CN114646693A

CN114646693A - Method for detecting polysorbate80 content in biological preparation

Info

Publication number: CN114646693A
Application number: CN202011505810.4A
Authority: CN
Inventors: 张婷婷; 袁杰; 王超; 刘颖; 靳征; 苏冬梅; 娄竞
Original assignee: Shenyang Sunshine Pharmaceutical Co ltd
Current assignee: Shenyang Sunshine Pharmaceutical Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-06-21

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of polysorbate80 in a biological preparation. The detection method comprises the following steps: step 1, preparing a reference substance solution and a test solution; step 2, detecting the reference solution and the test solution respectively by using a high performance liquid chromatography-evaporative light scattering device to obtain chromatograms; and 3, calculating the content of polysorbate80 in the test solution by an external standard method according to the chromatograms of the reference solution and the test solution. The detection method has the advantages of high accuracy, good precision, good durability, simple and quick operation and high detection efficiency. The detection method is suitable for quality control of polysorbate80 in biological preparations.

Description

Method for detecting polysorbate80 content in biological preparation

Technical Field

The invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content of polysorbate80 in a biological preparation.

Background

Polyoxyethylene sorbitan monooleate, polysorbate80 for short, is an organic compound. Polysorbate80 is a yellowish to orange-yellow viscous liquid; is easily soluble in water, ethanol, vegetable oil, ethyl acetate, methanol, and toluene, and insoluble in mineral oil; gelatinizing at low temperature, and recovering after heating; it is very smelly and slightly bitter.

Polysorbate80 is a nonionic surfactant, is often used as an emulsifier in biological agents, and can reduce the original interaction between proteins without destroying the protein structure. Due to its special production process and mode of use, the composition of ingredients of biological agents needs to be strictly controlled to ensure the safety, effectiveness and stability of the product. Polysorbate80 is used as a pharmaceutical adjuvant, and excessive use of polysorbate80 can cause adverse effects such as hemolysis, so that strict control of the dosage of polysorbate80 in the biological preparation is of great importance.

The content of polysorbate80 is determined by a colorimetric method by using 3203 polysorbate80 residual quantity determination method of three biological products in the 2020 edition of Chinese pharmacopoeia. The method has complicated pretreatment steps and long time for the test sample. Polysorbate80 contains more hydrophilic groups and does not contain any chromophore, so that conventional ultraviolet detection is not suitable for detection of the substances. An Evaporative Light Scattering Detector (ELSD) is a universal detector, and eliminates the difficulties commonly found in conventional High Performance Liquid Chromatography (HPLC) detection methods. The response of ELSD is independent of the optical characteristics of the sample, and any sample with volatility lower than that of the mobile phase can be detected without being influenced by the functional group. The method has the greatest advantage that the method can detect compounds without chromophores, and is an effective means for determining polysorbate 80. Therefore, the development of an HPLC-ELSD method suitable for detecting the polysorbate80 content in biological agents is urgently needed.

Disclosure of Invention

The invention aims to provide an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) detection method for the polysorbate80 content in a biological preparation. The invention also aims to provide the application of the detection method in the quality control of biological agents.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

the invention provides a method for detecting the polysorbate80 content in a biological preparation,

the detection method comprises the following steps: step 1, preparing a reference substance solution and a test solution; step 2, detecting the reference solution and the test solution respectively by using a high performance liquid chromatography-evaporative light diffuser to obtain chromatograms; step 3, calculating the content of polysorbate80 in the test solution by an external standard method according to the chromatograms of the reference solution and the test solution; the reference solution is polysorbate80 solution with known concentration; the test solution is a biological agent solution;

the chromatographic conditions include: the chromatographic column is a quaternary amine polymer solid phase extraction column; the mobile phase is a binary mobile phase system consisting of a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid isopropanol solution; the elution mode is gradient elution;

operating conditions of the evaporative light diffuser include any one or combination of the following i) to iii): i) the gain value is 100-500; ii) an air pressure of 20 to 30 psi; iii) the temperature of the drift tube is 90-100 ℃;

the elution procedure for the gradient elution was: 0.00-2.40min, 40-90% of mobile phase A + 60-10% of mobile phase B, and flowing to waste liquid by the flow path switching valve; 2.40-3.40min, 40-60% of mobile phase A + 60-40% of mobile phase B, and a flow path switching valve flows to a detector; 3.50-4.60min, 100% mobile phase B, flow direction detector of flow path switching valve; 4.70-6.60min, 40-90% of mobile phase A + 60-10% of mobile phase B, and the flow direction of the flow path switching valve to the detector; the flow path switching valve flowed to waste liquid for 6.60 min.

In a preferred embodiment, the mobile phase A is 1.8-2.2% (v/v) formic acid in water, and the mobile phase B is 1.8-2.2% (v/v) formic acid in isopropanol; more preferably, the mobile phase A is 2.0% (v/v) formic acid water solution, and the mobile phase B is 2.0% (v/v) formic acid isopropanol solution.

In a preferred embodiment, the quaternary amine polymer solid phase extraction column is an Oasis MAX solid phase extraction column.

In a preferred embodiment, the Oasis MAX solid phase extraction column employs a mixed mode mechanism.

In a preferred embodiment, the column temperature of the quaternary amine macromolecule solid phase extraction column is 25-35 ℃; preferably, the column temperature of the quaternary amine polymer solid phase extraction column is 30 ℃.

In a preferred embodiment, the flow rate of the mobile phase is 1.13 to 1.38 mL/min; preferably, the mobile phase flow rate is 1.25 mL/min.

In a preferred embodiment, the operating conditions of the evaporative light diffuser include any one or combination of the following i) to iii): i) the gain value is 250; ii) a gas pressure of 25 psi; iii) the drift tube temperature is 95 ℃.

In a preferred embodiment, the elution procedure of the gradient elution is: 0.00min, 90% mobile phase A + 10% mobile phase B, flow to waste liquor by the flow path switching valve; 1.00-2.40min, 60% of mobile phase A + 40% of mobile phase B, and the flow path switching valve flows to waste liquid; 2.40-3.40min, 60% mobile phase A + 40% mobile phase B, flow direction detector of flow path switching valve; 3.50-4.60min, 100% mobile phase B, flow direction detector of flow path switching valve; 4.70-6.60min, 90% mobile phase A + 10% mobile phase B, flow direction detector of flow path switching valve; the flow path switching valve flowed to waste liquid for 6.60 min.

In a preferred embodiment, the hplc-evaporative light diffuser is a hplc Waters e2695 system.

In a preferred embodiment, the external standard method comprises: (1) performing linear regression on the main peak area logarithm values and the concentration logarithm values of the chromatograms of a plurality of reference substance solutions with different concentrations to obtain a linear equation; (2) substituting the peak area of the main peak of the chromatogram of the test solution into the linear equation to calculate the concentration of polysorbate80 in the test solution; preferably, the plurality of different concentrations is at least 5 different concentrations.

In a preferred embodiment, the biological agent comprises an antibody and a fusion protein.

Preferably, the antibodies include monoclonal antibodies and bispecific/multispecific antibodies.

Preferably, the antibody comprises an anti-human TNF α monoclonal antibody.

Preferably, the biological agent is an injection.

Preferably, the content of polysorbate80 in the biological preparation is 0.025 mg/mL-0.5 mg/mL.

In a second aspect, the invention provides the use of the above detection method in the quality control of biological agents.

The invention has the beneficial effects that: the invention provides an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) detection method for detecting polysorbate80 content in a biological preparation. The detection method disclosed by the invention is high in accuracy, and the recovery rate is 85-105%; the accuracy is good, and the RSD value does not exceed 9%; the durability is good, and the relative difference is between 0.10 and 6.76 percent; the method is simple and quick to operate, the test sample does not need special pretreatment, the removal of the protein and the detection of polysorbate80 can be carried out simultaneously, the single-needle sample injection time only needs 6.6min, and the detection efficiency is high. The detection method is suitable for quality control of polysorbate80 in biological preparations.

Drawings

The abscissa in the following figures is the unit of minutes and the ordinate is the unit of LSU (unit of intensity of scattered light signal). Fig. 6-33 are representative chromatogram plots for fig. 1 provided.

FIG. 1: the ultrapure water chromatogram in example 2.

FIG. 2: a0.025 mg/mL PS80 chromatogram of example 2.

FIG. 3: a0.025 mg/mL PS80 chromatogram of example 3.

FIG. 4: a0.025 mg/mL PS80 chromatogram of example 4.

FIG. 5: a0.025 mg/mL PS80 chromatogram of example 5.

FIG. 6: STD1 in example 6 is directed to the chromatogram of fig. 1.

FIG. 7: STD2 in example 6 is directed to the chromatogram of fig. 1.

FIG. 8: STD3 in example 6 is directed to the chromatogram of fig. 1.

FIG. 9: STD4 in example 6 is directed to the chromatogram of fig. 1.

FIG. 10: STD5 in example 6 is directed to the chromatogram of fig. 1.

FIG. 11: sample 1 of example 6 is presented in chromatogram 1.

FIG. 12: sample 2 of example 6 is presented in chromatogram of item 1.

FIG. 13: sample 3 of example 6 is presented in chromatogram of 1 st hand.

FIG. 14 is a schematic view of: sample 4 of example 6 is presented in chromatogram 1.

FIG. 15 is a schematic view of: sample 5 of example 6 is presented in chromatogram of item 1.

FIG. 16: sample 1, in example 7 is chromatogram 1.

FIG. 17: sample 5 of example 7 is presented in chromatogram of item 1.

FIG. 18: sample 1 of example 8 is presented in chromatogram 1.

FIG. 19: sample 5 of example 8 is presented in chromatogram 1.

FIG. 20: sample 1 of example 9 is presented in chromatogram 1.

FIG. 21: sample 5 of example 9 is presented in chromatogram of item 1.

FIG. 22: sample 1 of example 10 is presented in chromatogram 1.

FIG. 23: sample 5 of example 10 is presented in chromatogram of item 1.

FIG. 24: sample 1 of example 11 is presented in chromatogram 1.

FIG. 25: sample 5 of example 11 is presented in chromatogram 1.

FIG. 26: sample 1 of example 12 is directed to the chromatogram of 1 st.

FIG. 27 is a schematic view showing: sample 5 of example 12 is presented in chromatogram 1.

FIG. 28: sample 1 of example 13 is presented in chromatogram 1.

FIG. 29: sample 5 in example 13 is presented in chromatogram of item 1.

FIG. 30: sample 1 of example 14 is presented in chromatogram 1.

FIG. 31: sample 5 of example 14 is directed to the chromatogram of 1 st.

FIG. 32: sample 1, in example 15 is chromatogram 1.

FIG. 33: sample 5 of example 15 is presented in chromatogram of item 1.

Detailed Description

The present invention will be explained in further detail with reference to specific examples.

The experimental methods in the examples, in which the specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer. General equipment, materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

1.1 obtaining monoclonal antibodies

For exemplary purposes, humanized anti-human TNF α monoclonal antibodies were selected as experimental samples in this study. The sequence and preparation method of the antibody are shown in Chinese patent CN 102037012B.

1.2 preparation of control

200. + -.10 mg of polysorbate80 (manufacturer: Nanjing Will; lot No. 20171001) was weighed into a 100mL volumetric flask, and about 80mL of ultrapure water was added and dissolved with stirring, diluted with ultrapure water and fixed to volume, followed by mixing for about 30 minutes to obtain a stock solution (2mg/mL) of polysorbate 80. Control solutions were prepared as in table 1.

TABLE 1 Polysorbate80 control solution formulation

Reference substance	Theoretical concentration (mg/mL)	Polysorbate80 stock solution (2mg/mL)	Ultrapure water
				STD 1	0.025	12.5μL	987.5μL
STD 2	0.05	25μL	975μL
				STD 3	0.1	50μL	950μL
STD 4	0.25	125μL	875μL
				STD 5	0.5	250μL	750μL

1.3 preparation of test articles

Monoclonal antibody (mAb-1) (protein content 72mg/mL) and Polysorbate80 (Polysorbate80, PS80) in 1.1 were diluted with ultrapure water to the corresponding concentrations as in Table 2.

TABLE 2 preparation of the test articles

1.4 preparation of the Mobile phase

Transferring 20mL of formic acid and 980mL of ultrapure water into a 1L reagent bottle, and uniformly mixing to obtain a 2% formic acid aqueous solution (v/v); transfer 20mL formic acid and 980mL isopropanol to a 1L reagent bottle and mix well to give a 2% formic acid isopropanol solution (v/v).

Transferring 18mL of formic acid and 982mL of ultrapure water into a 1L reagent bottle, and uniformly mixing to obtain a 1.8% formic acid aqueous solution (v/v); transfer 18mL formic acid and 982mL isopropanol to a 1L reagent bottle and mix well to give a 1.8% formic acid isopropanol solution (v/v).

Transferring 22mL of formic acid and 978mL of ultrapure water into a 1L reagent bottle, and uniformly mixing to obtain a 2.2% formic acid aqueous solution (v/v); transfer 22mL formic acid and 978mL isopropanol to a 1L reagent bottle and mix well to give a 2.2% formic acid isopropanol solution (v/v).

Example 2

2.1 conditions of the experiment

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 3); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 3 gradient elution procedure

Numbering	Time (min)	Flow rate (mL/min)	A％	B％	Flow direction of the flow path switching valve
						1	0.00	1.25	40	60	Waste liquid
2	2.40	1.25	40	60	Detector
						3	3.40	1.25	40	60
4	3.50	1.25	0	100
						5	4.60	1.25	0	100
6	4.70	1.25	40	60
						7	6.60	1.25	40	60	Waste liquid

2.2 data analysis

The ultrapure water and the PS80 with the concentration of 0.025mg/mL are subjected to sample injection analysis, the ultrapure water spectrum is shown in figure 1, and the PS80 spectrum with the concentration of 0.025mg/mL is shown in figure 2. No characteristic polysorbate80 peak could be detected.

Example 3

3.1 conditions of the experiment

High performance liquid chromatograph: the Waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 4); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant is as follows: others, 1.0000 seconds; data acquisition rate: 10 pps; sample injection amount: 25 μ L.

TABLE 4 gradient elution procedure

Numbering	Time (min)	Flow rate (mL/min)	A％	B％	Flow direction of the flow path switching valve
						1	0.00	1.25	90	10	Waste liquid
2	1.00	1.25	60	40
						3	2.40	1.25	60	40	Detector
4	3.40	1.25	60	40
						5	3.50	1.25	0	100
6	4.60	1.25	0	100
						7	4.70	1.25	90	10
8	6.60	1.25	90	10	Waste liquid

3.2 data analysis

When the sample is analyzed by using PS80 with the concentration of 0.025mg/mL, the map of PS80 with the concentration of 0.025mg/mL is shown in figure 3. And a polysorbate80 characteristic peak can be detected after the mobile phase gradient is optimized.

Example 4

4.1 conditions of the experiment

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 5); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 100, respectively; temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample injection amount: 25 μ L.

TABLE 5 gradient elution procedure

Number of	Time (min)	Flow rate (mL/min)	A％	B％	Flow direction of the flow path switching valve
						1	0.00	1.25	90	10	Waste liquid
2	1.00	1.25	60	40
						3	2.40	1.25	60	40	Detector
4	3.40	1.25	60	40
						5	3.50	1.25	0	100
6	4.60	1.25	0	100
						7	4.70	1.25	90	10
8	6.60	1.25	90	10	Waste liquid

4.2 data analysis

When the sample is analyzed by using PS80 with the concentration of 0.025mg/mL, the map of PS80 with the concentration of 0.025mg/mL is shown in FIG. 4. When the gain value is set as 100, a characteristic peak of polysorbate80 can be detected.

Example 5

5.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 6); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 500, a step of; temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample injection amount: 25 μ L.

TABLE 6 gradient elution procedure

5.2 data analysis

When the sample is analyzed by using PS80 with the concentration of 0.025mg/mL, the map of PS80 with the concentration of 0.025mg/mL is shown in FIG. 5. When the gain value is set to 500, a characteristic peak of polysorbate80 can be detected.

Example 6

6.1 Experimental conditions

A high performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 7); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 7 gradient elution procedure

6.2 data analysis

And (3) injecting samples of a reference substance and a test substance for analysis, wherein 3 needles are respectively injected into each reference substance and each test substance. A calibration curve y of 1.18x +5.08 and a correlation coefficient of 0.9972 were obtained by using a double-log linear equation of the peak area average value of the main peak of 3 needles of each control (STD1-5) and the polysorbate80 concentration through chromatographic software. And (3) making a double-log linear equation by using the peak area average value of the main peak of 3 needles of each test sample (test samples 1-5) and the concentration of polysorbate80 to obtain a calibration curve y which is 1.23x +5.03 and a correlation coefficient 0.9981.

And (3) making a double-logarithm linear equation by using the average value of the main peak area of each reference product (STD1-5) and the concentration of the polysorbate80, substituting the average value of the main peak area of each test product into the linear equation y of 1.18x +5.08 by using Empower 3 software, calculating the actually measured concentration of the polysorbate80 of each test product, calculating the recovery rate of each test product, and inspecting the accuracy. As a result, as shown in Table 8, the recovery rates at 5 concentration points were 95.7%, 88.8%, 85.6%, 99.1%, and 105.0%, respectively, and the accuracy was good. The map of the 1 st needle of the control article is shown in FIGS. 6-10, and the map of the 1 st needle of the test article is shown in FIGS. 11-15.

TABLE 8 accuracy results

And substituting the main peak area of each test sample into a linear equation y of 1.18x +5.08 by using Empower 3 software, calculating the actually measured concentration of polysorbate80 of each test sample, calculating the Relative Standard Deviation (RSD) of the actually measured concentration, and inspecting repeatability. As a result, the measured concentrations RSD of 5 concentration points were 8.77%, 0.80%, 3.44%, 1.62%, and 2.17%, respectively, as shown in Table 9, and the reproducibility was good.

TABLE 9 repeatability results

Example 7

7.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 10); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 (c); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample injection amount: 25 μ L.

TABLE 10 gradient elution procedure

7.2 data analysis

And (3) feeding a reference substance (STD1-5), a test sample 1 and a test sample 5 for analysis, wherein 1 needle is respectively fed into each reference substance, and 2 needles are respectively fed into the test sample. Performing double-log linear equation (1.21 x + 5.24) on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 through chromatographic software, wherein the correlation coefficient is 0.9978; and substituting the main peak areas of the test sample 1 and the test sample 5 into a linear equation by using Empower 3 software, calculating the actual measurement concentration of the polysorbate80 of the test sample 1 and the test sample 5, and calculating the average value of the actual measurement concentration. The results are shown in Table 11. The 1 st map of the sample 1 is shown in fig. 16, and the 1 st map of the sample 5 is shown in fig. 17.

TABLE 11 actual measurement results of polysorbate80 concentrations for test article 1 and test article 5

Example 8

8.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 12); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 25 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 12 gradient elution procedure

8.2 data analysis

A reference sample (STD1-5), a test sample 1 and a test sample 5 are injected and analyzed, wherein 1 needle is inserted into each reference sample, and 2 needles are inserted into each test sample. Performing double-logarithmic linear equation (1.21 x + 5.23) on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 by using chromatographic software, wherein the correlation coefficient is 0.9984; the Empower 3 software is used for substituting the main peak areas of the test sample 1 and the test sample 5 into a linear equation, calculating the actual measurement concentration of the polysorbate80 of the test sample 1 and the test sample 5 at the column temperature of 25 ℃, calculating the average value of the actual measurement concentration, and calculating the relative difference with the average value of the actual measurement concentration at the column temperature of 30 ℃ in the embodiment 7. The results are shown in Table 13. The 1 st map of the sample 1 is shown in fig. 18, and the 1 st map of the sample 5 is shown in fig. 19.

TABLE 13 actual measurement results of polysorbate80 concentrations of test sample 1 and test sample 5 at a column temperature of 25 deg.C

Example 9

9.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; and (3) mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 14); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 35 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 14 gradient elution procedure

9.2 data analysis

A reference sample (STD1-5), a test sample 1 and a test sample 5 are injected and analyzed, wherein 1 needle is inserted into each reference sample, and 2 needles are inserted into each test sample. Performing double logarithm linear equation y of 1.25x +5.11 and correlation coefficient 0.9983 on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 by using chromatographic software; the Empower 3 software is used for substituting the main peak areas of the test sample 1 and the test sample 5 into a linear equation, calculating the actual measurement concentration of the polysorbate80 of the test sample 1 and the test sample 5 at the column temperature of 35 ℃, calculating the average value of the actual measurement concentration, and calculating the relative difference with the average value of the actual measurement concentration at the column temperature of 30 ℃ in the embodiment 7. The results are shown in Table 15. The map of the 1 st needle of the test sample 1 is shown in fig. 20, and the map of the 1 st needle of the test sample 5 is shown in fig. 21.

TABLE 15 actual concentration results of polysorbate80 of test article 1 and test article 5 at a column temperature of 35 deg.C

Example 10

10.1 Experimental conditions

High performance liquid chromatograph: the Waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 1.8% aqueous formic acid; mobile phase B: 1.8% formic acid in isopropanol; gradient elution (procedure see table 16); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 16 gradient elution procedure

10.2 data analysis

And (3) feeding a reference substance (STD1-5), a test sample 1 and a test sample 5 for analysis, wherein 1 needle is respectively fed into each reference substance, and 2 needles are respectively fed into the test sample. Performing double-log linear equation y of 1.23x +5.22 and correlation coefficient 0.9988 on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 by using chromatographic software; the Empower 3 software is used for substituting the main peak areas of the test sample 1 and the test sample 5 into a linear equation, the actual measurement concentration of polysorbate80 of the test sample 1 and the test sample 5 is calculated when the mobile phase contains 1.8% of formic acid, the average value of the actual measurement concentration is calculated, and the relative difference between the average value of the actual measurement concentration and the average value of the actual measurement concentration when the mobile phase contains 2% of formic acid in the embodiment 7 is calculated. The results are shown in Table 17. The 1 st needle map of the test article 1 is shown in fig. 22, and the 1 st needle map of the test article 5 is shown in fig. 23.

Table 17 results of actual concentration measurement of polysorbate80 in test sample 1 and test sample 5 when the mobile phase contains 1.8% formic acid

Example 11

11.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2.2% aqueous formic acid; mobile phase B: 2.2% formic acid in isopropanol; gradient elution (procedure see table 18); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 18 gradient elution procedure

11.2 data analysis

And (3) feeding a reference substance (STD1-5), a test sample 1 and a test sample 5 for analysis, wherein 1 needle is respectively fed into each reference substance, and 2 needles are respectively fed into the test sample. Performing double-logarithmic linear equation y of 1.22x +5.23 by using the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 through chromatographic software, wherein the correlation coefficient is 0.9985; the main peak areas of the test sample 1 and the test sample 5 are substituted into a linear equation by using Empower 3 software, the actual measured concentrations of the polysorbate80 of the test sample 1 and the test sample 5 when the mobile phase contains 2.2% of formic acid are obtained through calculation, the average value of the actual measured concentrations is calculated, and the relative difference between the average value of the actual measured concentrations and the average value of the actual measured concentrations when the mobile phase contains 2% of formic acid in the embodiment 7 is calculated. The results are shown in Table 19. The 1 st map of the sample 1 is shown in fig. 24, and the 1 st map of the sample 5 is shown in fig. 25.

TABLE 19 actual measurement of polysorbate80 concentrations in test 1 and test 5 when the mobile phase contains 2.2% formic acid

Example 12

12.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); a mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 20); sample room temperature: 5 ℃; flow rate: 1.13 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 20 gradient elution procedure

Numbering	Time (min)	Flow rate (mL/min)	A％	B％	Flow direction of the flow path switching valve
						1	0.00	1.13	90	10	Waste liquid
2	1.00	1.13	60	40
						3	2.40	1.13	60	40	Detector
4	3.40	1.13	60	40
						5	3.50	1.13	0	100
6	4.60	1.13	0	100
						7	4.70	1.13	90	10
8	6.60	1.13	90	10	Waste liquid

12.2 data analysis

A reference sample (STD1-5), a test sample 1 and a test sample 5 are injected and analyzed, wherein 1 needle is inserted into each reference sample, and 2 needles are inserted into each test sample. Performing double-log linear equation (1.25 x + 5.24) on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 through chromatographic software, wherein the correlation coefficient is 0.9992; the main peak areas of the test sample 1 and the test sample 5 are substituted into a linear equation by using Empower 3 software, the actual measurement concentrations of the polysorbate80 of the test sample 1 and the test sample 5 at the flow rate of 1.13mL/min are calculated, the average value of the actual measurement concentrations is calculated, and the relative difference between the actual measurement concentrations and the average value of the flow rate of 1.25mL/min in the embodiment 7 is calculated. The results are shown in Table 21. The map of the 1 st needle of the test sample 1 is shown in FIG. 26, and the map of the 1 st needle of the test sample 5 is shown in FIG. 27.

TABLE 21 actual measurement results of Polysorbate80 concentration in test article 1 and test article 5 at a flow rate of 1.13mL/min

Example 13

13.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; and (3) chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 22); sample room temperature: 5 ℃; flow rate: 1.38 mL/min; column temperature: 30 ℃; ELSD gas pressure: 25 psi; ELSD gain: 250 of (a); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant is as follows: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 22 gradient elution procedure

Numbering	Time (min)	Flow rate (mL/min)	A％	B％	Flow direction of the flow path switching valve
						1	0.00	1.38	90	10	Waste liquid
2	1.00	1.38	60	40
						3	2.40	1.38	60	40	Detector
4	3.40	1.38	60	40
						5	3.50	1.38	0	100
6	4.60	1.38	0	100
						7	4.70	1.38	90	10
8	6.60	1.38	90	10	Waste liquid

13.2 data analysis

A reference sample (STD1-5), a test sample 1 and a test sample 5 are injected and analyzed, wherein 1 needle is inserted into each reference sample, and 2 needles are inserted into each test sample. Performing a double-log linear equation of 1.16x +5.18 by using the peak area of the main peak of each control (STD1-5) and the concentration of polysorbate80 through chromatographic software, wherein the correlation coefficient is 0.9966; the main peak areas of the test sample 1 and the test sample 5 are substituted into a linear equation by using Empower 3 software, the actual measured concentrations of the polysorbate80 of the test sample 1 and the test sample 5 at the flow rate of 1.38mL/min are calculated, the average value of the actual measured concentrations is calculated, and the relative difference between the actual measured concentrations and the average value of the flow rate of 1.25mL/min in the embodiment 7 is calculated. The results are shown in Table 23. The 1 st map of the sample 1 is shown in fig. 28, and the 1 st map of the sample 5 is shown in fig. 29.

TABLE 23 actual measurement results of polysorbate80 concentrations of test sample 1 and test sample 5 at a flow rate of 1.38mL/min

Example 14

14.1 Experimental conditions

A high performance liquid chromatograph: waters e2695 system; and (3) chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); mobile phase A: 2% aqueous formic acid; and (3) mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 24); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 20 psi; ELSD gain: 250 (c); temperature of the drift tube: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 24 gradient elution procedure

14.2 data analysis

And (3) feeding a reference substance (STD1-5), a test sample 1 and a test sample 5 for analysis, wherein 1 needle is respectively fed into each reference substance, and 2 needles are respectively fed into the test sample. Performing a double-log linear equation of 1.19x +5.32 by using the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 through chromatographic software, wherein the correlation coefficient is 0.9990; the main peak areas of the test sample 1 and the test sample 5 are substituted into a linear equation by using Empower 3 software, the actual measured concentrations of the polysorbate80 of the test sample 1 and the test sample 5 at an ELSD air pressure of 20psi are calculated, the average value of the actual measured concentrations is calculated, and the relative difference between the actual measured concentration and the average value of the ELSD air pressure of 25psi in the embodiment 7 is calculated. The results are shown in Table 25. The 1 st map of the sample 1 is shown in FIG. 30, and the 1 st map of the sample 5 is shown in FIG. 31.

TABLE 25 actual polysorbate80 concentration results for test 1 and test 5 at ELSD pressure of 20psi

Example 15

15.1 Experimental conditions

High performance liquid chromatograph: waters e2695 system; a chromatographic column: oasis MAX chromatography column (2.1 × 20mm, 30 μm); a mobile phase A: 2% aqueous formic acid; mobile phase B: 2% formic acid in isopropanol; gradient elution (procedure see table 26); sample room temperature: 5 ℃; flow rate: 1.25 mL/min; column temperature: 30 ℃; ELSD gas pressure: 30 psi; ELSD gain: 250 of (a); drift tube temperature: 95 ℃; atomizer mode: heating at a power level of 50%; time constant: others, 1.0000 seconds; data acquisition rate: 10 pps; sample introduction amount: 25 μ L.

TABLE 26 gradient elution procedure

15.2 data analysis

A reference sample (STD1-5), a test sample 1 and a test sample 5 are injected and analyzed, wherein 1 needle is inserted into each reference sample, and 2 needles are inserted into each test sample. Performing a double-log linear equation of 1.28x +5.10 on the peak area of the main peak of each reference substance (STD1-5) and the concentration of polysorbate80 by using chromatographic software, wherein the correlation coefficient is 0.9994; the main peak areas of the test sample 1 and the test sample 5 are substituted into a linear equation by using Empower 3 software, the actual measured concentrations of the polysorbate80 of the test sample 1 and the test sample 5 at an ELSD air pressure of 30psi are calculated, the average value of the actual measured concentrations is calculated, and the relative difference between the actual measured concentration and the average value of the ELSD air pressure of 25psi in the embodiment 7 is calculated. The results are shown in Table 27. The 1 st map of the sample 1 is shown in FIG. 32, and the 1 st map of the sample 5 is shown in FIG. 33.

TABLE 27 actual concentration results of polysorbate80 for test 1 and test 5 at ELSD pressure of 30psi

The results in examples 8-15 show that the relative difference between the measured concentration of the test sample and the measured concentration of the preferred embodiment is between 0.10% and 6.76% and the durability is good when the column temperature is 5 ℃ for the preferred embodiment, the mobile phase composition is 10% for the preferred embodiment, the flow rate is 10% for the preferred embodiment, and the ELSD pressure is 20% for the preferred embodiment.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that various changes and substitutions can be made without departing from the spirit of the invention.

Claims

1. A method for detecting the polysorbate80 content in a biological preparation is characterized in that,

operating conditions of the evaporative light diffuser include any one or combination of i) through iii) as follows: i) the gain value is 100-500; ii) an air pressure of 20 to 30 psi; iii) the temperature of the drift tube is 90-100 ℃;

the elution procedure for the gradient elution was: 0.00-2.40min, 40-90% of mobile phase A + 60-10% of mobile phase B, and the flow path switching valve flows to waste liquid; 2.40-3.40min, 40-60% of mobile phase A + 60-40% of mobile phase B, and a flow path switching valve flows to a detector; 3.50-4.60min, 100% mobile phase B, flow direction detector of flow path switching valve; 4.70-6.60min, 40-90% of mobile phase A + 60-10% of mobile phase B, and the flow direction of the flow path switching valve to the detector; the flow path switching valve flowed to waste liquid for 6.60 min.

2. The detection method according to claim 1, wherein the mobile phase a is a 1.8% to 2.2% (v/v) formic acid aqueous solution, and the mobile phase B is a 1.8% to 2.2% (v/v) formic acid isopropyl alcohol solution; preferably, the mobile phase A is 2.0% (v/v) formic acid water solution, and the mobile phase B is 2.0% (v/v) formic acid isopropanol solution.

3. The detection method of claim 1 or 2, wherein the quaternary amine polymer solid phase extraction column is an Oasis MAX solid phase extraction column.

4. The detection method of any one of claims 1 to 3, wherein the column temperature of the quaternary amine polymer solid phase extraction column is 25 to 35 ℃; preferably, the column temperature of the quaternary ammonium macromolecule solid phase extraction column is 30 ℃.

5. The detection method according to any one of claims 1 to 3, wherein the flow rate of the mobile phase is 1.13 to 1.38 mL/min; preferably, the mobile phase flow rate is 1.25 mL/min.

6. The detection method according to any one of claims 1 to 5, wherein the operating conditions of the evaporative light diffuser include any one or a combination of the following i) to iii): i) the gain value is 250; ii) a gas pressure of 25 psi; iii) the drift tube temperature was 95 ℃.

7. The detection method according to any one of claims 1 to 6, wherein the gradient elution is carried out by an elution procedure comprising: 0.00min, 90% mobile phase A + 10% mobile phase B, flow to waste liquor by the flow path switching valve; 1.00-2.40min, 60% of mobile phase A + 40% of mobile phase B, and the flow path switching valve flows to waste liquid; 2.40-3.40min, 60% mobile phase A + 40% mobile phase B, flow direction detector of flow path switching valve; 3.50-4.60min, 100% mobile phase B, flow direction detector of flow path switching valve; 4.70-6.60min, 90% mobile phase A + 10% mobile phase B, flow direction detector of flow path switching valve; the flow path switching valve flowed to waste liquid for 6.60 min.

8. The detection method of any one of claims 1 to 6, wherein the external standard method comprises: (1) performing linear regression on the main peak area logarithm values and the concentration logarithm values of the chromatograms of a plurality of reference substance solutions with different concentrations to obtain a linear equation; (2) substituting the peak area of the main peak of the chromatogram of the test solution into the linear equation to calculate the concentration of polysorbate80 in the test solution; preferably, the plurality of different concentrations is at least 5 different concentrations.

9. The assay of any one of claims 1 to 8 wherein the biological agent comprises an antibody and a fusion protein.

10. Use of the assay method of any one of claims 1 to 9 for the quality control of a biological agent.