquots from each sample according to the method of Example 1.

[0287] At least one blank was applied after the standards to assess carryover.

[0288] All results met the method system criteria in Table 3. A stable baseline was observed for the blank before the first standard was injected, and the blank chromatogram resembled Figure 1. No peaks at levels higher than the lowest standard were observed in the blank. No chromatographic interference was demonstrated for each sample. No carryover was observed.

[0289] The results are shown in Table 20.

[0290] [Table 20]

[0291] The theoretical percentage difference criteria for the day 0 check standard was ±10%.

[0292] Specificity was assessed by chromatographic non-interference, which was demonstrated by analyzing a blank, a local reference standard, and STD 1 according to the method of Example 1.

[0293] The blank produced a peak at the retention time of polysorbate 80. However, the calibration curve compensated for this interference. The baseline was consistent.

[0294] Any interfering peaks rising above baseline from the local reference standard were no greater than the corresponding peaks in STD 1.

[0295] The chromatograms were as shown in Figures 1 to 3. Figure 1 shows the blank, Figure 2 shows the local reference standard, and Figure 3 shows STD1.

[0296] Example 6 The precision and reproducibility of the method of Example 1 was determined by preparing six replicate drug substance samples and six replicate drug product samples.

[0297] The acceptance criterion required that the percentage relative standard deviation for six replicates did not exceed 10.

[0298] As shown in Table 21, the mean percentage relative standard deviation for the drug substance was 0.

[0299] [Table 21]

[0300] As shown in Table 22, the average percentage relative standard deviation for the formulations was 1.

[0301] [Table 22]

[0302] This example demonstrates that the method of Example 1 was reproducible for both the drug substance and the drug product with a percentage relative standard deviation of less than or equal to 10. Representative chromatograms for the drug substance and drug product were shown in Figures 4a and 4b, respectively.

[0303] Example 7 The stability of the standard solutions was evaluated to identify when new concentration standards should be generated.

[0304] Duplicate calibration curves were generated on day 0. One set of standards was stored at ambient conditions and the other set of standards was stored under refrigerated conditions (5°C ± 3°C). "Ambient conditions" was room temperature.

[0305] Formulation samples were analyzed on days 1, 2, and 3 using a calibration curve constructed from both sets of standards and using freshly prepared standards.

[0306] The criterion for the old standard to be considered stable was that the sample results calculated from the new and old standards differed by no more than 20%.

[0307] All standards remained stable over the 3-day test period, with the results shown in Table 23.

[0308] [Table 23]

[0309] The percentage difference was calculated according to the following formula:

[0310]

number

[0311] Example 8 To assess the specificity of the disclosed method and to ensure that the robustness of the method is consistent across multiple molecules, different antibody molecules were evaluated.

[0312] There are different antibody molecules in different matrices. The matrix compositions from the antibody molecules are listed in Table 24. Each polysorbate was polysorbate 80.

[0313] [Table 24]

[0314] By implementing this method, it was demonstrated that specificity for polysorbate 80 was achieved across all antibody molecules tested. Evaporative light scattering detection identified a single peak signal at approximately 6.5 minutes for all samples (FIG. 8). No significant matrix effect was observed. Therefore, the method of the present invention is suitable for measuring polysorbate 80 concentrations in different samples.

[0315] Example 9 Further testing was performed to demonstrate the robustness of the method of Example 1 with different polysorbates and different laboratories.

[0316] Two different laboratories applied the example method to samples containing polysorbate 20 (Table 25) or polysorbate 80 (Table 26).

[0317] [Table 25]

[0318] [Table 26]

[0319] This study demonstrated that the disclosed method is robust to different polysorbates and that robust results are achieved by the method in different laboratories.

[0320] Further robustness testing for polysorbate 20 identified a percentage relative standard deviation of less than 10 for two samples (Table 27).

[0321] [Table 27]

[0322] Two further studies identified acceptable robustness across the percentage relative standard deviations of the three samples containing polysorbate 80 (Tables 28 and 29).

[0323] [Table 28]

[0324] Sample S1 in Table 28 is a stressed sample. The disclosed method is also robust to stressed samples.

[0325] [Table 29]

[0326] D1 was a super stressed sample that was kept at 40° C. for 5 months. The sample showed high viscosity.

[0327] Thus, the methods of the present disclosure are robust to different polysorbates.

[0328] equivalent Those skilled in the art will recognize many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Detailed Description, but rather is as set forth in the claims.

Claims

1. 1. A method for measuring polysorbate levels in a sample, the method comprising: applying an aliquot of the sample to a high performance liquid chromatography system equipped with a mixed-mode column; eluting the sample using a gradient from a first mobile phase to a second mobile phase; applying evaporative light scattering detection to the eluate after it exits the column, thereby determining the polysorbate level; the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or a mixture thereof, and water; The method of claim 1, wherein the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

2. The method of 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 claims 1 to 3, wherein the first mobile phase and / or the second mobile phase comprises 0 to 2% acid.

5. The method of any one of claims 1 to 4, wherein the organic solvent in the first mobile phase is different from the organic solvent in the second mobile phase.

6. 6. The method of claim 1, wherein the first mobile phase comprises 2% formic acid, 20% acetonitrile, and 78% purified water.

7. 7. The method of claim 1, wherein the second mobile phase comprises 2% formic acid and 98% isopropyl alcohol.

8. 8. The method of any one of claims 1 to 7, wherein the polysorbates measured include polysorbate 20, polysorbate 40, polysorbate 60, and / or polysorbate 80.

9. 9. The method of any one of claims 1 to 8, wherein the measured polysorbates include polysorbate 20 and / or polysorbate 80.

10. 10. The method of any one of claims 1 to 9, wherein the measured polysorbate comprises polysorbate 20.

11. The method of any one of claims 1 to 10, wherein the polysorbate measured comprises polysorbate 80.

12. Before applying the aliquot to be measured to the column, applying a blank to a high performance liquid chromatography system equipped with a mixed-mode column; eluting the blank using a gradient from a first mobile phase to a second mobile phase; applying evaporative light scattering detection to the blank after it exits the column; the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or a mixture thereof, and water; 12. The method of any one of claims 1 to 11, wherein the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

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

14. After applying the first blank to the column and before applying the aliquot to be measured to the column, applying a second blank to a high performance liquid chromatography system equipped with a mixed-mode column; eluting the blank using a gradient from a first mobile phase to a second mobile phase; applying evaporative light scattering detection to the blank after it exits the column; the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or a mixture thereof, and water; the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water; 14. The method of claim 12 or claim 13, wherein a stable baseline signal is generated by applying evaporative light scattering detection to the second blank.

15. The method according to any one of claims 1 to 14, wherein the first mobile phase and / or the second mobile phase have been prepared within two weeks of carrying out the method.

16. 16. The method of any one of claims 1 to 15, further comprising quantifying the measured level of polysorbate in the sample.

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

18. 18. The method of claim 17, wherein the amount of polysorbate in the aliquot is determined by comparing the measured amount of polysorbate to the calibration curve.

19. The calibration curve is applying one or more concentration standards containing known amounts of polysorbate separately to a high performance liquid chromatography system equipped with a mixed-mode column; eluting the concentration standards using a gradient from a first mobile phase to a second mobile phase; applying evaporative light scattering detection to the concentrated standards after each exits the column, thereby determining polysorbate levels; by generating a calibration curve from the measured polysorbate levels in the concentration standards; the first mobile phase comprises 0-20% acid, 1-70% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or a mixture thereof, and water; 19. The method of claim 17 or claim 18, wherein the second mobile phase comprises 0-20% acid, 80-100% organic solvent selected from acetonitrile, methanol, tetrahydrofuran, isopropyl alcohol, or mixtures thereof, and optionally water.

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

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

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

23. 23. The method of any one of claims 19 to 22, wherein applying evaporative light scattering detection to a blank produces a signal that is lower than the signal produced by the concentration standard comprising the least amount of polysorbate.

24. 24. The method of any one of claims 19 to 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 by a gravimetric method.

26. The calibration curve has a coefficient of determination (R ２ 26. The method according to any one of claims 17 to 25, wherein

27. 27. The method of claim 26, wherein the coefficient of determination is 0.998 or greater.

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

29. 29. The method of any one of claims 1 to 28, wherein the percentage relative standard deviation of two or more aliquots of the sample is 10 or less.

30. The gradient is 100% first mobile phase and 0% second mobile phase from 0.0 min to 2.0 min; 2.1 min to 5.0 min: 85% first mobile phase and 15% second mobile phase; 5.1 min to 7.6 min: 30% of the first mobile phase and 70% of the second mobile phase; 0% of the first mobile phase and 100% of the second mobile phase from 7.7 minutes to 9.0 minutes; and / or 30. The method of any one of claims 1 to 29, comprising 100% of the first mobile phase and 0% of the second mobile phase from 9.1 minutes to 10.0 minutes.

31. 31. The method of any one of claims 1 to 30, wherein the sample is applied to the mixed-mode column at a temperature of 22 to 28°C.

32. 32. The method of any one of claims 1 to 31, wherein the eluent is eluted from the column at a flow rate of 0.9 to 1.1 mL / min.

33. 33. The method of any one of claims 1 to 32, wherein the evaporative light scattering detection utilizes an Alltech 3300 detector.

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

35. 35. The method of claim 33 or claim 34, wherein the method is carried out using a gas flow rate for the detector of 2.3 to 2.7 L / min.

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

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

38. 38. The method of claim 36 or claim 37, wherein the method is carried out using a heater tube temperature of the detector of from 70 to 90°C.

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

40. 40. The method of any one of claims 1 to 39, wherein the sample is an active pharmaceutical ingredient.

41. The method of any one of claims 1 to 39, wherein the sample is a formulation.

42. 1. A method for quantifying polysorbate levels in a sample, the method comprising: applying a first polysorbate concentration standard to a high performance liquid chromatography system equipped with a mixed-mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby determining the polysorbate level; applying one or more additional polysorbate concentration standards containing different known amounts of polysorbate compared to the first concentration standard to a high performance liquid chromatography system equipped with a reversed-phase mixed-mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby determining 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 equipped with a mixed-mode column, eluting the sample using a gradient from a first mobile phase to a second mobile phase, and applying evaporative light scattering detection to the eluent after it exits the column, thereby measuring the polysorbate level; and quantifying the amount of the polysorbate in the aliquot of the sample by comparison with the calibration curve; The method of claim 1, 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% isopropyl alcohol.

43. 41. The method of claim 40, wherein the method is performed according to any one of claims 8 to 15 or claims 20 to 41.

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