1. A method for detecting the Tween 80 content in a monoclonal antibody injection based on solid phase extraction is characterized by comprising the steps of pretreatment of the monoclonal antibody injection and high performance liquid chromatography detection of flow injection;

the pretreatment comprises the following specific steps:

dispersing a filler of a solid phase extraction column into ultrapure water, wherein the volume ratio of the ultrapure water to the filler is (4-6): 1, and then centrifuging to remove the ultrapure water;

the filler is an affinity chromatography medium taking Protein A as a ligand;

loading the monoclonal antibody injection to a balanced solid-phase extraction column, collecting initial effluent, eluting by using methanol with the volume fraction of 100% as eluent, collecting subsequent effluent, mixing the obtained initial effluent and the subsequent effluent, concentrating, drying and redissolving to obtain a sample to be detected;

the monoclonal antibody is an IgG antibody and comprises IgG1, IgG2, IgG3 and IgG4 antibodies, the concentration of protein in the monoclonal antibody injection is 60-200 mg/mL, and the content of Tween 80 is 0.005-0.015 wt%; (ii) a

The mobile phase used for the high performance liquid chromatography detection comprises:

0.1-0.15M of sodium chloride, 0.025-0.03M of sodium tetraborate decahydrate, 3-5 wt% of acetonitrile, 4-6M of N-phenyl-1-naphthylamine and 802-3 mg/L of tween;

the concentration method is vacuum centrifugal concentration, wherein the rotating speed of the vacuum centrifugal concentration is 1000-1500 rpm, the temperature is 25-50 ℃, and the time is 3-4 hours.

2. The method of claim 1, further comprising the step of washing the solid phase extraction column;

the cleaning solution used for cleaning comprises an acetic acid aqueous solution with the molar mass of 20-50 mM.

3. The method according to claim 1, wherein the column temperature of the high performance liquid chromatography is 25-26 ℃;

the sample injection amount of the high performance liquid chromatography is 8-10 mu L.

4. The method of claim 1, wherein the detector of the high performance liquid chromatography detection is a fluorescence detector;

the fluorescence maximum excitation wavelength of the fluorescence detector is 350 nm;

the fluorescence maximum emission wavelength of the fluorescence detector is 420 nm.

5. The method of claim 1, further comprising the steps of preparing a standard working fluid and plotting a standard curve;

the final concentration of the Tween 80 in the standard working solution is 0.00125 wt% -1 wt%.

6. The method according to claim 5, wherein the standard curve uses the concentration of Tween 80 as an input variable and the peak area of the HPLC detection spectrum as an output variable.

7. The method of claim 6, further comprising: and calculating the content of the Tween 80 in the solution to be detected according to the standard curve and the peak area obtained by the high performance liquid chromatography detection.

8. Method according to claim 1, characterized in that it comprises the following steps:

(1) pretreatment of the monoclonal antibody injection:

loading the monoclonal antibody injection to a balanced solid-phase extraction column, collecting initial effluent, leaching with 100% methanol by volume fraction, collecting subsequent effluent, mixing the initial effluent and the subsequent effluent, concentrating, drying, and re-dissolving to obtain a sample to be detected;

the filler of the solid phase extraction column is an affinity chromatography medium taking Protein A as a ligand, and the filler is washed and stored by using an acetic acid aqueous solution with the molar mass of 20-50 mM after being used;

(2) and (3) high performance liquid chromatography detection:

formulating a mobile phase comprising: sodium chloride with the molar concentration of 0.1-0.15M, sodium tetraborate decahydrate with the molar concentration of 0.025-0.03M, acetonitrile with the mass percent of 3-5%, N-phenyl-1-naphthylamine with the molar concentration of 4-6M and tween 80 with the mass concentration of 2-3 mg/L, wherein the flow rate of the mobile phase is 1-2 mL/min;

the sample injection amount of the high performance liquid chromatography is 8-10 mu L, the column temperature is 25-26 ℃, the detector is a fluorescence detector, the maximum excitation wavelength of fluorescence is 350nm, and the maximum emission wavelength of fluorescence is 420 nm;

(3) preparing a standard working solution:

the final concentration of the Tween 80 in the standard working solution is 0.00125 wt% -1 wt%, the concentration of the Tween 80 is used as an input variable, and the peak area of a high performance liquid chromatography detection spectrum is used as an output variable to draw a standard curve;

(4) calculating the concentration of Tween 80 in the sample to be detected:

and calculating the content of the Tween 80 in the solution to be detected according to the standard curve and the peak area obtained by the high performance liquid chromatography detection.

Background

Tween 80(Tween-80), also known as polysorbate 80, is known chemically as polyoxyethylene 20 sorbitan monooleate. Tween 80 is used as cosolvent, emulsifier and stabilizer, and is commonly used in therapeutic monoclonal antibody injection preparation. In recent years, more and more reports about the allergic reaction induced by tween 80 are provided, so that the establishment of an accurate and effective method for measuring the content of tween 80 is particularly important.

At present, methods for detecting the content of tween 80 mainly include a colorimetric method, an HPLC-Evaporative Light Scattering (ELSD) detection method, a hydrolysis method, a mass spectrometry method, an HPLC-Fluorescence (FLD) method, and the like.

The colorimetric method has a wide application range due to low requirements on equipment, for example, CN107300555A discloses a colorimetric method for rapidly and efficiently determining the content of tween 80. In an aqueous medium, polyethoxy of Tween 80 and cobalt ammonium thiocyanate react quickly under the heating condition of a boiling water bath to generate a water-insoluble blue chromogenic product quantitatively, an organic solvent is added quantitatively to extract the chromogenic product after the product is cooled to room temperature, and the content of Tween 80 is measured by a colorimetric method. The method is suitable for rapid quantitative determination of Tween 80 serving as an auxiliary material for various medicinal preparations and traditional Chinese medicines, but the method is time-consuming, poor in repeatability and poor in applicability to samples with high protein concentration.

The HPLC-ELSD method has more accurate result and wider application range, but the ELSD system is easily polluted by protein to influence the detection result; the hydrolysis method is to hydrolyze Tween 80 into oleic acid, and high performance liquid chromatography-ultraviolet detector is used for detection, and the method is long in time consumption and is easily influenced by the stability of the oleic acid; mass spectrometry requires the use of expensive instruments and therefore has a limited range of use; the HPLC-FLD method has poor detection accuracy because the sample is directly injected for analysis and the protein is interfered at the Tween 80 peak position.

In addition, CN103940773A discloses a method for rapidly determining the content of tween-80 in a Xuebijing injection. The method comprises the following steps: (1) adding different qualities of Tween-80 into the Xuebijing injection to prepare at least 15 Xuebijing injection standard products containing Tween-80 with different concentrations; (2) collecting a near infrared spectrogram of the Xuebijing injection standard substance; (3) establishing a correction model between the content of tween-80 in the standard product of the Xuebijing injection and the near-infrared spectrogram by adopting a chemometrics method; (4) and collecting a near infrared spectrogram of the Xuebijing injection to be detected, and inputting the spectrogram into the correction model to obtain the Tween-80 content in the Xuebijing injection to be detected. The method is used as a rapid determination method, can also be used for rapid online determination of an intermediate of a key production process point of the Xuebijing injection in the production process, and provides a real-time online rapid detection method for quality control in the production process. This method requires expensive instruments as well as mass spectrometry and also does not exclude protein interference.

Therefore, it is important to the art to provide an accurate and effective detection method capable of detecting the tween 80 content of the monoclonal antibody injection without being interfered by protein.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for detecting the Tween 80 content in a monoclonal antibody injection based on solid-phase extraction. The method has the advantages of convenience in operation, short detection period, good data reproducibility, high result accuracy and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for detecting the Tween 80 content in a monoclonal antibody injection based on solid-phase extraction, which comprises the steps of pretreatment of the monoclonal antibody injection and high performance liquid chromatography detection of flow injection;

the pretreatment comprises the following specific steps:

loading the monoclonal antibody injection to a balanced solid-phase extraction column, collecting initial effluent, eluting with an eluent, collecting subsequent effluent, mixing the initial effluent and the subsequent effluent, concentrating, drying, and re-dissolving to obtain a sample to be detected;

wherein, the filler of the solid phase extraction column is an affinity chromatography medium taking Protein A as ligand; the leacheate is a 40-100% methanol aqueous solution;

the mobile phase used for the high performance liquid chromatography detection comprises:

0.1-0.15M of sodium chloride, 0.025-0.03M of sodium tetraborate decahydrate, 3-5 wt% of acetonitrile, 4-6M of N-phenyl-1-naphthylamine and 802-3 mg/L of tween.

The monoclonal antibody of the invention is mostly an IgG antibody, and comprises four subtypes of IgG1, IgG2, IgG3 and IgG4, and the Fc fragment of the monoclonal antibody has good binding effect with Protein A. The solid phase extraction column used in the invention is filled with a medium with Protein A as a ligand, after the monoclonal antibody injection is pretreated by solid phase extraction, the monoclonal antibody is combined on the solid phase extraction column, Tween 80 flows out along with the washing of leacheate and is collected, and after drying and redissolving, liquid chromatography detection is carried out, so that the detection result is not interfered by the monoclonal antibody.

In the present invention, the concentration of the protein in the monoclonal antibody injection is 60 to 200mg/mL, and may be, for example, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 180mg/mL, or 200 mg/mL; the content of tween 80 is 0.005 to 0.015 wt%, and may be, for example, 0.005 wt%, 0.006 wt%, 0.008 wt%, 0.01 wt%, 0.012 wt%, or 0.015 wt%.

The method is particularly suitable for the monoclonal antibody injection with higher protein content and lower Tween concentration, and for the monoclonal antibody, compared with the conventional method for detecting Tween 80 after protein is precipitated by adding an ethanol solution of saturated sodium chloride, the method has obviously better effect; the method is also applicable to monoclonal antibody injection with lower protein content and tween content, but the improvement degree of the improvement effect is not obvious compared with injection with higher protein content and lower tween concentration.

In the invention, the requirement on the eluent is high, and ultrapure water, methanol water solution or ethanol water solution can be used in the pretreatment process of the sample, but when acetonitrile is used as the eluent, the interference is great at the peak position of Tween 80; compared with leacheate such as ultrapure water and ethanol aqueous solution, the leacheate is methanol aqueous solution, and especially when a sample is treated by using methanol aqueous solution with volume fraction of 40-100% (for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, and the like), peaks of tween 80 in the obtained high performance liquid chromatogram are obvious, impurity interference is avoided, and quantification is facilitated.

In the invention, the mobile phase contains a proper amount of Tween 80, which can ensure that micelles are formed for quantification, and the quantitative determination of the Tween 80 in a sample is not influenced. However, the amount of tween 80 added affects the quantification of samples with very low tween 80 content.

According to the method for detecting the Tween 80 content in the monoclonal antibody injection by the solid phase extraction-flow injection HPLC fluorescence analysis method, on one hand, interference of a monoclonal antibody on a Tween 80 detection result is eliminated by a solid phase extraction technology with Protein A as a medium, on the other hand, the solid phase extraction and flow injection HPLC are combined, compared with the traditional method, the method is more convenient to operate, better in data reproducibility, shorter in detection period, and capable of obtaining the detection result within 5 hours, and the method only specifically aims at the Tween 80, and has no obvious detection and quantification effects on other impurities in the monoclonal antibody injection.

In the present invention, the flow injection HPLC is based on the flow injection concept proposed by j.ruzicka and eh.hansen of the technical university of danish, namely: according to the continuous flow method, a reaction reagent and a sample to be detected are proportionally injected into a closed and continuous flow current carrier through a peristaltic pump, a color reaction is generated in a chemical reaction unit, a signal value of the color reaction unit is measured in a detector, and the concentration of the sample to be detected is measured according to a standard curve method.

Preferably, the solvent used for reconstitution is ultrapure water.

As a preferred technical scheme of the invention, the equilibrium method of the solid phase extraction column comprises the following steps: and dispersing the filler of the solid phase extraction column into the equilibrium liquid, and then centrifuging to remove the equilibrium liquid.

Preferably, the balancing liquid comprises ultrapure water.

Preferably, the volume ratio of the equilibrium liquid to the packing of the solid phase extraction column is (4-6: 1), and may be, for example, 4:1, 4.2:1, 4.5:1, 4.8:1, 5:1, 5.2:1, 5.4:1, 5.5:1, 5.8:1, or 6: 1.

As a preferable technical scheme, the method also comprises a step of cleaning the solid phase extraction column.

Preferably, the cleaning solution used for cleaning comprises an acetic acid aqueous solution with a molar mass of 20-50 mM (for example, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM).

As a preferred technical solution of the present invention, the concentration method includes any one of vacuum centrifugal concentration, nitrogen concentration or vacuum freeze concentration, preferably vacuum centrifugal concentration; in the nitrogen concentration, a nitrogen gas is blown in to concentrate a sample, but a nitrogen gas making device is needed, and if the nitrogen gas is impure, impurities are easy to introduce; the vacuum freezing concentration needs to freeze a sample to a lower temperature (-about 100 ℃), then vacuum pumping is carried out for concentration, and the method needs an additional freeze-drying device and has a longer period; the invention selects centrifugal concentration, properly increases the temperature and accelerates the concentration of the sample through vacuum and centrifugation, and the concentration method does not introduce impurities, is convenient to operate and has shorter concentration time.

Preferably, the rotation speed of the vacuum centrifugal concentration is 1000-1500 rpm, for example, 1000rpm, 1050rpm, 1100rpm, 1150rpm, 1200rpm, 1300rpm, 1400rpm or 1500rpm, etc.

Preferably, the temperature of the vacuum centrifugal concentration is 25-50 ℃, for example, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 48 ℃ or 50 ℃.

Preferably, the time for vacuum centrifugal concentration is 3-4 h, for example, 3h, 3.1h, 3.2h, 3.3h, 3.5h, 3.6h, 3.8h, 3.9h or 4h, etc.

As a preferred technical scheme of the invention, the mobile phase used for the high performance liquid chromatography detection comprises sodium chloride, sodium tetraborate decahydrate, acetonitrile, N-phenyl-1-naphthylamine and Tween 80.

Preferably, the molar concentration of sodium chloride in the mobile phase is 0.1-0.15M, and may be, for example, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, or 0.15M.

Preferably, the molar concentration of the sodium tetraborate decahydrate in the mobile phase is 0.025-0.03M, and may be, for example, 0.025M, 0.026M, 0.027M, 0.028M, 0.029M, or 0.03M.

Preferably, the acetonitrile in the mobile phase is 3 to 5% by mass, and may be, for example, 3%, 3.2%, 3.4%, 3.5%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.5%, 4.6%, 4.8%, 5%, or the like.

Preferably, the molar concentration of the N-phenyl-1-naphthylamine in the mobile phase is 4-6M, and can be 4M, 4.2M, 4.4M, 4.5M, 4.6M, 4.8M, 5M, 5.2M, 5.4M, 5.6M or 6M, for example.

Preferably, the mass concentration of tween 80 in the mobile phase is 2-3 mg/L, and may be, for example, 2mg/mL, 2.2mg/mL, 2.5mg/mL, 2.6mg/mL, 2.8mg/mL or 3 mg/mL.

Preferably, the flow rate of the mobile phase is 1 to 2mL/min, and may be, for example, 1mL/min, 1.2mL/min, 1.4mL/min, 1.5mL/min, 1.6mL/min, 1.8mL/min, 2mL/min or the like.

In a preferred embodiment of the present invention, the column temperature of the HPLC assay is 25 to 26 ℃, and may be, for example, 25 ℃, 25.2 ℃, 25.4 ℃, 25.5 ℃, 25.6 ℃, 25.8 ℃ or 26 ℃.

Preferably, the sample volume detected by the high performance liquid chromatography is 8-10 μ L, for example, 8 μ L, 8.2 μ L, 8.4 μ L, 8.5 μ L, 8.8 μ L, 9 μ L, 9.2 μ L, 9.5 μ L, 9.6 μ L, 9.8 μ L or 10 μ L.

Preferably, the detector for high performance liquid chromatography detection is a fluorescence detector.

Preferably, the fluorescence detector has a fluorescence maximum excitation wavelength of 350 nm.

Preferably, the fluorescence detector has a fluorescence maximum emission wavelength of 420 nm.

As a preferable technical scheme of the invention, the method further comprises the steps of preparing a standard working solution and drawing a standard curve.

Preferably, the final concentration of tween 80 in the standard working solution is 0.00125 wt% to 1 wt% (i.e. 0.0125 mg/mL), and may be, for example, 0.00125 wt%, 0.0025 wt%, 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt%, 0.8 wt%, or 1 wt%.

Accurately weighing 1.0g of Tween 80 standard, using ultrapure water to fix the volume to 100mL to prepare a Tween 80 stock solution (1 wt%, namely 10mg/mL), then accurately transferring a certain volume of Tween 80 stock solution, and diluting to obtain standard working solutions with certain concentrations, wherein the concentrations are respectively 0.0025 wt%, 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.04 wt% and 0.08 wt%.

Preferably, the standard curve takes the concentration of tween 80 as an input variable and takes the peak area of a high performance liquid chromatography detection spectrum as an output variable.

As a preferred technical solution of the present invention, the method further comprises: and calculating the content of the Tween 80 in the solution to be detected according to the standard curve and the peak area obtained by the high performance liquid chromatography detection.

As a preferred technical scheme of the invention, the method comprises the following steps:

(1) pretreatment of the monoclonal antibody injection:

loading the monoclonal antibody injection to a balanced solid-phase extraction column, collecting initial effluent, leaching with 40-100% methanol aqueous solution by volume fraction, collecting subsequent effluent, mixing the obtained initial effluent and the subsequent effluent, concentrating, drying and re-dissolving to obtain a sample to be detected;

the filler of the solid phase extraction column is an affinity chromatography medium taking Protein A as a ligand, and the filler is washed and stored by using an acetic acid aqueous solution with the molar mass of 20-50 mM after being used;

(2) and (3) high performance liquid chromatography detection:

formulating a mobile phase comprising: sodium chloride with the molar concentration of 0.1-0.15M, sodium tetraborate decahydrate with the molar concentration of 0.025-0.03M, acetonitrile with the mass percent of 3-5%, N-phenyl-1-naphthylamine with the molar concentration of 4-6M and tween 80 with the mass concentration of 2-3 mg/L, wherein the flow rate of the mobile phase is 1-2 mL/min;

the sample injection amount of the high performance liquid chromatography is 8-10 mu L, the column temperature is 25-26 ℃, the detector is a fluorescence detector, the maximum excitation wavelength of fluorescence is 350nm, and the maximum emission wavelength of fluorescence is 420 nm;

(3) preparing a standard working solution:

the final concentration of the Tween 80 in the standard working solution is 0.00125 wt% -1 wt%, the concentration of the Tween 80 is used as an input variable, and the peak area of a high performance liquid chromatography detection spectrum is used as an output variable to draw a standard curve;

(4) calculating the concentration of Tween 80 in the sample to be detected:

and calculating the content of the Tween 80 in the solution to be detected according to the standard curve and the peak area obtained by the high performance liquid chromatography detection.

Illustratively, the following method can be used in the present invention for testing the content of tween 80:

(1) pretreatment of the monoclonal antibody injection:

taking the solid phase extraction column filled with the filler, and balancing the solid phase extraction column by using 2.0-5.0 mL of balancing liquid. And (3) sampling 0.1-0.5 mL of monoclonal antibody injection to a solid phase extraction column, simultaneously starting to collect effluent liquid, then leaching with 1.2-3.0 mL of eluent, collecting all the effluent liquid, centrifuging, concentrating, drying, and redissolving with pure water to be tested. The solid phase extraction column can be repeatedly used after being washed by 2.0-5.0 mL of washing liquid.

(2) Determining operating parameters of a liquid chromatograph

Reaction coil: specification 750 μ L; or a reaction coil of equivalent performance;

mobile phase: 0.15M sodium chloride, 0.025M sodium tetraborate decahydrate, 5.0% acetonitrile, 5.0M N-phenyl-1-naphthylamine, 2.5mg/L Tween 80;

flow rate: 1.5 mL/min; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: a fluorescence detector, λ ex-350 nm, λ em-420 nm;

(3) drawing a standard working curve

Preparing a standard working solution, injecting the standard working solution into a liquid chromatograph, measuring a corresponding peak area according to the working parameters in the step (2), drawing a standard working curve by taking the concentration of the Tween 80 as a horizontal coordinate and the corresponding peak area as a vertical coordinate, and obtaining a linear equation;

(4) detecting the content of Tween 80 in the monoclonal antibody injection:

injecting the re-dissolved sample into a liquid chromatograph according to the step (1), determining according to the working parameters of the step (2), positioning a chromatographic peak by retention time, measuring the peak area of the chromatographic peak, and calculating the content of the Tween 80 in the monoclonal antibody injection according to a linear equation of a standard working curve.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.

Compared with the prior art, the invention has the beneficial effects that:

(1) the detection method is used for detecting the content of the Tween 80 in the monoclonal antibody injection, the monoclonal antibody contains Fc fragments, the monoclonal antibody is combined on a solid-phase extraction column after the monoclonal antibody injection is pretreated by solid-phase extraction by utilizing the combination action of the Fc fragments and Protein A, the Tween 80 flows out along with the washing of leacheate, and the interference of the monoclonal antibody on the Tween 80 detection result is eliminated after the Tween 80 is dried and redissolved and then is detected by liquid chromatography; when methanol aqueous solution is used as leacheate, the obtained result is the most accurate;

(2) the method combines solid phase extraction and flow injection HPLC, is more convenient to operate, has better data reproducibility and shorter detection period compared with the traditional method, and can obtain a detection result within 5 hours.

Drawings

Figure 1 is a tween 80 chromatogram determined by the method provided in example 1.

Figure 2 is a graph of a standard operating curve obtained according to the method provided in example 1.

Figure 3 is a tween 80 chromatogram determined by the method provided in example 2.

Fig. 4 is a graph of a standard operating curve obtained according to the method provided in example 2.

Figure 5 is a tween 80 chromatogram determined by the method provided in example 3.

Fig. 6 is a graph of a standard operating curve obtained according to the method provided in example 3.

Figure 7 is a tween 80 chromatogram determined by the method provided in example 4.

Figure 8 is a tween 80 chromatogram determined by the method provided in example 5.

Figure 9 is a tween 80 chromatogram determined by the method provided in example 6.

Fig. 10 is a graph of a standard operating curve obtained according to the method provided in example 6.

Figure 11 is a tween 80 chromatogram determined by the method provided in example 7.

Figure 12 is a tween 80 chromatogram determined by the method provided in example 8.

Fig. 13 is a standard operating graph obtained according to the method provided in comparative example 1.

Fig. 14 is a standard operating graph obtained according to the method provided in comparative example 2.

Fig. 15 is a standard operating graph obtained according to the method provided in comparative example 3.

Fig. 16 is a standard operating graph obtained according to the method provided in comparative example 4.

Fig. 17 is a standard operating graph obtained according to the method provided in comparative example 5.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, reagents and consumables used were obtained from conventional reagent manufacturers in the field unless otherwise specified; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.

Example 1

The embodiment provides a method for detecting the content of tween 80 in a monoclonal antibody injection, which is used for detecting the content of tween 80 in a recombinant anti-EGFR human mouse chimeric monoclonal antibody injection and comprises the following specific steps:

(1) pretreatment: taking a solid phase extraction column filled with 0.5mL of Protein A filler, and balancing with 2.5mL of ultrapure water; loading 0.1mL of recombinant anti-EGFR human-mouse chimeric monoclonal antibody injection (with the protein concentration of 5mg/mL) to a solid-phase extraction column, collecting effluent, then leaching with 1.5mL of 100% methanol (volume fraction) aqueous solution, and collecting all effluent;

washing the solid phase extraction column with 2.5mL of aqueous solution containing 50mM acetic acid, and discarding the washing solution;

and (3) placing all the collected effluent liquid into a centrifugal concentrator, rotating at 1300 ℃ for 3 hours, and redissolving with ultrapure water to serve as a sample to be detected.

(2) And (3) high performance liquid chromatography detection: the reaction coil is 750 mu L reaction coil;

using a solution of sodium chloride with a molar concentration of 0.15M, sodium tetraborate decahydrate with a molar concentration of 0.025M, acetonitrile with a mass percentage of 5.0%, phenyl-1-naphthylamine with a molar concentration of 5.0M N and tween 80 with a mass concentration of 2.5mg/L as a mobile phase;

the flow rate is 1.5 mL/min; the column temperature is 25 ℃; the sample volume is 10 mu L; the wavelength λ ex of the fluorescence detector is 350nm, and the wavelength λ em of the fluorescence detector is 420 nm;

injecting the standard working solution into a high performance liquid chromatograph for analysis, wherein the preparation method of the standard working solution comprises the following steps:

accurately weighing 1.0g of Tween 80 standard, adding ultrapure water to a constant volume of 100mL to prepare a Tween 80 stock solution (1 wt%, namely 10mg/mL), then accurately transferring a certain volume of Tween 80 stock solution, adding a certain volume of ultrapure water, and diluting to obtain standard working solutions with concentrations of 0.0025 wt%, 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.04 wt% and 0.08 wt%, wherein the specific dilution steps are shown in the following table 1:

TABLE 1

Drawing a standard working curve by taking the concentration (wt%) of the standard working solution as an abscissa (X) and the peak area of a Tween 80 chromatographic peak as an ordinate (Y), wherein the linear equation of the obtained standard working curve is that Y is 1.07 multiplied by 10⁹X-2.72×10⁶Coefficient of correlation R²0.999638; the detection range is 0.0025 wt% to 0.08 wt% (namely 0.025-0.8 mg/mL).

FIG. 1 is a chromatogram of Tween 80, and the retention time of 0.512min is Tween 80 chromatogram peak;

FIG. 2 is a standard operating curve; and calculating the detection result of the Tween 80 content in the sample to be detected according to the standard working curve to be 0.0094% (namely 0.094mg/mL) and meet the range requirement of 0.005-0.015% (namely 0.05-0.15 mg/mL) (wherein the standard quantity of the Tween 80 in the recombinant anti-EGFR human-mouse chimeric monoclonal antibody injection is 0.01%).

Example 2

The embodiment provides a method for detecting the content of tween 80 in a monoclonal antibody injection, which is used for detecting the content of tween 80 in a specific targeting human IL-4Ra humanized monoclonal antibody injection and comprises the following specific steps:

(1) taking a solid phase extraction column filled with 1mL of Protein A filler, and balancing with 5.0mL of ultrapure water; loading 0.1mL of specific targeting human IL-4Ra humanized monoclonal antibody injection (with the protein concentration of 150mg/mL) to a solid phase extraction column, collecting effluent, eluting with 3.0mL of 100% methanol (volume fraction) aqueous solution, and collecting all effluent;

washing the solid phase extraction column with 5.0mL of aqueous solution containing 50mM acetic acid, and discarding the washing solution;

all the collected effluent is placed in a centrifugal concentrator, dried for 4 hours at 1300rpm and 40 ℃, and then re-dissolved by ultrapure water to be used as a sample to be detected.

(2) And (3) high performance liquid chromatography detection: the reaction coil is 750 mu L reaction coil;

using a solution of sodium chloride with a molar concentration of 0.15M, sodium tetraborate decahydrate with a molar concentration of 0.025M, acetonitrile with a mass percentage of 5.0%, phenyl-1-naphthylamine with a molar concentration of 5.0M N and tween 80 with a mass concentration of 2.5mg/L as a mobile phase;

the flow rate is 1.5 mL/min; the column temperature is 25 ℃; the sample volume is 10 mu L; the wavelength λ ex of the fluorescence detector is 350nm, and the wavelength λ em of the fluorescence detector is 420 nm.

And injecting the standard working solution into a high performance liquid chromatograph for analysis, then injecting a sample to be detected, and detecting the content of the Tween 80 in the sample.

Drawing a standard working curve by taking the concentration (%) of the standard working solution as an abscissa (X) and the peak area of a Tween 80 chromatographic peak as an ordinate (Y), wherein the linear equation of the obtained standard working curve is that Y is 1.23 multiplied by 10⁹X-2.78×10⁶Coefficient of correlation R²＝0.999711。

FIG. 3 is a chromatogram of measured Tween 80, and the retention time of 0.512min is Tween 80 chromatogram peak;

FIG. 4 is a standard operating curve; according to the standard working curve, the detection result of the Tween 80 content in the sample to be detected is calculated to be 0.0086% (namely 0.086mg/mL), and the range requirement of 0.005-0.015% (namely 0.05-0.15 mg/mL) is met.

Example 3

The embodiment provides a method for detecting the content of tween 80 in a monoclonal antibody injection, which detects the content of tween 80 in a fully human anti-PD-L1 antibody injection, and comprises the following specific steps:

(1) the solid phase extraction column, which had been loaded with 0.5mL of Protein A packing, was equilibrated with 2.5mL of ultrapure water. Loading 0.1mL of fully human anti-PD-L1 antibody injection (protein concentration is 20mg/mL) to a solid phase extraction column, collecting the effluent, then eluting with 1.5mL of 50% methanol (volume fraction) aqueous solution, and collecting the whole effluent. The solid phase extraction column was washed with 2.5mL of an aqueous solution containing 50mM acetic acid and the wash was discarded. And (3) placing all the collected effluent liquid into a centrifugal concentrator, rotating at 1300 ℃ for 4 hours, and redissolving with ultrapure water to serve as a sample to be detected.

(2) And (3) high performance liquid chromatography detection: the reaction coil has the same specification and performance as 750 mu L;

using a solution of sodium chloride with a molar concentration of 0.15M, sodium tetraborate decahydrate with a molar concentration of 0.025M, acetonitrile with a mass percentage of 5.0%, phenyl-1-naphthylamine with a molar concentration of 5.0M N and tween 80 with a mass concentration of 2.5mg/L as a mobile phase;

the flow rate is 1.5 mL/min; the column temperature is 25 ℃; the sample volume is 10 mu L; the wavelength λ ex of the fluorescence detector is 350nm, and the wavelength λ em of the fluorescence detector is 420 nm.

And injecting the standard working solution into a high performance liquid chromatograph for analysis, then injecting a sample to be detected, and detecting the content of the Tween 80 in the sample.

Drawing a standard working curve by taking the concentration (%) of the standard working solution as an abscissa (X) and the peak area of a Tween 80 chromatographic peak as an ordinate (Y), wherein the linear equation of the obtained standard working curve is that Y is 1.46 multiplied by 10⁹X-2.21×10⁶Coefficient of correlation R²＝0.999951。

FIG. 5 is a chromatogram of Tween 80, and the retention time of 0.512min is Tween 80 chromatogram peak;

FIG. 6 is a standard operating curve; according to the standard working curve, the detection result of the Tween 80 content in the sample to be detected is calculated to be 0.0101% (namely 0.101mg/mL), and the range requirement of 0.005-0.015% (namely 0.05-0.15 mg/mL) is met

Example 4

The difference from the embodiment 1 is that in the embodiment, ethanol with the mass fraction of 100% is used as eluent to carry out leaching;

the remaining steps were as in example 1.

FIG. 7 is the chromatogram map of Tween 80, with Tween 80 peak at retention time of 0.514 min;

according to the standard working curve (same as example 1), the detection result of the Tween 80 content in the sample to be detected is calculated to be 0.0062% (namely 0.062mg/mL), and the theoretical value is 0.01%.

Example 5

The difference from the example 1 is that in the present example, methanol with a mass fraction of 40% is used as an eluent for elution;

the remaining steps were as in example 1.

FIG. 8 is the chromatogram of Tween 80, and the retention time of 0.512min is Tween 80 chromatogram peak;

according to the standard working curve (same as example 1), the detection result of the Tween 80 content in the sample to be detected is 0.0105% (namely 0.105mg/mL), and the theoretical value is 0.01%.

Example 6

The difference from the embodiment 1 is that in the embodiment, acetonitrile with the mass fraction of 40% is used as an eluent for leaching;

the remaining steps were as in example 1.

FIG. 9 is a chromatogram of measured Tween 80, and the retention time of 0.461min is a Tween 80 chromatographic peak;

FIG. 10 is a standard operating curve; the linear equation of the standard working curve is that Y is 1.54 multiplied by 10⁹X-3.75×10⁶Coefficient of correlation R²0.999336. And calculating the detection result of the Tween 80 content in the sample to be detected to be 0.0621% (namely 0.621mg/mL) and the theoretical value to be 0.01% according to the standard working curve.

Example 7

The difference from example 1 is that the flow rate of the mobile phase in this example is 0.5 mL/min; the remaining steps were as in example 1.

Fig. 11 is a chromatogram of measured tween 80, the retention time 1.557min is a tween 80 chromatographic peak, the detection result of the tween 80 content in the sample is 0.0107%, and the decrease of the flow rate does not affect the quantification of tween 80, so that the retention time of the chromatographic peak is increased and the chromatographic peak is widened.

Example 8

The difference from example 1 is that the flow rate of the mobile phase in this example is 2.5 mL/min; the remaining steps were as in example 1.

FIG. 12 is a chromatogram of measured Tween 80, where the retention time is Tween 80 chromatogram peak at 0.316min, and the detection result of Tween 80 content in the sample is 0.0106%, and the increase in flow rate does not affect the quantification of Tween 80, so that the retention time of the chromatogram peak is shortened, and the chromatogram peak is narrowed.

Comparative example 1

Compared with the example 2, the monoclonal antibody injection (protein concentration is 150mg/mL) in the comparative example is directly loaded without pretreatment, and the detection parameters of the high performance liquid chromatography are consistent with those of the example 2;

meanwhile, a standard working solution is prepared, and a standard curve (fig. 13) obtained by the method is drawn, wherein Y is 1.31 × 10⁹X-2.73×10⁶Coefficient of correlation R²＝0.999752；

According to the obtained standard curve, the content of Tween 80 in the monoclonal antibody injection is 0.0168% (namely 0.168mg/mL), and the theoretical value is 0.01%.

Comparative example 2

Compared with the embodiment 1, the monoclonal antibody injection (protein concentration is 5mg/mL) in the comparative example is directly loaded without pretreatment, and the detection parameters of the high performance liquid chromatography are consistent with those of the embodiment 2;

meanwhile, a standard working solution is prepared, and a standard curve (figure 14) obtained by the method is drawn, wherein Y is 1.49 multiplied by 10⁹X-3.87×10⁶Coefficient of correlation R²＝0.998711；

The content of Tween 80 in the monoclonal antibody injection is 0.0104% (namely 0.104mg/mL) and the theoretical value is 0.01% according to the obtained standard curve.

Comparative example 3

Compared with the embodiment 3, the monoclonal antibody injection (protein concentration is 20mg/mL) in the comparative example is directly loaded without pretreatment, and the detection parameters of the high performance liquid chromatography are consistent with those of the embodiment 2;

meanwhile, a standard working solution is prepared, and a standard curve (fig. 15) obtained by the method is drawn, wherein Y is 1.29 × 10⁹X-2.36×10⁶Coefficient of correlation R²＝0.999798；

The content of Tween 80 in the monoclonal antibody injection is 0.0114% (namely 0.114mg/mL) according to the obtained standard curve, and the theoretical value is 0.01%.

Comparative example 4

Compared with example 1, the filler adopted by the solid phase extraction column in the comparative example is hydrophilic bonding filler (GLYCOCLEANTM S CARTRIDGES from Agilent);

the remaining steps were as in example 1.

Meanwhile, a standard working solution is prepared, and a standard curve (fig. 16) obtained by the method is drawn, wherein Y is 1.48 × 10⁹X-3.15×10⁶Coefficient of correlation R²0.999433, the content of Tween 80 in the monoclonal antibody injection was 0.0035% (i.e., 0.035mg/mL), theoretical value 0.01%, according to the obtained standard curve.

Comparative example 5

The injection of example 2 was assayed by methods conventional in the art, the protein was precipitated with an ethanol solution of saturated sodium chloride, the supernatant was dried, and the remaining steps were identical to those of example 2.

Meanwhile, a standard working solution is prepared, and a standard curve (fig. 17) obtained by the method is drawn, wherein Y is 1.77 × 10⁹X-3.54×10⁶Coefficient of correlation R²0.999717, the content of tween 80 in the monoclonal antibody injection was 0.0061% (i.e., 0.061mg/mL), theoretical 0.01% according to the obtained standard curve.

Wherein, the peak areas and the concentrations obtained in the examples 1, 4 to 8 and the comparative example 4 for detecting the recombinant anti-EGFR human-mouse chimeric monoclonal antibody injection are shown in the table 2:

TABLE 2

As can be seen from the results in the above table, when the eluent in the present invention adopts acetonitrile, the detection result of the tween 80 content in the sample to be tested is 0.0621%, which is far beyond the calibration value of the sample to be tested, and the reliability of the result is poor in comparison with the examples 1 and 6;

as can be seen from the comparison among the embodiments 1, 4 and 5, when the leacheate is 100% methanol, 100% ethanol and 40% methanol, respectively, the detection result obtained by using 100% methanol or 40% methanol is closer to the calibration value of the sample to be detected, and when 100% ethanol is used as the leacheate, the detection result obtained by using 100% ethanol is different from the calibration value;

as can be seen from comparison among examples 1, 7 and 8, in the present invention, the flow rate of the mobile phase affects the retention time and width of the chromatographic peak, and when the flow rate is 1-2 mL/min, the peak profile of the obtained peak is more standard;

as can be seen from the comparison between example 1 and comparative example 4, the affinity chromatography medium using Protein A as ligand is used as the filler of the extraction column in the present invention, and the obtained detection result is more accurate.

In addition, as can be seen from comparing example 2 with comparative example 1, if the monoclonal antibody injection is directly loaded without pretreatment, the content of tween 80 in the obtained monoclonal antibody injection is significantly higher under the condition of higher protein content.

Comparing example 2 and comparative example 5, it can be seen that the method provided by the present invention (detection value of 0.0086%) has smaller deviation and higher accuracy under the condition of higher protein content compared with the conventional precipitation method in the art (detection value of 0.0061%).

And comparing examples 1-3 with comparative examples 1-3 (table 3), it can be seen that compared with 5mg/mL and 20mg/mL injection, the method of the present invention has more obvious advantages when treating 150mg/mL monoclonal antibody injection: for the sample with high protein concentration and low Tween 80 content, the detection deviation is obviously reduced.

TABLE 3

Note: deviation | theoretical value-measured value |/theoretical value × 100%; the theoretical value of the tween 80 content of each experimental group sample is 0.01% (namely 0.1mg/mL, the lower content level in the technical field).

In conclusion, the method for detecting the Tween 80 content in the monoclonal antibody injection provided by the invention has the advantages of high result accuracy, short detection period and convenience in operation, and has higher application value for detecting impurities in the monoclonal antibody injection.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.