CN113424057A

CN113424057A - Method for detecting poloxamer 188 in composition

Info

Publication number: CN113424057A
Application number: CN202080012695.9A
Authority: CN
Inventors: 曹宜力; 李镭; 王海彬; 叶银萍; 郭芮
Original assignee: Haizheng Biopharmaceutical Co ltd
Current assignee: Haizheng Biopharmaceutical Co ltd
Priority date: 2019-04-19
Filing date: 2020-03-17
Publication date: 2021-09-21
Anticipated expiration: 2040-03-17
Also published as: CN113424057B; WO2020211584A1

Abstract

The method for detecting the poloxamer 188 in the composition utilizes a reversed phase chromatographic column and an electrospray detector to detect the poloxamer 188 content in the composition, and has the advantages of rapidness, simplicity, high sensitivity and good repeatability.

Description

Method for detecting poloxamer 188 in composition

Technical Field

The invention belongs to the field of pharmaceutical preparations, and relates to a method for detecting poloxamer 188 in a composition.

Background

Poloxamer 188 is polyoxyethylene polyoxypropylene ether block copolymer, is a high molecular nonionic surfactant, is an excellent new auxiliary material of a pharmaceutical preparation, is non-toxic, non-irritant and allergic to skin and mucous membranes, is widely applied to cell culture and fermentation processes of biological products, and is mainly used as a defoaming agent, a solubilizer, a dispersing agent, an emulsifier and a stabilizer.

Poloxamer 188 was reported to have a safety factor (ISF) of 314 relative to humans, and the concentration of producer cell culture medium was no greater than 1 mg/ml. The median lethal dose is 90g per 90kg adult, and the intake of each dose is not more than 286mg, and the lethal transfusion of surfactant in disinfectant has been reported in Japan. Therefore, the detection of the content of poloxamer 188 in the pharmaceutical composition is very necessary for the safety of the use of the medicine.

Due to the fact that poloxamer 188 does not absorb ultraviolet rays and the residual quantity is too low, a conventional detector (UV) cannot effectively detect poloxamer 188. The differential detector (RID) has low detection sensitivity, cannot adopt gradient elution and is not suitable for gradient elution of an inverse phase method. The evaporative light detector (ELSD) has a detection sensitivity of ng order, but its reproducibility is poor. Therefore, the conventional methods are not suitable for the detection of poloxamer 188. On the other hand, in the antibody culture process, poloxamer 188 serving as an antifoaming agent in the purified solution is continuously removed through a plurality of purification means, so that the concentration of the target analyte is extremely low, and the detection difficulty is increased. Meanwhile, the detection of the antibody is further interfered due to the existence of the macromolecular antibody. In the existing literature reports, a Poly RP-100 chromatographic column is combined with an ELSD detector to determine the content of poloxamer 188 in the injection, and the detection sensitivity is about 400ng (Libang et al, an evaporative light scattering detection method is used for determining poloxamer 188 in the recombinant human IL-12 injection, and the drug evaluation research is carried out in 2018, stage 09). The molecular weight of the monoclonal antibody drug is generally about 150KDa, the conventional C18 chromatographic column cannot be separated from poloxamer 188, and the monoclonal antibody drug molecules entering the conventional C18 chromatographic column can cause the blockage of the chromatographic column and damage the chromatographic column. Size exclusion chromatography columns, however, can perform better separations on high purity samples, but can interfere with sample detection for complex samples, such as culture fluids and partially purified fluids, due to the presence of other proteins. The existing ELSD detector has low detection sensitivity, and cannot detect sample detection with the content of below 200ng or lower, so that certain potential safety hazard exists when the medicine containing the auxiliary material poloxamer 188 is used in a clinical period. Therefore, the method for sensitively and rapidly detecting the poloxamer 188 in the antibody composition is invented, and the quality of the antibody medicine is better improved.

Disclosure of Invention

Aiming at the problem of insufficient sensitivity in the prior art, the invention provides a method for detecting poloxamer 188 in a composition. The method is rapid, simple and convenient, good in repeatability, higher in detection sensitivity and good in application prospect.

The specific technical scheme of the invention is as follows: the method for detecting poloxamer 188 in the composition adopts a reversed-phase chromatographic column combined with an electrospray detector (hereinafter referred to as CAD detector) for detection, wherein the chromatographic column is a Cadenza HS-C18 reversed-phase chromatographic column.

The composition described herein is a pharmaceutical composition and can be any pharmaceutical composition containing poloxamer 188. In particular antibody compositions, such as adalimumab compositions. The adalimumab composition is a mixture containing adalimumab, poloxamer 188 or/and other impurities, such as adalimumab culture solution, adalimumab purification solution, adalimumab antigen solution, adalimumab preparation, etc.

Preferably, the Cadenza HS-C18 reverse phase chromatography column has a specification of 150 x 3mm, 3 μm.

Preferably, the detection method adopts an external standard method to quantitatively detect the content of poloxamer 188 in the sample.

Preferably, the mobile phase system adopted by the reversed phase chromatographic column comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1M ammonium acetate solution, and the mobile phase B is acetonitrile; the mobile phase was filtered through a 0.22 μm filter before entering the HPLC.

Specifically, the mobile phase A is 0.1M ammonium acetate solution, and the pH value is between 5.0 and 6.0, preferably 5.6.

Preferably, the elution procedure of the mobile phase adopted by the reverse phase chromatographic column is as follows: 0-5min, mobile phase B0%, 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%.

Specifically, the column temperature of the reversed phase chromatographic column is 25-40 ℃, and preferably 35 ℃.

Specifically, the flow rate of the mobile phase is 0.4ml/min to 0.8ml/min, preferably 0.5 ml/min.

Specifically, the sample injection volume of the reverse phase chromatographic column is 10-100 μ l, and preferably 20 μ l.

Specifically, the parameter setting of the CAD detector includes: the gas used by the CAD detector is high-purity nitrogen, and the purity of the nitrogen is more than or equal to 99.9 percent; the gas pressure is 0.5MPa +/-0.05 MPa; the temperature mode of the CAD detector is a High mode; the CAD detector temperature is 50 ℃; the sampling frequency is 5Hz, and the filtering constant is 3.6S; starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100. mu.l.

The invention develops a method for detecting the content of poloxamer 188 in a composition by using a reversed-phase chromatographic column and a CAD detector. Centrifuging the solution to be detected, and taking the supernatant to directly enter a chromatographic system for detection. The method has the advantages of rapidness, convenience, good repeatability, high sensitivity, good selectivity, environmental protection and the like, and culture solution or purification solution does not need pretreatment for removing protein, so that the condition that the detection result is not credible due to the loss of the detected components caused by sample pretreatment is reduced. The method can be used for cell culture of drugs, such as adalimumab, in cell culture, purification, and detection of poloxamer 188 residue in final product. Under the best condition, the target analyte is rapidly detected within 22min, the detection limit of the obtained poloxamer 188 is less than 100ng, which is obviously lower than that of the existing reported method, and the quality control of adalimumab is more strict.

Drawings

FIG. 1 is a graph of adalimus stock solution to which poloxamer 188 standard solution was added, as detected by Candenza HS-C18 chromatography.

FIG. 2 is a chromatogram of poloxamer 188 standard and a blank control obtained by detecting with Candenza HS-C18 chromatographic column.

FIG. 3 is a spectrum of a ZORBAX SB-C18 column detecting adalimus stock solution to which poloxamer 188 standard solution has been added.

FIG. 4 is a diagram of a PolyRP-NP3 column detecting adalimus stock solution to which a standard solution of poloxamer 188 is added.

FIG. 5 is a spectrum of a standard poloxamer 188 and a blank control obtained by detecting with a ZORBAX SB-C18 chromatographic column.

FIG. 6 is a chromatogram of poloxamer 188 standard substance and blank control obtained by detecting through a polyRP-NP3 chromatographic column.

FIG. 7 is a graph obtained by detecting adalimus stock solution to which poloxamer 188 standard solution is added by CAPCELL PAK MF C8 chromatographic column.

FIG. 8 is a detection map of adalimumab culture solution test sample solution.

FIG. 9 is a detection spectrum of the adalimumab purification solution C1P test sample solution.

FIG. 10 is a detection spectrum of adalimumab purification solution C2P test sample solution.

FIG. 11 is a detection spectrum of adalimumab purification solution C3P test sample solution.

FIG. 12 is a detection map of the adalimumab solution test sample solution.

FIG. 13 is a detection spectrum of the adalimumab finished product test solution.

Detailed Description

The principles and advantages of the present invention are explained and illustrated below by specific embodiments in order to provide a better understanding of the present invention to those skilled in the art. The following description is exemplary in nature and is not intended to limit the scope of the disclosure.

Unless explicitly stated, the reagents and instrumentation used in the examples below are conventional and available from commercial sources; the methods used in the following examples are conventional methods, and those skilled in the art can unambiguously repeat the experiments and obtain corresponding results based on the description thereof.

Instrumentation and equipment

High performance liquid chromatograph, model: thermo U3000;

CAD detector, model: dionex Corona Veo;

a chromatographic column: imtakt Cadenza HS-C18, Specification: 150 x 3mm, 3 μm;

high-speed centrifuge, model: LEGEND MICRO 17R.

Reagent

Poloxamer 188 standard, source: BASF corporation;

ammonium acetate, analytical grade reagent, source Merck KGaA;

acetonitrile, HPLC grade reagent, source: merck KGaA company;

acetic acid, assay grade reagent, source: chemical agents of the national drug group, ltd.

Mobile phase composition

Mobile phase a was a 0.1M ammonium acetate solution, pH 5.6; mobile phase B was acetonitrile.

Standard Linear preparation

Weighing 25.0mg of poloxamer 188 standard, adding an appropriate amount of ultrapure water to dissolve, and fixing the volume to 25ml to avoid bubble formation, and using the standard stock solution. The stock solutions were diluted with ultrapure water as required to give standard linear solutions of 50. mu.g/ml, 40. mu.g/ml, 30. mu.g/ml, 20. mu.g/ml, 10. mu.g/ml and 5. mu.g/ml, respectively, and the blank solution was ultrapure water.

Source of test sample

Chinese Hamster Ovary (CHO) cells containing stable and high-efficiency expression adalimumab light and heavy chain genes are cultured for 12 days at 37 ℃ by a Permab basal medium and a Cell boost fed culture medium, and adalimumab culture solution is obtained after deep filtration; carrying out MabSelect SuRe affinity chromatography separation on the adalimumab culture solution, and eluting under the condition of pH3.5 to obtain adalimumab purified solution C1P; performing low pH virus inactivation (pH3.5, 120min) on the adalimumab purification solution C1P, performing QFF anion exchange chromatography separation, and obtaining adalimumab purification solution C2P in a flow-through mode; then, allowing the adalimumab purified liquid C2P to enter SPFF cation exchange chromatography for separation, and performing linear elution to obtain adalimumab purified liquid C3P; then, the adalimumab purification solution C3P is subjected to virus removal filtration and ultrafiltration displacement buffer solution to obtain adalimumab solution; adding appropriate amount of adjuvants into the stock solution, and performing two-stage sterilization and filtration with 0.22 μm filter membrane to obtain adalimumab final product. The amino acid sequence of adalimumab and the light and heavy chain coding sequences are conventional in the art. The materials and the specific operating modes involved in the procedures of cell culture, various chromatographic separations, filtration, inactivation and the like which occur in the above-mentioned processes are all conventional in the art.

In the following examples, the gas used by the CAD detector was high purity nitrogen, the nitrogen purity being 99.9% or greater; the gas pressure is 0.5MPa +/-0.05 MPa.

EXAMPLE 1 screening of chromatography columns

1. Chromatographic conditions

Mobile phase a was a 0.1M ammonium acetate solution, pH 5.6. Mobile phase B was acetonitrile. The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume is 20 mul; the temperature mode of the CAD detector is a High mode; the detector temperature was 50 ℃. The sampling frequency was 5Hz and the filter constant was 3.6S. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100 μ l; the gas pressure is 0.5MPa +/-0.05 MPa.

2. Preparation of standards

Weighing poloxamer 188 standard, and diluting the standard stock solution with ultrapure water to obtain poloxamer 188 standard solution with the concentration of 50 micrograms per ml.

3. Preparation of adalimus stock solution to which poloxamer 188 standard solution is added

Taking 1ml of adalimumab antigen solution, centrifuging for 10min in a high-speed centrifuge of 12000r/min, sucking 500 ul of supernatant, adding 500 ul of poloxamer 188 standard solution of 50 ug/ml, and mixing uniformly. Waiting for sample injection.

4. HPLC-CAD detection

Taking the adalimus stock solution added with the poloxamer 188 standard solution, ultrapure water and 50 mug/ml poloxamer 188 standard solution, respectively sampling by a microsyringe, entering a high performance liquid chromatography system for HPLC analysis, and detecting by a CAD detector.

5. Design of experiments

To obtain a better separation, different columns were tested, and the best column was selected, see table 1 below.

Table 1 comparison of different chromatographic columns

Numbering	Chromatographic column	Specification of	Mobile phase A	Mobile phase B
1	Candenza HS-C18	150*3mm，3μm	0.1M ammonium acetate solution pH5.6	Acetonitrile
2	ZORBAX SB-C18	250*4.6mm，5μm	0.1M ammonium acetate solution pH5.6	Acetonitrile
3	CAPCELL PAK MF C8	150*4.6mm，5μm	0.1M ammonium acetate solution pH5.6	Acetonitrile
4	PolyRP-NP3	250*4.6mm，3μm	0.1M ammonium acetate solution pH5.6	Acetonitrile

6. Results of the experiment

According to the chromatographic conditions in the table 1, the adalimumab stock solution added with the poloxamer 188 standard solution, the ultrapure water and the 50 mug/ml poloxamer 188 standard solution are subjected to detection analysis, and the result shows that only the Candenza HS-C18 chromatographic column shows the best separation effect (figure 1), and the adalimumab is completely separated from the poloxamer 188; while no blank peaks interfered with poloxamer 188 detection (fig. 2); ZORBAX SB-C18 and Poly RP-NP3 also completely separated adalimumab from poloxamer 188 (FIGS. 3 and 4); however, both ZORBAX SB-C18 and Poly RP-NP3 columns had blank solution hollow white peaks interfering with poloxamer 188 detection (FIGS. 5 and 6), and the ZORBAX SB-C18 column pressure was significantly increased after the adalimumab column was run, from the first 36bar to 125bar, resulting in irreversible column damage. CAPCELL PAK MF C8 when the sample containing adalimumab was tested, there was already a substance interfering with poloxamer 188, and it was not sufficient to completely separate adalimumab from poloxamer 188 (FIG. 7). Thus, the most preferred column is Candenza HS-C18.

EXAMPLE 2 screening of mobile phase compositions

1. Chromatographic conditions

A chromatographic column: candenza HS-C18(150 × 3mm, 3 μm). The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume is 20 mul; the temperature mode of the CAD detector is a High mode; the detector temperature was 50 ℃. The sampling frequency was 5Hz and the filter constant was 3.6S. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100 μ l; the gas pressure is 0.5MPa +/-0.05 MPa.

2. Preparation of standards

Weighing poloxamer 188 standard, and diluting the standard stock solution with ultrapure water to obtain a standard solution with the concentration of 50 mu g/ml.

3. HPLC-CAD detection

Taking ultrapure water and a 50 mu g/ml standard solution, respectively sampling by a microsyringe, entering a high performance liquid chromatography system for HPLC analysis, and detecting by a CAD detector.

4. Design of experiments

For better separation, different mobile phase compositions were tested, as shown in table 2 below. The optimum mobile phase composition is selected.

TABLE 2 comparison of different mobile phase compositions

Numbering	Mobile phase A	Mobile phase B
1	0.05M ammonium acetate solution pH5.6	Acetonitrile
2	0.1M ammonium acetate solution pH5.6	Acetonitrile
3	0.2M ammonium acetate solution pH5.6	Acetonitrile
4	0.05M ammonium formate solution pH5.6	Acetonitrile

5	0.1M ammonium formate solution pH5.6	Acetonitrile
6	0.2M ammonium formate solution pH5.6	Acetonitrile
7	0.1M ammonium acetate solution pH5.6	Methanol
8	0.1M ammonium formate solution pH5.6	Methanol
9	0.1% aqueous TFA	0.1% TFA acetonitrile

5. Results of the experiment

According to the chromatographic conditions in the table 2, the ultrapure water and the 50 mu g/ml poloxamer 188 standard solution are subjected to detection analysis, and the result shows that the baseline noise fluctuation of the ammonium acetate and the baseline noise fluctuation of the ammonium formate are compared, so that the baseline noise range of the ammonium acetate is 0.3-1.8, and the baseline noise fluctuation of the 0.1M ammonium acetate solution is the minimum and is 0.5-1.3. And the baseline noise of ammonium formate is about 0.9-2.3, and the baseline noise fluctuation is larger than that of an ammonium acetate solution, so that the selection of ammonium acetate is more favorable for high-sensitivity detection requirements, and the signal noise interference caused by a mobile phase is reduced.

Comparing methanol and acetonitrile, see table 3, poloxamer 188 was found to respond more to the acetonitrile system than to methanol. Whereas poloxamer 188 was found to respond lower than the ammonium acetate acetonitrile system in 0.1% aqueous TFA and 0.1% TFA acetonitrile. Therefore, we finally chose mobile phase a as 0.1M ammonium acetate solution and mobile phase B as acetonitrile according to the experimental results.

TABLE 3 comparison of different mobile phase test conditions

Numbering	Mobile phase A	Mobile phase B	Peak height	Peak area	Baseline noise
1	0.05M ammonium acetate solution pH5.6	Acetonitrile	24.190	2.593	0.3～1.5
2	0.1M ammonium acetate solution pH5.6	Acetonitrile	24.689	2.838	0.5～1.3
3	0.2M ammonium acetate solution pH5.6	Acetonitrile	24.430	2.621	0.6～1.8
4	0.05M ammonium formate solution pH5.6	Acetonitrile	27.141	3.360	0.9～1.9
5	0.1M ammonium formate solution pH5.6	Acetonitrile	27.541	3.430	1.2～2.1
6	0.2M ammonium formate solution pH5.6	Acetonitrile	26.974	3.213	1.3～2.3
7	0.1M ammonium acetate solution pH5.6	Methanol	7.072	2.019	0.2～1.1
8	0.1M ammonium formate solution pH5.6	Methanol	8.802	2.575	0.2～1.4
9	0.1% aqueous TFA	0.1% TFA acetonitrile	18.101	1.844	0.2～1.4

Example 3 screening of CAD Detector parameters

1. Chromatographic conditions

A chromatographic column: candenza HS-C18(150 × 3mm, 3 μm). Mobile phase a was 0.1M ammonium acetate solution ph5.6 and mobile phase B was acetonitrile. The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume was 20. mu.l. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100 μ l; the gas pressure is 0.5MPa +/-0.05 MPa.

2. Preparation of standards

3. HPLC-CAD detection

4. Design of experiments

To obtain better detection, the parameters of the CAD detector were tested and the optimal CAD detector parameter settings were selected, see table 4 below.

TABLE 4 CAD Detector comparison of different parameters

Numbering	Temperature mode	Sampling frequency (Hz)	Filtration constant (S)
1	Low(35℃)	10	1
2	Low(35℃)	10	2
3	Low(35℃)	10	3.6
4	High(50℃)	10	1
5	High(50℃)	10	2
6	High(50℃)	10	3.6
7	High(50℃)	5	1
8	High(50℃)	5	2
9	High(50℃)	5	3.6

5. Results of the experiment

The results of the assay performed on ultrapure water and 50 μ g/ml poloxamer 188 standard solution according to the chromatographic conditions of table 4 above show that the data are shown in table 5 below. The higher the temperature selection, the more complete the mobile phase atomization, and the smaller the detection signal baseline noise fluctuation, therefore 50 ℃ was selected as the atomization temperature. The smaller the sampling frequency, the larger the filter constant, the smaller the signal noise, and the smoother the baseline. Therefore, the sampling frequency of 5Hz and the filter constant of 3.6S are chosen as CAD detector parameters for the method.

TABLE 5 comparison of different parameters of CAD detector

Numbering	Temperature mode	Sampling frequency (Hz)	Filtration constant (S)	Baseline noise
1	Low(35℃)	10	1	0.8～2.6

2	Low(35℃)	10	2	0.9～2.1
3	Low(35℃)	10	3.6	0.7～1.8
4	High(50℃)	10	1	0.7～1.9
5	High(50℃)	10	2	0.8～1.6
6	High(50℃)	10	3.6	0.7～1.6
7	High(50℃)	5	1	0.6～1.6
8	High(50℃)	5	2	0.5～1.6
9	High(50℃)	5	3.6	0.5～1.3

EXAMPLE 4 screening of the Detector

1. Chromatographic conditions

A chromatographic column: candenza HS-C18(150 × 3mm, 3 μm). Mobile phase a was 0.1M ammonium acetate solution ph5.6 and mobile phase B was acetonitrile. The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume was 20. mu.l.

2. Preparation of standards

3. Experimental design and method

To compare the detection effect with the current detection method, two different detectors, a CAD detector and an ELSD detector (Alltech 3300ELSD), were compared, see table 6 below. A detector selection is determined.

TABLE 6 CAD DETECTOR and ELSD DETECTOR COMPARATIVE TESTS

3.1 HPLC-CAD detection

Taking ultrapure water and a 50 mu g/ml standard solution, respectively sampling by a microsyringe, entering a high performance liquid chromatography system for HPLC analysis, and detecting by a CAD detector. CAD detector parameter setting: the temperature mode of the CAD detector is a High mode; the detector temperature was 50 ℃. The sampling frequency was 5Hz and the filter constant was 3.6S. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100 μ l; the gas pressure is 0.5MPa +/-0.05 MPa.

3.2 HPLC-ELSD detection

Taking ultrapure water and 50 mu g/ml standard solution, respectively sampling by a microsyringe, entering a high performance liquid chromatography system for HPLC analysis, and detecting by an ELSD detector. ELSD detector parameter settings: the gas flow rate is 1.0SLM, the atomization temperature is 60 ℃, the evaporation light temperature is 105 ℃, the data acquisition frequency is 10H, and the detector gain PTM 10.

4. Results of the experiment

The results of the detection analysis of ultrapure water and 50 μ g/ml poloxamer 188 standard solution with a CAD detector and an ELSD respectively under the same chromatographic conditions of a chromatographic column, a mobile phase composition, an elution gradient and the like show that the data are shown in the following Table 7. The detection limit using the CAD detector method is below 100ng, while the detection limit using the ELSD detector method is below 200 ng. The CAD detector is selected as the detector of the method, which shows that the detection sensitivity of the CAD detector is higher, and the lower limit analysis is facilitated.

TABLE 7 CAD Detector and ELSD Detector comparison results

Example 5 method verification

1. Chromatographic conditions

A chromatographic column: candenza HS-C18(150 × 3mm, 3 μm). Mobile phase a was 0.1M ammonium acetate solution ph5.6 and mobile phase B was acetonitrile. The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume is 20 mul; the temperature mode of the CAD detector is a High mode; the detector temperature was 50 ℃. The sampling frequency was 5Hz and the filter constant was 3.6S. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100 μ l; the gas pressure is 0.5MPa +/-0.05 MPa.

2. Standard Linear solution preparation

Weighing 25.0mg of poloxamer 188 standard, adding an appropriate amount of ultrapure water to dissolve, and fixing the volume to 25ml to avoid bubble formation, and using the standard stock solution. The stock solutions were diluted as required to give standard linear solutions of 50. mu.g/ml, 40. mu.g/ml, 30. mu.g/ml, 20. mu.g/ml, 10. mu.g/ml and 5. mu.g/ml, respectively.

3. The linearity, limit of detection (LOD), Limit of Quantitation (LQD), reproducibility, precision and accuracy of the method of the invention were verified and are shown in Table 8. The result shows that when the concentration of poloxamer 188 is within the range of 5-50 mug/ml, the peak area and the concentration of poloxamer 188 standard solution are in a good linear range, and the square of the correlation coefficient is higher than 0.997. The detection limit (signal to noise ratio is 2-4) and the quantification limit (signal to noise ratio is 8-10) are respectively 100ng and 280ng, and the sensitivity is high. Experimental repeatability was expressed by the Relative Standard Deviation (RSD) of peak area and retention time obtained by repeating 6 times, peak area repeatability RSD was 2.7%, retention time repeatability RSD was 0.06%; RSD in the daytime and the daytime is respectively 1.1% and 1.7%, and the precision is good; the method has the accuracy of 101.3-104.8%, and the related results are shown in Table 8.

Table 8 method verification results

Example 6 adalimumab sample detection

1. Chromatographic conditions

A chromatographic column: candenza HS-C18(150 × 3mm, 3 μm). Mobile phase a was 0.1M ammonium acetate solution ph5.6 and mobile phase B was acetonitrile. The gradient elution mode was used, and the elution procedure was as follows: 0-5min, mobile phase B0%; 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%; the column temperature was 35 ℃; the injection volume is 20 mul; the temperature mode of the CAD detector is a High mode; the detector temperature was 50 ℃. The sampling frequency was 5Hz and the filter constant was 3.6S. Starting a gradient compensation function by a CAD detector, wherein the highest liquid flow rate of the detector is not higher than 2 ml/min; the gradient compensation volume was 100. mu.l. The gas pressure is 0.5MPa +/-0.05 MPa.

2. Standard Linear solution preparation

3. Preparation of test sample supernatant solution

Taking 1ml of adalimumab culture solution, purified solution C1P, purified solution C2P, purified solution C3P, stock solution and finished product respectively, centrifuging for 10min at a high-speed centrifuge of 12000r/min, and sucking supernatant to obtain adalimumab culture solution supernatant, purified solution C1P supernatant, purified solution C2P supernatant, purified solution C3P supernatant, stock solution supernatant and finished product supernatant respectively.

4. Preparation of test solution

Diluting adalimumab culture solution supernatant solution by 10 times with ultrapure water, and uniformly mixing to obtain adalimumab culture solution test solution; taking 200 mul of each of the purified solution C1P supernatant solution, the purified solution C2P supernatant solution, the purified solution C3P supernatant solution, the stock solution supernatant solution and the finished product supernatant solution as a purified solution C1P sample solution, a purified solution C2P sample solution, a purified solution C3P sample solution, a stock solution sample solution and a finished product sample solution.

5. Sample application recovery solution preparation

Taking 50 mu g/ml standard linear solution to be respectively mixed with adalimumab culture solution sample solution, purified solution C1P sample solution, purified solution C2P sample solution, purified solution C3P sample solution, stock solution sample solution and finished product sample solution 1:1(v: v) to be respectively used as adalimumab culture solution sample recovery solution, purified solution C1P sample recovery solution, purified solution C2P sample recovery solution, purified solution C3P sample recovery solution, stock solution sample recovery solution and finished product sample recovery solution.

6. HPLC-CAD detection

Sampling ultrapure water, each linear standard solution, each sample solution and each sample recovery solution by a microsyringe respectively, performing HPLC analysis in a high performance liquid chromatography system, and detecting by a CAD detector.

7. Results of the experiment

The detection results are shown in Table 9, the sample solution (figure 8) of the adalimumab culture solution contains 17.3 mug/ml of poloxamer 188, and the recovery rate is 96.8%. The content of poloxamer 188 in adalimumab purified solution C1P test solution (figure 9), C2P test solution (figure 10), C3P test solution (figure 11), stock solution test solution (figure 12) and finished product test solution (figure 13) is lower than the detection limit by 5 mug/ml.

TABLE 9 detection data of Poloxamer 188 in adalimumab test solutions

Test solution	Content (wt.)	Adding the concentration	Measured concentration	Sample recovery rate
Culture solution	17.3μg/ml	25μg/ml	41.5μg/ml	96.8％
Purified liquid C1P	/*	25μg/ml	25.4μg/ml	101.6％
Purified liquid C2P	/*	25μg/ml	25.8μg/ml	103.2％
Purified liquid C3P	/*	25μg/ml	24.4μg/ml	97.6％
Stock solution	/*	25μg/ml	24.1μg/ml	96.4％
Finished product	/*	25μg/ml	25.6μg/ml	102.4％

/*: no detection was detected, which was 5. mu.g/ml below the detection limit.

Claims

A method for detecting poloxamer 188 in a composition, comprising: detecting by adopting a reversed-phase chromatographic column combined with an electrospray detector, wherein the reversed-phase chromatographic column is a Cadenza HS-C18 reversed-phase chromatographic column; preferably, the composition is a pharmaceutical composition; more preferably, the composition is an adalimumab composition.
The assay of claim 1 wherein said Cadenza HS-C18 reverse phase chromatography column is 150 x 3mm, 3 μm in size.
The assay of claim 1 or 2, wherein the amount of poloxamer 188 in the sample is determined quantitatively using an external standard method.
The detection method according to any one of claims 1 to 3, wherein the reverse phase chromatography column employs a mobile phase system comprising mobile phase A and mobile phase B, wherein the mobile phase A is 0.1M ammonium acetate solution, and the mobile phase B is acetonitrile.
The detection method according to claim 4, wherein the mobile phase A is a 0.1M ammonium acetate solution, and the pH is between 5.0 and 6.0, preferably pH 5.6.
The detection method according to any one of claims 1 to 5, wherein the elution procedure of the mobile phase used in the reverse phase chromatography column is: 0-5min, mobile phase B0%, 5-10min, mobile phase B10%; 10-18min, mobile phase B45%; 18-22min, and 100% of mobile phase B; 22-28min, mobile phase B0%.
The detection method according to any one of claims 1 to 6, characterized in that the reverse phase chromatography column employs a column temperature of 25 ℃ to 40 ℃, preferably 35 ℃.
The detection method according to claim 6, characterized in that the flow rate of the mobile phase is between 0.4ml/min and 0.8ml/min, preferably 0.5 ml/min.
The detection method according to any one of claims 1 to 8, wherein the reverse phase chromatography column has a sample introduction volume of 10 to 100 μ l, preferably 20 μ l.
The detection method according to any one of claims 1 to 9, wherein the parameter setting of the CAD detector comprises: the gas used by the detector is high-purity nitrogen, and the purity of the nitrogen is more than or equal to 99.9 percent; the gas pressure is 0.5MPa +/-0.05 MPa; the detector temperature mode is a High mode; the temperature of the detector is 50 ℃; the sampling frequency is 5Hz, and the filtering constant is 3.6S; the detector starts the gradient compensation function, and the liquid flow rate of the detector is not higher than 2ml/min at most; the gradient compensation volume was 100. mu.l.