Polysorbate separation and measurement methods

Abstract

The present invention relates to a polysorbate separation method, comprising a polysorbate-containing solution to be measured flowing through a mixed-mode chromatograph and a reversed-phase chromatograph which are coupled in series, the use of the separation method in measuring the polysorbate content of said polysorbate-containing solution, a related apparatus, and a method for measuring the polysorbate content by using the separation method.

Description

Methods for the separation and detection of polysorbate Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for the separation and detection of polysorbate. Background Technology

[0002] Polysorbate is a nonionic surfactant commonly used as an emulsifier in biopharmaceuticals. It reduces existing protein-protein interactions without disrupting protein structure. Due to their unique manufacturing processes and usage methods, the composition of biopharmaceuticals requires strict control to ensure product safety, efficacy, and stability. As a pharmaceutical excipient, excessive use of polysorbate can lead to adverse effects such as hemolysis; therefore, strictly controlling the amount of polysorbate used in biopharmaceuticals is crucial. Summary of the Invention

[0003] The purpose of this invention is to provide a method for separating polysorbate and a method for detecting polysorbate content.

[0004] In some embodiments, the present invention provides a method for separating polysorbate, comprising subjecting a polysorbate-containing test solution to tandem mixed-mode chromatography and reversed-phase chromatography. In some embodiments, the chromatographic column for the reversed-phase chromatography is a polymer-based reversed-phase column. In some embodiments, the chromatographic column for the reversed-phase chromatography is a macropore polymer resin-based reversed-phase column.

[0005] In some embodiments, the present invention provides a method for detecting polysorbate content, comprising subjecting a test solution containing polysorbate and polysorbate standard solutions of different concentration gradients to tandem mixed-mode chromatography and reversed-phase chromatography, respectively, with the eluent detected by a detector and the polysorbate content in the test solution calculated. In some embodiments, the chromatographic column of the reversed-phase chromatography is a polymer-based reversed-phase column. In some embodiments, the chromatographic column of the reversed-phase chromatography is a macropore polymer resin-based reversed-phase column. In some embodiments, the polysorbate content in the test solution is calculated using a standard curve. In some embodiments, the calculation of the polysorbate content in the test solution includes plotting a standard curve with the polysorbate content of the polysorbate standard solution as the abscissa and the corresponding peak area as the ordinate, obtaining a linear regression equation; and substituting the polysorbate peak area of ​​the test solution into the linear regression equation to calculate the polysorbate content in the test solution.

[0006] In some embodiments, the present invention provides a method for detecting polysorbate content, comprising the following steps:

[0007] Step 1: Polysorbate standard solutions of different concentration gradients and test solutions containing polysorbate are subjected to mixed-mode chromatography and reversed-phase chromatography in series, respectively. The eluent is detected by a detector, and the chromatograms are recorded.

[0008] Step 2: Plot a standard curve with the polysorbate content of the polysorbate standard solution as the abscissa and the corresponding peak area as the ordinate to obtain the linear regression equation;

[0009] Step 3: Substitute the peak area of ​​polysorbate in the test solution into the linear regression equation to calculate the content of polysorbate in the test solution.

[0010] In some embodiments, the mixed-mode chromatography is a mixed-mode anion exchange / reversed-phase chromatography. In some embodiments, the column used in the mixed-mode chromatography is an Oasis MAX. In some embodiments, the column specifications are: 30 μm, 2.1 × 20 mm. In some embodiments, the column used in the mixed-mode chromatography is an Oasis MAX; 30 μm, 2.1 × 20 mm.

[0011] In some embodiments, the reversed-phase chromatography column is a polymer-based reversed-phase column. In some embodiments, the reversed-phase chromatography column is a large-pore polymer resin-based reversed-phase column. In some embodiments, the pore size of the polymer resin is approximately 1500 angstroms.

[0012] In some embodiments, the chromatographic column of the reversed-phase chromatography is MAb Pac. TM RP. In some embodiments, the column specifications are: 4 μm, 3.0 × 100 mm. In some embodiments, the reversed-phase chromatography column is MAb Pac. TM RP; 4μm, 3.0×100mm.

[0013] In some embodiments, the mixed-mode chromatography and / or reversed-phase chromatography is high-performance liquid chromatography.

[0014] In some embodiments, mobile phase A of the tandem mixed-mode chromatography and reversed-phase chromatography is an aqueous solution containing formic acid, and mobile phase B is an acetonitrile solution containing formic acid. In some embodiments, mobile phase A is an aqueous solution containing 0.01%-1% formic acid. In some embodiments, mobile phase A is an aqueous solution containing 0.05%-0.5% formic acid. In some embodiments, mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid. In some embodiments, mobile phase A is an aqueous solution containing about 0.1% formic acid. In some embodiments, mobile phase B is an acetonitrile solution containing 0.01%-1% formic acid. In some embodiments, mobile phase B is an acetonitrile solution containing 0.05%-0.5% formic acid. In some embodiments, mobile phase B is an acetonitrile solution containing 0.05%-0.2% formic acid. In some embodiments, mobile phase B is an acetonitrile solution containing about 0.1% formic acid. In some embodiments, mobile phase A is an aqueous solution containing 0.01%-1% formic acid, and mobile phase B is an acetonitrile solution containing 0.01%-1% formic acid. In some embodiments, mobile phase A is an aqueous solution containing about 0.1% formic acid, and mobile phase B is an acetonitrile solution containing about 0.1% formic acid.

[0015] In some embodiments, the column temperature of the tandem mixed-mode chromatography and reversed-phase chromatography is 4-80°C. In some embodiments, the column temperature is 50-70°C. In some embodiments, the column temperature is 55-65°C. In some embodiments, the column temperature is about 4°C, about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 80°C, or a range (including endpoints) of any two of these values, or any value therein.

[0016] In some embodiments, the flow rate of the tandem mixed-mode chromatography and reversed-phase chromatography is 0.05-0.5 mL / min. In some embodiments, the flow rate is 0.1-0.3 mL / min. In some embodiments, the flow rate is about 0.05 mL / min, about 0.1 mL / min, about 0.2 mL / min, about 0.3 mL / min, about 0.4 mL / min, about 0.5 mL / min, or a range (including endpoints) of any two of these values, or any value therein.

[0017] In some embodiments, the injection volume of the tandem mixed-mode chromatography and reversed-phase chromatography is 1-5 μL. In some embodiments, the injection volume is 1-3 μL. In some embodiments, the injection volume is about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, or a range (including endpoints) of any two of these values, or any value therein.

[0018] In some embodiments, the chromatographic conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are as follows: column temperature 4-80℃; flow rate 0.05-0.5mL / min; injection volume 1-5μL; mobile phase: mobile phase A is an aqueous solution containing formic acid, and mobile phase B is an acetonitrile solution containing formic acid.

[0019] In some embodiments, the chromatographic conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are as follows: column temperature of about 60°C; flow rate of about 0.2 mL / min; injection volume of about 2 μL; mobile phase: mobile phase A is an aqueous solution containing about 0.1% formic acid, and mobile phase B is an acetonitrile solution containing about 0.1% formic acid.

[0020] In some embodiments, the elution conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are as follows: decreasing mobile phase A from 99% to 10% over a period of time; maintaining mobile phase A at 10% for a period of time and then increasing mobile phase A to 99%; the total elution of mobile phase A and mobile phase B in each elution stage is 100%.

[0021] In some embodiments, the elution conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are as follows: 0-2 min, 99% mobile phase A; 2.1-4 min, 80% mobile phase A; 4.1-6 min, 70% mobile phase A; 6.1-9 min, 60% mobile phase A; 9-12 min, 60%-10% mobile phase A; 12-28 min, 10% mobile phase A; 28.1-33 min, 60% mobile phase A; 33.1-35 min, 99% mobile phase A; the total of mobile phase A and mobile phase B in each elution stage is 100%.

[0022] In some embodiments, the elution conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are: 0-2 min, 99% mobile phase A; 2-2.1 min, 99%–80% mobile phase A; 2.1-4 min, 80% mobile phase A; 4-4.1 min, 80%–70% mobile phase A; 4.1-6 min, 70% mobile phase A; 6-6.1 min, 70%–60% mobile phase A; 6.1-9 min… n, 60% mobile phase A; 9-12 min, 60%-10% mobile phase A; 12-28 min, 10% mobile phase A; 28-28.1 min, 10%-60% mobile phase A; 28.1-33 min, 60% mobile phase A; 33-33.1 min, 60%-99% mobile phase A; 33.1-35 min, 99% mobile phase A; the total of mobile phase A and mobile phase B in each elution stage is 100%.

[0023] In some embodiments, when the proportion of mobile phase A decreases from more than about 60% to about 60% and is maintained at about 60% for a period of time, the eluent of mixed-mode chromatography is switched from flowing through waste liquid to flowing through reversed-phase chromatography; when the proportion of mobile phase A decreases from about 60% to less than about 60% and then rises back to about 60% and is maintained at about 60% for a period of time, the eluent of mixed-mode chromatography is switched from flowing through reversed-phase chromatography to flowing through waste liquid. In some embodiments, when the proportion of mobile phase A decreases from about 99% to about 60% and is maintained at about 60% for a period of time, the eluent of mixed-mode chromatography is switched from flowing through waste liquid to flowing through reversed-phase chromatography; when the proportion of mobile phase A decreases from about 60% to about 10% and then rises back to about 60% and is maintained at about 60% for a period of time, the eluent of mixed-mode chromatography is switched from flowing through reversed-phase chromatography to flowing through waste liquid; the total of mobile phase A and mobile phase B in each elution stage is 100%.

[0024] In some implementations, when the elution time is less than 8 min, the eluent of the mixed-mode chromatography flows through the waste liquid; when the elution time is greater than or equal to 8 min and less than 32 min, the eluent of the mixed-mode chromatography flows through the reversed-phase chromatography; when the elution time is greater than or equal to 32 min, the eluent of the mixed-mode chromatography flows through the waste liquid.

[0025] In some implementation schemes, the elution conditions are shown in Table 3.

[0026] In some embodiments, the detector is an electrospray detector. In some embodiments, the detection conditions of the electrospray detector are: a sampling frequency of 2-10 Hz; and an atomization temperature of 30-80°C. In some embodiments, the sampling frequency is 3-8 Hz. In some embodiments, the sampling frequency is approximately 5 Hz. In some embodiments, the atomization temperature is 35-60°C. In some embodiments, the atomization temperature is approximately 50°C. In some embodiments, the detection conditions of the electrospray detector are: a sampling frequency of approximately 5 Hz; and an atomization temperature of approximately 50°C.

[0027] In some embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 (PS80), or polysorbate 85. In some embodiments, the polysorbate is polysorbate 80.

[0028] In some embodiments, the present invention provides a method for separating polysorbate 80, comprising subjecting a polysorbate-containing test solution to tandem mixed-mode chromatography and reversed-phase chromatography; the chromatographic conditions being: Oasis MAX column; 30 μm, 2.1 × 20 mm and MAb Pac TM RP; 4 μm, 3.0 × 100 mm; column temperature: approximately 60 °C; flow rate: approximately 0.2 mL / min; injection volume: approximately 2 μL; mobile phase: mobile phase A is an aqueous solution containing approximately 0.1% formic acid, mobile phase B is an acetonitrile solution containing approximately 0.1% formic acid; elution conditions: 0–2 min, 99% mobile phase A; 2–2.1 min, 99%–80% mobile phase A; 2.1–4 min, 80% mobile phase A; 4–4.1 min, 80%–70% mobile phase A; 4.1–6 min, 70% mobile phase A; 6–6.1 min, 70%–60% mobile phase A; 6.1–9 min, 60% mobile phase A; 9–12 min… n, 60%–10% mobile phase A; 12–28 min, 10% mobile phase A; 28–28.1 min, 10%–60% mobile phase A; 28.1–33 min, 60% mobile phase A; 33–33.1 min, 60%–99% mobile phase A; 33.1–35 min, 99% mobile phase A; the total of mobile phase A and mobile phase B in each elution stage is 100%; when the elution time is less than 8 min, the eluent of the mixed-mode chromatography flows through the waste liquid; when the elution time is greater than or equal to 8 min and less than 32 min, the eluent of the mixed-mode chromatography flows through the reversed-phase chromatography; when the elution time is greater than or equal to 32 min, the eluent of the mixed-mode chromatography flows through the waste liquid.

[0029] In some embodiments, the present invention provides a method for detecting the content of polysorbate 80, comprising the following steps:

[0030] Step 1: Polysorbate 80 standard solutions with different concentration gradients and test solutions containing polysorbate were subjected to mixed-mode chromatography and reversed-phase chromatography in series, respectively. The eluent was detected by an electrospray detector, and the chromatograms were recorded.

[0031] Chromatographic conditions were as follows: Oasis MAX column; 30 μm, 2.1 × 20 mm and MAb Pac TM RP; 4 μm, 3.0 × 100 mm; column temperature: approximately 60 °C; flow rate: approximately 0.2 mL / min; injection volume: approximately 2 μL; mobile phase: mobile phase A is an aqueous solution containing approximately 0.1% formic acid, mobile phase B is an acetonitrile solution containing approximately 0.1% formic acid; elution conditions: 0–2 min, 99% mobile phase A; 2–2.1 min, 99%–80% mobile phase A; 2.1–4 min, 80% mobile phase A; 4–4.1 min, 80%–70% mobile phase A; 4.1–6 min, 70% mobile phase A; 6–6.1 min, 70%–60% mobile phase A; 6.1–9 min, 60% mobile phase A; 9–12 min… n, 60%–10% mobile phase A; 12–28 min, 10% mobile phase A; 28–28.1 min, 10%–60% mobile phase A; 28.1–33 min, 60% mobile phase A; 33–33.1 min, 60%–99% mobile phase A; 33.1–35 min, 99% mobile phase A; the total of mobile phase A and mobile phase B in each elution stage is 100%; when the elution time is less than 8 min, the eluent of the mixed-mode chromatography flows through the waste liquid; when the elution time is greater than or equal to 8 min and less than 32 min, the eluent of the mixed-mode chromatography flows through the reversed-phase chromatography; when the elution time is greater than or equal to 32 min, the eluent of the mixed-mode chromatography flows through the waste liquid;

[0032] Step 2: Plot a standard curve with the polysorbate 80 content of the polysorbate 80 standard solution as the abscissa and the corresponding peak area as the ordinate to obtain the linear regression equation.

[0033] Step 3: Substitute the peak area of ​​polysorbate 80 in the test solution into the linear regression equation to calculate the content of polysorbate 80 in the test solution.

[0034] In some implementation schemes, the elution conditions are shown in Table 3.

[0035] In some embodiments, the concentration range of the polysorbate standard solutions with different concentration gradients is 40–180 μg / mL. In some embodiments, the concentrations of the polysorbate standard solutions with different concentration gradients are 40, 70, 100, 130, 160, and 180 μg / mL, respectively.

[0036] In some embodiments, the detection range of the detection method is 10–200 μg / mL. In some embodiments, the detection range of the detection method is 40–180 μg / mL.

[0037] In some implementations, the electrospray detector is a Corona Veo electrospray detector.

[0038] In some embodiments, the test solution is an antibody solution. In some embodiments, the test solution is an antibody preparation. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is an anti-IL-12 / 23 antibody. In some embodiments, the antibody preparation comprises an anti-IL-12 / 23 antibody. In some embodiments, the anti-IL-12 / 23 antibody is ustekinumab or a biosimilar thereof. In some embodiments, the antibody preparation is an injectable formulation.

[0039] The separation method of this invention can effectively separate polysorbate from other components (e.g., proteins or other excipients) in a test solution containing polysorbate (e.g., a protein solution containing polysorbate, such as an antibody preparation), eliminating interference from other components, especially protein interference, and is suitable for detecting the polysorbate content in a sample. The detection method of this invention has advantages such as good specificity, good system adaptability, good accuracy, good repeatability, and good precision. It has good linearity and a wide detection range, eliminates protein interference, and improves the detection accuracy of polysorbate (e.g., polysorbate 80), thereby effectively controlling the quality of test solutions containing polysorbate (e.g., polysorbate 80) (e.g., ustekinumab injection). Attached Figure Description

[0040] Figure 1 is a schematic diagram of the column connection method; where 100 is a mixed-mode column; 110 is a switching valve; 120 is a waste liquid collection container; 130 is a reversed-phase column; and 140 is a detector.

[0041] Figure 2 shows the specificity test confirmation spectrum. A is the vertical overlay spectrum and B is the tilted overlay spectrum. Among them, sample 1 is the excipient solution, sample 2 is ustekinumab injection 5, sample 3 is PS80 quality control solution, sample 4 is ustekinumab injection 2, sample 5 is ustekinumab injection 4, sample 6 is the excipient solution (without PS80), and samples 7 and 8 are blank solutions. Detailed Implementation

[0042] the term

[0043] The term "about" refers to a typical range of error for a given value that is readily known to those skilled in the art. In some embodiments, "about" is used herein to refer to the described value and its range of ±10%, ±5%, ±1%.

[0044] The terms "comprising" or "including" mean that a composition and method, etc., include the listed elements (such as components in the composition, steps in the method, etc.), but do not exclude others. When "consisting substantially of..." is used to define a composition and method, it means excluding other elements that have a fundamental effect on the combination for the intended use, but does not exclude elements that do not substantially affect the characteristics of the composition or method. "Containing..." means excluding elements not specifically listed. Embodiments defined by each of these transitional terms are within the scope of the invention. For example, when a composition is described as comprising components A, B, and C, compositions consisting substantially of A, B, and C and compositions consisting of A, B, and C are independently within the scope of the invention.

[0045] The term "polysorbate" (also abbreviated as PS) refers to PEGylated sorbitols esterified with fatty acids, including polysorbate 20 (polyoxyethylene (20) sorbitol monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitol monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitol monostearate), polysorbate 80 (polyoxyethylene (20) sorbitol monooleate), and polysorbate 85 (polyoxyethylene (20) sorbitol trioleate). Polysorbates are also known as Tween (e.g., polysorbate 20 is called Tween 20, and polysorbate 80 is called Tween 80).

[0046] The term "chromatography" refers to any kind of technique for separating an analyte of interest from other molecules present in a mixture or during binding and elution, wherein the analyte of interest is separated from other molecules as a result of the different rates at which the molecules in the mixture flow through a stationary medium under the influence of the mobile phase. Non-limiting examples of chromatography include conventional reversed-phase chromatography (RP), ion-exchange chromatography (IEX), mixed-mode chromatography, and normal-phase chromatography (NP).

[0047] The term "liquid chromatography" refers to the process in which a biological / chemical mixture carried by a liquid can be separated by means of the differential distribution of components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography.

[0048] The term "high performance liquid chromatography" or "HPLC" refers to a chromatographic process that uses a liquid as the mobile phase and employs a high-pressure delivery system to pump a single solvent or a mixture of solvents or buffer solutions with different polarities into a chromatographic column packed with a stationary phase, where the components are separated.

[0049] The term "stationary phase" refers to the phase that remains stationary during chromatographic separation and retains the sample. The term "mobile phase" refers to the substance that carries the analyte forward during chromatographic processing; it is another phase that is in equilibrium with the stationary phase and drives the sample forward.

[0050] The terms "reversed-phase chromatography," "reversed-phase liquid chromatography," or "RPLC" refer to chromatographic methods that use a hydrophobic stationary phase. Reversed-phase chromatography typically employs a polar (aqueous) initial mobile phase; therefore, hydrophobic molecules in the polar mobile phase tend to adsorb onto the hydrophobic stationary phase, while hydrophilic molecules pass through the column and are eluted first. Organic (non-polar) solvents that reduce hydrophobic interactions can be used to decrease the polarity of the mobile phase, thereby eluting the hydrophobic molecules from the column. The greater the hydrophobicity of the analyte, the stronger its binding force to the stationary phase, and the higher the concentration of organic solvent required to elute the analyte. Based on the different packing materials (stationary phase matrix), reversed-phase chromatography columns can be classified into silica-based, polymer-based, and other inorganic packing materials. "Polymer-based" refers to chromatographic column packing materials with a cross-linked organic polymer framework, such as polymethyl methacrylate, polystyrene / polymethyl methacrylate, or polystyrene-divinylbenzene packing materials. Unlike silica-based reversed-phase columns with C4, C8, and C18 functional groups, polymer-based reversed-phase columns can withstand mobile phases and regeneration conditions across the entire pH range (pH 0-14), exhibiting a longer lifespan compared to other matrix columns under the same conditions. Large-pore polymer resin matrices (e.g., 1500 Å) can help address peak tailing and baseline elevation issues caused by strong adsorption.

[0051] The term "mixed-mode chromatography" refers to a method using a chromatographic solid phase carrying at least two different types of functional groups (each capable of interacting with the molecule of interest). Mixed-mode chromatography typically uses ligands with more than one interaction mode with one or more components in the sample. Alternatively, the ligands typically contain at least two distinct but cooperative sites that interact with the substance to be bound. For example, one of these sites may have a charge-charge type interaction with the substance of interest, while other sites may have electron acceptor-donor type interactions and / or hydrophobic and / or hydrophilic interactions. Electron donor-acceptor type interactions include hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, and induced dipole interactions.

[0052] The terms "ion exchange" and "ion exchange chromatography" refer to the separation of substances based on the affinity between the oppositely charged components of the chromatographic stationary phase and the reversible binding of their charges. Ion exchange chromatography includes cation exchange, anion exchange, and mixed-mode ion exchange chromatography. For example, a negatively charged stationary phase in cation exchange chromatography can bind positively charged components in the sample, or it can primarily bind impurities, allowing target proteins to "flow through" the column (FT-CIEX). A positively charged stationary phase in anion exchange chromatography can bind negatively charged components in the sample. Based on the charged groups on the cation or anion exchange stationary phase, cation exchange chromatography can be classified as strong or weak cation or anion exchange chromatography.

[0053] The term "electrospray detector" is a general-purpose detector that can detect any non-volatile and partially semi-volatile substances. It is also gradient compatible and suitable for quantitative and qualitative analysis of compounds without UV absorption in liquid chromatography. Its principle is based on the fact that the mass of the analyte in the sample is proportional to the signal current. It can detect all non-volatile and partially semi-volatile substances, regardless of the molecular structure of the analyte.

[0054] The abbreviations used in this article have the following meanings:

[0055] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0056] The instruments and equipment, reagents and samples, and detection methods used in specific embodiments of the present invention are as follows:

[0057] 1. Instruments and equipment:

[0058] High performance liquid chromatograph: Waters e2695;

[0059] Column: Oasis MAX (30 μm, 2.1 × 20 mm; Waters, catalog number 186002052); MAb Pac TM RP (4μm, 3.0×100mm; Thermo, part number 088644);

[0060] Detector: Corona Veo detector (Thermo);

[0061] Switching valve: EV700-100-WA (Waters).

[0062] 2. Reagents and Samples:

[0063] Reagents: Polysorbate 80 (PS80) standard (Nanjing Weier); Formic acid (Thermo); Acetonitrile (CNW);

[0064] sample:

[0065] Table 1. Sample Formulation

[0066] Standard solution:

[0067] Accurately weigh 1g of PS80, dissolve it in an appropriate amount of ultrapure water, and transfer it to a 1000mL volumetric flask. Make up to volume with ultrapure water and sonicate for 10min to obtain PS80 standard stock solution (1000μg / mL). Transfer an appropriate volume of the above PS80 standard stock solution to a 1000mL volumetric flask, make up to volume with ultrapure water, and mix well to prepare PS80 standard solutions with different concentration gradients (40, 70, 100, 130, 160, 180μg / mL).

[0068] Mobile phase:

[0069] Mobile phase A (0.1% formic acid aqueous solution): Measure 500 mL of ultrapure water, add 0.5 mL of formic acid, mix well, and sonicate for 10 min to obtain the mobile phase A.

[0070] Mobile phase B (0.1% formic acid acetonitrile solution): Measure 500 mL of acetonitrile, add 0.5 mL of formic acid, mix well, and sonicate for 10 min to obtain the mobile phase B.

[0071] 3. Detection Method

[0072] 1) Chromatographic conditions

[0073] Software: Empower data analysis system;

[0074] Injection volume: 2 μL;

[0075] Column temperature: 60℃;

[0076] Electrospray detector (CAD detector) acquisition frequency: 5Hz;

[0077] CAD detector atomization temperature: High (50℃).

[0078] 2) Column connection method:

[0079] As shown in Figure 1, chromatographic columns 100 and 130 are connected to switching valve 110. When switch 110 is open, the eluent from column 100 flows out through switch 110 into waste collection container 120. When switch 110 is closed, the eluent from column 100 flows through switch 110 to column 130, and then to CAD detector 140. Column 100 is an Oasis MAX column, and column 130 is an MAb Pac column. TM RP.

[0080] 3) Switching valve event

[0081] Table 2. Switching Valve Event Table

[0082] 4) Elution conditions

[0083] Table 3. Elution gradient table

[0084] In the following embodiments, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the art and can be obtained commercially; the methods used are conventional technical methods in the art, and those skilled in the art can undoubtedly implement the methods and obtain the corresponding results based on the content of the embodiments.

[0085] Example 1: Specificity Test

[0086] Utekinumab injection solution 2, Utekinumab injection solution 4, excipient solution, excipient solution (excluding PS80), Utekinumab injection solution 5, PS80 quality control solution (PS80 concentration of 0.1 mg / mL, prepared in the same way as 100 μg / mL polysorbate 80 standard solution, the same below), and blank solution (ultrapure water) were prepared and injected into the high performance liquid chromatograph. The eluent was detected by a CAD detector, the chromatogram was recorded, and specificity analysis was performed. The specific results are shown in Figure 2.

[0087] As shown in Figure 2, the PS80 control solution, ustekinumab injection 2, ustekinumab injection 5, and excipient solution all showed six distinct absorption peaks in the range of 20-28 min, while ustekinumab injection 4, excipient solution (without PS80), and blank solution did not show any peaks in this range, indicating that the method has good specificity.

[0088] Example 2: System Suitability Test

[0089] Step 1: Prepare polysorbate 80 standard solutions with different concentration gradients (concentrations of 40, 70, 100, 130, 160, and 180 μg / mL), PS80 quality control solution (PS80 concentration of 0.1 mg / mL), and blank solution (ultrapure water). Inject these solutions into a high-performance liquid chromatograph (HPLC). Detect the eluents using a CAD detector, record the chromatograms, and perform system suitability analysis.

[0090] Step 2: Plot a standard curve with the PS80 content of the polysorbate 80 standard solution as the abscissa and the corresponding peak area as the ordinate, to obtain the linear regression equation: Y = 1.22e 3 X 2 +1.83e 6 X+4.42e 7 ;R 2 =0.9999. The sum of the peak areas corresponding to the six absorption peaks of PS80 (see Figure 2) is taken as the total peak area of ​​PS80.

[0091] Step 3: Substitute the total peak area of ​​PS80 into the linear regression equation to calculate the PS80 content. The specific results are shown in Table 4.

[0092] Table 4. Summary of System Applicability Results

[0093] As shown in Table 4, the blank solution showed no interference at the PS80 peak; the PS80 recovery rates of each PS80 standard and PS80 quality control solution were between 90-110%, indicating that the method has good system applicability.

[0094] Example 3: Linearity and Range

[0095] Step 1: Prepare polysorbate 80 standard solutions with different concentration gradients (40, 70, 100, 130, 160, and 180 μg / mL), PS80 quality control solution (PS80 concentration of 0.1 mg / mL), and blank solution (ultrapure water). Inject these solutions into a high-performance liquid chromatograph (HPLC). Detect the eluents using a CAD detector, record the chromatograms, and perform linearity confirmation analysis using a standard curve. Inject each standard solution three times.

[0096] Step 2: Plot a standard curve with the PS80 content of the polysorbate 80 standard solution as the abscissa and the corresponding peak area as the ordinate, to obtain the linear regression equation: Y = -1.13e 3 X 2 +2.46e 6 X+3.07e 6 ;R 2 =0.9990. The sum of the peak areas corresponding to the six absorption peaks of PS80 (see Figure 2) is taken as the total peak area of ​​PS80.

[0097] Step 3: Substitute the total peak area of ​​PS80 into the linear regression equation to calculate the PS80 content. The specific results are shown in Table 5.

[0098] Table 5. Summary of Linearity and Range Results

[0099] As shown in Table 5, PS80 exhibits good linearity within the standard curve range, indicating that the method has good linearity and range.

[0100] Example 4: Accuracy Test

[0101] Step 1: Prepare three samples with high, medium, and low PS80 concentrations: ustekinumab injection 1, ustekinumab injection 2, and ustekinumab injection 3. Prepare three copies of each sample. At the same time, prepare blank solution (ultrapure water), polysorbate 80 standard solutions with different concentration gradients (concentrations of 40, 70, 100, 130, 160, and 180 μg / mL, respectively), ustekinumab injection 5, and PS80 quality control solution (PS80 concentration of 0.1 mg / mL). Inject these solutions into a high-performance liquid chromatograph (HPLC). Detect the eluent using a CAD detector, record the chromatograms, and perform accuracy analysis.

[0102] Step 2: Plot a standard curve with the PS80 content of the polysorbate 80 standard solution as the x-axis and the corresponding peak area as the y-axis, obtaining the linear regression equation: Y = 5.29e 2 X 2 +1.94e 6 X+4.03e 6 ;R 2 =0.9995. The sum of the peak areas corresponding to the six absorption peaks of PS80 (see Figure 2) is taken as the total peak area of ​​PS80.

[0103] Step 3: Substitute the total peak area of ​​PS80 into the linear regression equation to calculate the PS80 content. The specific results are shown in Table 6.

[0104] Table 6. Summary of Accuracy Results

[0105] As shown in Table 6, the accuracy of this method is good.

[0106] Example 5: Precision Test

[0107] 5.1 Repeatability

[0108] Using ustekinumab injection 2 as the test sample, six test sample solutions were prepared in parallel and injected into a high performance liquid chromatograph. The eluent was detected by a CAD detector, the chromatograms were recorded, and repeatability analysis was performed.

[0109] Table 7. Summary of Repeatability Results

[0110] As shown in Table 7, the method has good repeatability.

[0111] 5.2 Intermediate Precision

[0112] One mobile phase and six test solutions of ustekinumab injection 2 were prepared by different analysts at different times and injected into a high performance liquid chromatograph. The eluent was detected by a CAD detector, the chromatogram was recorded, and intermediate precision analysis was performed.

[0113] Table 8. Summary of Intermediate Precision Results

[0114] As shown in Table 8, the intermediate precision of this method is good.

Claims

1. A method for separating polysorbate, comprising subjecting a test solution containing polysorbate to tandem mixed-mode chromatography and reversed-phase chromatography; wherein, The chromatographic column used in the reversed-phase chromatography is a polymer-based reversed-phase column.

2. A method for detecting polysorbate content, comprising subjecting a test solution containing polysorbate and polysorbate standard solutions of different concentration gradients to tandem mixed-mode chromatography and reversed-phase chromatography, respectively, wherein the eluent is detected by a detector and the polysorbate content in the test solution is calculated; wherein, The chromatographic column used in the reversed-phase chromatography is a polymer-based reversed-phase column.

3. A method for detecting polysorbate content, comprising the following steps: Step 1: Polysorbate standard solutions of different concentration gradients and test solutions containing polysorbate are subjected to mixed-mode chromatography and reversed-phase chromatography in series, respectively. The eluent is detected by a detector, and the chromatograms are recorded. Step 2: Plot a standard curve with the polysorbate content of the polysorbate standard solution as the abscissa and the corresponding peak area as the ordinate to obtain the linear regression equation; Step 3: Substitute the peak area of ​​polysorbate in the test solution into the linear regression equation to calculate the content of polysorbate in the test solution; The chromatographic column used in the reversed-phase chromatography is a polymer-based reversed-phase column.

4. The method according to any one of claims 1-3, wherein the mixed-mode chromatography is a mixed-mode anion exchange / reversed-phase chromatography.

5. The method according to any one of claims 1-4, wherein the mobile phase A of the tandem mixed-mode chromatography and reversed-phase chromatography is an aqueous solution containing formic acid, and the mobile phase B is an acetonitrile solution containing formic acid.

6. The method of claim 5, wherein the mobile phase A is an aqueous solution containing 0.01%-1% formic acid, and the mobile phase B is an acetonitrile solution containing 0.01%-1% formic acid.

7. The method of claim 6, wherein the mobile phase A is an aqueous solution containing about 0.1% formic acid, and the mobile phase B is an acetonitrile solution containing about 0.1% formic acid.

8. The method according to any one of claims 1-7, wherein the column temperature of the tandem mixed-mode chromatography and reversed-phase chromatography is 4-80°C.

9. The method according to any one of claims 1-7, wherein the column temperature of the tandem mixed-mode chromatography and reversed-phase chromatography is 50-70°C.

10. The method according to any one of claims 1-7, wherein the column temperature of the tandem mixed-mode chromatography and reversed-phase chromatography is about 60°C.

11. The method according to any one of claims 1-10, wherein the flow rate of the tandem mixed-mode chromatography and reversed-phase chromatography is 0.05-0.5 mL / min.

12. The method according to any one of claims 1-10, wherein the flow rate of the tandem mixed-mode chromatography and reversed-phase chromatography is 0.1-0.3 mL / min.

13. The method according to any one of claims 1-10, wherein the flow rate of the tandem mixed-mode chromatography and reversed-phase chromatography is about 0.2 mL / min.

14. The method according to any one of claims 1-13, wherein the injection volume of the tandem mixed-mode chromatography and reversed-phase chromatography is 1-5 μL.

15. The method according to any one of claims 1-13, wherein the injection volume of the tandem mixed-mode chromatography and reversed-phase chromatography is 1-3 μL.

16. The method according to any one of claims 1-13, wherein the injection volume of the tandem mixed-mode chromatography and reversed-phase chromatography is about 2 μL.

17. The method according to any one of claims 1-16, wherein the elution conditions for the tandem mixed-mode chromatography and reversed-phase chromatography are: 0-2 min, 99% mobile phase A; 2-2.1 min, 99% to 80% mobile phase A; 2.1-4 min, 80% mobile phase A; 4-4.1 min, 80%–70% mobile phase A; 4.1-6 min, 70% mobile phase A; 6-6.1 min, 70%–60% mobile phase A; 6.1-9 min, 60% mobile phase A; 9-12 min, 60%–10% mobile phase A; 12-28 min, 10% mobile phase A; 28-28.1 min, 10%–60% mobile phase A; 28.1-33 min, 60% mobile phase A; 33-33.1 min, 60%–99% mobile phase A; 33.1-35 min, 99% mobile phase A; the total of mobile phase A and mobile phase B in each elution stage is 100%.

18. The method of claim 17, wherein when the elution time is less than 8 min, the eluent of the mixed-mode chromatography flows through the waste liquid; when the elution time is greater than or equal to 8 min and less than 32 min, the eluent of the mixed-mode chromatography flows through the reversed-phase chromatography; and when the elution time is greater than or equal to 32 min, the eluent of the mixed-mode chromatography flows through the waste liquid.

19. The method according to any one of claims 1-18, wherein the chromatographic column of the mixed-mode chromatography is an Oasis MAX.

20. The method of claim 19, wherein the chromatographic column has the following specifications: 30 μm, 2.1 × 20 mm.

21. The method according to any one of claims 1-20, wherein the reversed-phase chromatographic column is MAb Pac. TM RP.

22. The method of claim 21, wherein the chromatographic column has the following specifications: 4 μm, 3.0 × 100 mm.

23. The method according to any one of claims 2-22, wherein the detector is an electrospray detector.

24. The method as described in claim 23, wherein the detection conditions of the electrospray detector are: a sampling frequency of 2-10 Hz; and an atomization temperature of 30-80 ℃.

25. The method of claim 24, wherein the sampling frequency is 3-8 Hz, or about 5 Hz.

26. The method of claim 24 or 25, wherein the atomization temperature is 35-60°C, or about 50°C.

27. The method according to any one of claims 1-26, wherein the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 85.

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