Method for simultaneously determining residual quantity of polyethylene glycol 6000 and Tween 80 in recombinant norovirus vaccine stock solution

Abstract

The invention relates to the technical field of detection, in particular to a high performance liquid chromatography (HPLC)-evaporative light scattering detector (ELSD) analysis method for simultaneously determining residual quantity of process-related impurities PEG6000 and Tween 80 in a recombinant norovirus vaccine stock solution. The detection method for simultaneously determining the residual quantity of PEG6000 and Tween 80 is established by exploring a gradient elution method, screening the sample size of HPLC and optimizing the evaporation and atomization temperature of ELSD, and the method is good in specificity, linearity and durability, high in accuracy and precision and capable of being exclusively used for detecting the residual quantity of process-related impurities PEG6000 and Tween 80 in the recombinant norovirus vaccine stock solution.

Description

Technical Field

[0001] The invention relates to the field of detection technology, in particular to a method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in a recombinant norovirus vaccine stock solution. Background Art

[0002] Norovirus (NOV) belongs to the Caliciviridae family and is the main pathogen of sporadic and epidemic non-bacterial gastroenteritis. Currently, there are no commercial drugs or vaccines for the treatment or blocking of Norovirus. Therefore, the development of Norovirus vaccines is of great significance to public health. The recombinant Norovirus bivalent (GI.1 / GII.4) vaccine (Hansen yeast) jointly applied by the Chinese Academy of Biological Products and Lanzhou Institute of Biological Products Co., Ltd. is the world's first NOV vaccine to enter Phase III clinical research. The vaccine is composed of virus-like particles (VLPs) composed of NOV VP1 protein expressed by recombinant Hansen yeast, which are purified and added with aluminum-containing adjuvants. It will be used to prevent moderate / severe acute gastroenteritis caused by GI.1 and GII.4 Norovirus infection.

[0003] PEG6000 is a water-soluble polyether obtained by the step-by-step addition polymerization of ethylene oxide and water or ethylene glycol. As an amphiphilic polymer, it is easily soluble in water and most organic solvents. It is widely used as a protein precipitant and pharmaceutical excipient due to its good biocompatibility and safety. It can also be used to modify proteins and peptides to enhance stability and prolong half-life. PEG6000 is used as a precipitant in the purification of the recombinant norovirus bivalent (GI.1 / GII.4) vaccine (Hansen yeast). Tween 80 is a polyol-type nonionic surfactant. Due to its strong hydrophilicity, it is widely used as a solubilizer, emulsifier, dispersant and stabilizer in vaccine production (D'SOUZAAA, SHEGOKARR. Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications. Expert Opin Drug Deliv [J]. 2016, 13 (9): 1257-1275. IBRAHIM M, RAMADANE, ELSADEK NE, et al. Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products [J]. J Control Release. 2022, 351: 215-230. KERWIN BA. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways [J]. J Pharm Sci. 2008, 97 (8): 2924-2935.). Tween 80 was used as a solubilizer in the purification of recombinant norovirus bivalent (GI.1 / GII.4) vaccine (Hansenula).

[0004] During the purification of the recombinant norovirus bivalent (GI.1 / GII.4) vaccine (Hansen yeast), PEG6000 and Tween80 were both added reagents. Studies have reported that PEG may be a hapten, and PEG6000 can induce the aggregation of macromolecules in aqueous solution, and excessive amounts can cause liver damage (Zhang P, Sun F, Liu S, Jiang S. Anti-PEG antibodies in the clinic: Current issues and beyond PEGylation [J]. J Control Release. 2016; 244 (PtB): 184-193.). The hydrophilic chain of polysorbate 80 contains ester bonds, alcohol hydroxyl groups and ether oxygen groups, which are easy to hydrolyze, form hydrogen bonds or complexes. Its polyoxyethylene chain is unstable and easily undergoes self-oxidation degradation when exposed to air, which poses a potential threat to the safety of drugs (Li Yue, Zhang Lumei, Duan Jinlian, et al. Impurities, safety issues and possible solutions of polysorbate 80 for injection [J]. Journal of Yunnan University of Traditional Chinese Medicine, 2018, 41(04): 93-97.). In the subsequent protein purification process of vaccine preparation, PEG6000 and Tween 80 will be gradually removed and diluted, but there is still a possibility that PEG6000 and Tween 80 will remain in the vaccine stock solution. Therefore, the determination of PEG6000 and Tween 80 residues in the recombinant norovirus bivalent (GI.1 / GII.4) vaccine (Hansen yeast) is of key significance to the quality standard of the vaccine.

[0005] Chinese patent document CN114295745B discloses a method for detecting the residual amount of dimethyl sulfoxide in a live attenuated varicella vaccine, comprising the following steps: S1, mixing the live attenuated varicella vaccine stock solution and acetonitrile in a volume ratio of 1: (10-20), and taking the supernatant after centrifugation as the test solution; S2, taking the test solution and using the gas chromatography external standard method for detection. Chinese patent document CN113406237A discloses a method for detecting the content of Tween 80 in a monoclonal antibody injection based on solid phase extraction. Chinese patent document CN110220859B discloses a method for detecting the residual amount of Tween 80 in hemostatic powder, which belongs to the technical field of residual detection of chemical substances in medicine. However, there is no report on a method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in a recombinant norovirus vaccine stock solution. Summary of the invention

[0006] The purpose of the present invention is to establish a high performance liquid chromatography (HPLC)-evaporative light scattering detector (ELSD) detection method for simultaneously detecting the residual amounts of process-related impurities polyethylene glycol 6000 (PEG6000) and Tween 80 (Tween80) in the recombinant norovirus vaccine stock solution, and to verify and preliminarily apply the method.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention establishes a detection method for simultaneously determining the residual amounts of PEG6000 and Tween 80 by exploring a gradient elution method, screening the injection amount of HPLC and optimizing the evaporation and atomization temperature of ELSD. The method adopts a NanoChrom Ghost-Remover (4.6*50mm) column to reduce baseline noise, adopts a ChromCore SAA (4.6*150mm, 5μm) column to separate the target components, and uses 0.1% acetic acid aqueous solution and 0.1% acetic acid isopropanol solution as mobile phases for gradient elution, the flow rate is 1.2mL / min, the column temperature is 25℃, the injection amount is 100μL, the ELSD evaporation temperature is 75℃, the atomization temperature is 65℃, the gas flow rate is 1.60SLM, the detection time is 20min, and the post-running time is 5min. The specificity, linearity and range, quantitative limit, accuracy and precision of the method are verified. The established method was used to detect the residual amounts of PEG6000 and Tween 80 in three batches of GI.1 and three batches of GII.4 recombinant Norovirus (NOV) vaccine stock solutions.

[0009] The target peak of PEG6000 appeared at about 6.7min, and the target peak of Tween 80 appeared at about 10.9min, and there was no interference between the two. There were no interference peaks in the water blank, solvent blank and stock solution buffer at about 6.7min and 10.9min. This method can exclusively determine the residual amount of PEG6000 and Tween80 in the vaccine stock solution. The concentrations of PEG6000 and Tween 80 were in the range of 5-50μg / mL, and the logarithms of their concentrations and the logarithms of their peak areas showed good linear relationships, R 2>0.990; the limits of quantification of PEG6000 and Tween 80 were both 3.125μg / mL; the recoveries of high, medium and low concentrations of PEG6000 and Tween 80 reference substances were all between 90% and 115%; the precision verification showed that the RSDs of the retention time, peak height and peak area of ​​PEG6000 and Tween 80 were all less than 5%. No PEG6000 and Tween 80 were detected in 3 batches of GI.1 and 3 batches of GII.4 recombinant NOV vaccine concentrates. The method of the present invention has good specificity, linearity and durability, high accuracy and precision, and can be used to detect the residual amount of PEG6000 and Tween 80 in the recombinant norovirus vaccine concentrate.

[0010] Based on the above technical scheme, the present invention provides a method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in a recombinant norovirus vaccine stock solution, comprising the following steps:

[0011] (A) Setting the test conditions

[0012] HPLC conditions: the mobile phase was 0.1% acetic acid aqueous solution and 0.1% acetic acid isopropanol solution in a volume ratio of (0-90): (10-100), gradient elution. The flow rate was 1.2 mL / min, the column temperature was 25°C, the injection volume was 5-100 μL. The detection time was 20 min, and the post-run time was 5 min.

[0013] ELSD conditions: evaporation temperature 60-75 °C, atomization temperature 55-70 °C, gas flow rate 1.60 SLM.

[0014] (B) Draw a standard curve

[0015] Prepare 100, 50, 25, 12.5 and 10 μg / mL PEG6000 and Tween80 reference substances respectively, mix the PEG6000 and Tween80 reference substances in an equal volume of 1:1 to prepare 50, 25, 12.5, 6.25 and 5 μg / mL PEG6000+Tween80 mixed reference substances, inject and detect under the conditions of step (A), obtain the peak areas of PEG6000 and Tween80, draw standard curves with the logarithm of the PEG6000 and Tween80 concentrations as the abscissa and the logarithm of the elution peak area as the ordinate, and obtain the regression equation of PEG6000 and Tween80.

[0016] (C) The vaccine stock solution is tested under the conditions of step (A), and the contents of PEG6000 and Tween80 in the vaccine stock solution are calculated based on the obtained peak area and the standard curve and regression equation obtained in step (B).

[0017] Furthermore, a NanoChrom Ghost-Remover column with an inner diameter of 4.6 mm and a column length of 50 mm was installed at the front end of the injector according to the liquid flow direction to reduce baseline noise.

[0018] Furthermore, the chromatographic column used in the method is a ChromCore SAA column with an inner diameter of 4.6 mm, a column length of 150 mm, and a particle size of 5 μm.

[0019] Further, the gradient elution method is as follows, recording the elution peak at 4.1-14.7 min in the chromatogram:

[0020]

[0021] Furthermore, in the method described, the injection volume is 100 μL.

[0022] Furthermore, in the method described above, the evaporation temperature of the evaporative light scattering detector is 75°C and the atomization temperature is 65°C.

[0023] Furthermore, in the method described above, the limits of quantification of PEG6000 and Tween 80 were both 3.125 μg / mL.

[0024] The advantages of the present invention are:

[0025] The present invention uses a ChromCore SAA surfactant-specific chromatographic column in a reverse / ion exchange mixed mode, and realizes baseline separation of antigen protein, PEG6000 and Tween 80 in the sample by optimizing the gradient elution method according to the difference in hydrophobicity of each component. The separation eluent is introduced into a universal detector ELSD that does not rely on the optical properties of the analyte, and the temperature and airflow controlled ELSD atomizer atomizes the eluent into a droplet plume of uniform size, and then the solvent component is removed by an independently temperature-controlled evaporation tube, leaving solute particles with low volatility, and finally the solute particles enter the optical detection cell for detection, and finally realizes simultaneous and efficient analysis of PEG6000 and Tween 80. This method has good specificity, linearity and durability, high accuracy and precision, and can be exclusively used for the detection of process-related impurities PEG6000 and Tween 80 residues in the recombinant norovirus vaccine stock solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Determination of injection volume.

[0027] Figure 2 .Determination of ELSD evaporation and atomization temperature.

[0028] Figure 3.Chromatogram of simultaneous detection of PEG6000 and Tween80 by HPLC-ELSD method; Note: A is 25μg / mL PEG6000 and Tween80 mixed reference; B is water blank; C is solvent blank.

[0029] Figure 4 . Specificity verification chromatogram; Note: A is a 25μg / mL PEG6000 and Tween80 mixed reference substance; B is a water blank; C is a solvent blank; D is a stock solution buffer; E is a vaccine stock solution; F is a vaccine stock solution containing a 25μg / mL PEG6000 and Tween80 mixed reference substance.

[0030] Figure 5 .The standard curve of PEG6000 and Tween80 was detected simultaneously by HPLC-ELSD method. DETAILED DESCRIPTION

[0031] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0032] Example:

[0033] 1 Materials and methods

[0034] 1.1 Sample

[0035] The recombinant norovirus vaccine concentrate was developed by the Chinese Academy of Biological Products and prepared and provided by the Fourth Laboratory of Lanzhou Institute of Biological Products Co., Ltd.

[0036] 1.2 Main reagents and instruments

[0037] Polyethylene glycol 6000 (8.17007.9050) and Tween 80 (8170611000) were purchased from Merck, USA; isopropanol was purchased from TEDIA, USA; acetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.; NanoChrom Ghost-Remover (4.6*50mm) and ChromCore SAA (4.6*150mm, 5μm) chromatographic columns were purchased from Suzhou Nanovita Technology Co., Ltd.; chromatographic bottles were purchased from Waters, USA; water purifier was purchased from Merck Millipore, Germany; high performance liquid chromatograph (1260 Infinity II) and evaporative light scattering detector (G4260B) were purchased from Agilent, USA.

[0038] 1.3 Solution preparation

[0039] 1.3.1 Mobile phase

[0040] Mobile phase A (0.1% acetic acid aqueous solution): take 2 mL of acetic acid and dilute to 2 L with ultrapure water, filter through 0.45 μm and degas for later use; Mobile phase B (0.1% acetic acid isopropanol solution): take 2 mL of acetic acid and dilute to 2 L with isopropanol, filter through 0.45 μm and degas for later use.

[0041] 1.3.2PEG6000 reference substance

[0042] 100mg / g PEG6000 stock solution: weigh 90.00±0.01g ultrapure water in a 250mL conical flask using an electronic balance, add PEG6000 to make the mass 100.00±0.01g, and stir at room temperature for 30 minutes; 1mg / g PEG6000 stock solution: weigh 99.00±0.01g ultrapure water in a 250mL conical flask using an electronic balance, add 100mg / g PEG6000 stock solution to make the mass 100.00±0.01g, and stir at room temperature for 30 minutes; Take 1mg / g PEG6000 stock solution and dilute it with ultrapure water to prepare PEG6000 reference substance according to Table 1.

[0043] Table 1 Dilution method of PEG6000 reference working solution

[0044]

[0045] 1.3.3 Tween80 reference

[0046] 100 mg / g Tween80 stock solution: weigh 90.00±0.01 g ultrapure water in a 250 mL conical flask using an electronic balance, add Tween80 to make the mass 100.00±0.01 g, and stir at room temperature for 60 minutes; 1 mg / g Tween80 stock solution: weigh 99.00±0.01 g ultrapure water in a 250 mL conical flask using an electronic balance, add 100 mg / g Tween80 stock solution to make the mass 100.00±0.01 g, and stir at room temperature for 60 minutes; take 1 mg / g Tween80 stock solution and dilute it with ultrapure water to prepare Tween80 reference substance according to Table 2.

[0047] Table 2 Dilution method of Tween80 reference substance working solution

[0048]

[0049]

[0050] 1.3.4PEG6000+Tween80 mixed reference

[0051] 100, 50, 25, 12.5, 10 and 6.25 μg / mL of PEG6000 and Tween80 reference substances were mixed in equal volumes of 1:1 to prepare 50, 25, 12.5, 6.25, 5 and 3.125 μg / mL of PEG6000+Tween80 mixed reference substances.

[0052] 1.4 Method development

[0053] 1.4.1 Screening of injection volume

[0054] The injection volumes were selected as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μL to investigate the retention behavior of the defoaming agent.

[0055] 1.4.2 Screening of ELSD evaporation and atomization temperature

[0056] The ELSD gas flow rate was set to 1.60 SLM, and the evaporation temperature and atomization temperature were selected to be 75°C / 70°C, 70°C / 65°C, 65°C / 60°C, 65°C / 60°C, 65°C / 55°C and 60°C / 55°C, respectively, to investigate the retention behavior of the defoaming agent.

[0057] 1.4.3 According to the liquid flow direction, NanoChrom Ghost-Remover (4.6*50mm) ghost peak removal column was installed at the front end of the injector, and ChromCore SAA (4.6*150mm, 5μm) chromatographic column was installed in the column oven. The chromatographic column was balanced with 90% mobile phase A + 10% mobile phase B at a flow rate of 1.2mL / min. The injection volume was 100μL, the column temperature was 25℃, the ELSD evaporation temperature was 75℃, the atomization temperature was 65℃, the gas flow rate was 1.60SLM, the analysis time was 20 minutes, the post-run time was 5 minutes, and the gradient elution method was shown in Table 3. The elution peaks at 4.1-14.7min in the chromatogram were recorded. Linear regression was performed on the logarithm of the PEG6000 concentration and the logarithm of the peak area of ​​the corresponding elution peak, and the logarithm of the Tween80 concentration and the logarithm of the peak area of ​​the corresponding elution peak, respectively, and the concentrations of PEG6000 and Tween80 in the test sample were calculated according to the regression equation.

[0058] Table 3 Reverse HPLC gradient elution method

[0059]

[0060]

[0061] 1.5 Method Validation

[0062] 1.5.1 Exclusivity

[0063] The water blank, solvent blank (90% mobile phase A + 10% mobile phase B), stock buffer, vaccine stock solution and vaccine stock solution containing 25ug / mL PEG6000 + Tween80 were injected and tested according to the method in 1.4.4 to verify the specificity of the method.

[0064] 1.5.2 Linearity and Range

[0065] Prepare 50, 25, 12.5, 6.25 and 5 μg / mL of PEG6000+Tween80 mixed reference substances according to the method in 1.3.4, and perform sampling and detection according to the method in 1.4.4. Perform linear regression on the logarithm of the PEG6000 concentration and the logarithm of the elution peak area, respectively, and obtain the regression equation and linear correlation coefficient, and determine the linear range.

[0066] 1.5.3 Minimum limit of quantification

[0067] Prepare 6.25, 5 and 3.125 μg / mL PEG6000+Tween80 mixed reference substances according to the method in 1.3.4, and inject each sample 6 times according to the method in 1.4.4 to obtain the signal / noise ratio (Signal / Noise, S / N). The minimum quantification limit of this method is determined by S / N>10.

[0068] 1.5.4 Accuracy

[0069] Prepare 50μg / mL, 12.5μg / mL and 5μg / mL PEG6000+Tween80 mixed reference substances according to the method in 1.3.4, inject each sample 3 times, and substitute into the regression equation to calculate the measured concentration. The recovery rate is obtained by dividing the measured concentration by the theoretical concentration.

[0070] 1.5.5 Precision

[0071] 1.5.5.1 Instrument repeatability

[0072] Prepare one portion of 25 μg / mL PEG6000+Tween80 mixed reference substance according to the method in 1.3.4, inject it six times continuously, and calculate the relative standard deviation (RSD) of retention time, peak height and peak area of ​​the six injections.

[0073] 1.5.5.2 Sample repeatability

[0074] Prepare three copies of 25 μg / mL PEG6000+Tween80 mixed reference substances according to the method in 1.3.4. Inject each sample twice and calculate the RSD of retention time, peak height and peak area of ​​the six injections.

[0075] 1.5.5.3 Intermediate precision

[0076] The effects of different analysts and different dates on precision were investigated. The experimental scheme is shown in Table 4. For each experiment, one 25 μg / mL PEG6000+Tween80 mixed reference was prepared according to the method in 1.3.4, and each sample was injected twice. The RSD of retention time, peak height and peak area of ​​6 injections was calculated.

[0077] Table 4 Intermediate precision test scheme

[0078]

[0079] 1.6 Preliminary application of the method

[0080] The established method was used to detect the residual amount of PEG6000+Tween80 in three batches of GI.1 and three batches of GII.4 recombinant NOV vaccine stock solutions.

[0081] 2 Results

[0082] 2.1 Method development

[0083] 2.1.1 Overlapping spectra of injection volumes of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μL are shown in Figure 1 As the injection volume increased, the peak area and peak height of the target peaks of PEG6000 and Tween80 increased. In order to improve the sensitivity of the residue detection method, an injection volume of 100 μL was selected.

[0084] 2.1.2 Determination of injection volume

[0085] The peak height, peak area and signal-to-noise ratio of PEG6000 and Tween80 under different ELSD evaporation and atomization temperature conditions are shown in Figure 2. Figure 2 When the evaporation temperature is 75℃ and the atomization temperature is 65℃, the peak height, peak area and signal-to-noise ratio of PEG6000 and Tween80 are the highest. Therefore, 75℃ / 65℃ are selected as the evaporation and atomization temperatures of ELSD.

[0086] 2.1.2 Method determination

[0087] Through gradient elution, the target peak of PEG6000 appeared at about 6.7min, and the target peak of Tween80 appeared at about 10.9min, and there was no interference between the target peaks of PEG6000 and Tween80. Figure 3 .

[0088] 2.2 Validation of the method

[0089] 2.2.1 Exclusivity

[0090] The 25μg / mL PEG6000 and Tween80 mixed reference substance and the vaccine stock solution containing 25μg / mL PEG6000 and Tween80 mixed reference substance showed absorption peaks at around 6.7min and 10.9min, respectively, while the water blank, solvent blank, stock solution buffer and vaccine stock solution had no interference peaks at around 6.7min and 10.9min. Therefore, this method can exclusively determine the residual amount of PEG6000 and Tween80 in the vaccine stock solution, see Figure 4 .

[0091] 2.2.2 Linearity and range

[0092] The concentration of PEG6000 and Tween80 mixed reference substances was in the range of 5-50 μg / mL. The logarithm of the concentration and the logarithm of the peak area showed a good linear relationship. The regression equation of PEG6000 was Ig(peak area)=

[0093] 1.8434*Ig(concentration)+0.2055, R 2 is 0.997; Tween80 regression equation is Ig(peak area)=1.6164*Ig(concentration)+0.3819, R 2 is 0.999. Figure 5 .

[0094] 2.2.3 Minimum limit of quantification

[0095] The S / N of 6.250μg / mL, 5.000μg / mL and 3.125μg / mL defoamer reference substances are shown in Table 5. The minimum quantification limit of this method was determined to be 3.125μg / mL.

[0096] Table 5 Signal-to-noise ratio

[0097]

[0098] 2.2.4 Accuracy

[0099] The recoveries of 50 μg / mL, 12.5 μg / mL and 5 μg / mL PEG6000 and Tween80 reference substances were all in the range of 90% to 115%, as shown in Table 6.

[0100] Table 6 Accuracy verification results

[0101]

[0102] 2.2.5 Precision

[0103] 2.2.5.1 Instrument repeatability

[0104] The retention time, peak height and peak area of ​​PEG6000 and Tween80 were measured six times with 1 portion of 25 μg / mL mixed reference substance, and the RSDs were all less than 5%, as shown in Table 7.

[0105] Table 7 Instrument repeatability

[0106]

[0107] 2.2.5.2 Intermediate precision

[0108] The RSDs of retention time, peak height and peak area of ​​PEG6000 and Tween80 measured by 25 μg / mL PEG6000 and Tween80 mixed reference substances prepared by different analysts on the same date and by the same analyst on different dates were all <5%, see Table 8.

[0109] Table 8 Intermediate precision verification results

[0110]

[0111] 2.3 Application of the method

[0112] The test results showed that the residual amounts of PEG6000 and Tween80 in the recombinant NOV vaccine concentrates of batches GI.1 of B-20231001 (GI.1), B-20231101 (GI.1) and B-20231102 (GI.1) and batches GII.4 of B-20230801 (GII.4), B-20230901 (GII.4) and B-20230902 (GII.4) were all below the limit of quantification.

[0113] 3 Discussions

[0114] The three volumes of the Chinese Pharmacopoeia include colorimetric methods for detecting the content of PEG and Tween in biological products (Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (Volume III) [S]. Beijing: China Medical Science and Technology Press. 2020: 114.), but Wang Jin et al. found that Tween has a significant impact on the detection of PEG content (Wang Jin, He Peng, Zhao Ran, et al. Establishment and validation of HPLC-evaporative light scattering detector detection method for polyethylene glycol 6000 residue in hepatitis B vaccine [J]. Chinese Journal of Biological Products, 2017, 30(07): 742-746+750.). Since both PEG6000 and Tween 80 are used in the purification of recombinant Norovirus bivalent vaccine, this method should not be used for detection.

[0115] At present, PEG6000 is commonly measured by HPLC-differential refractive index detection, gel chromatography and other methods, but the differential refractive index detector has low sensitivity and cannot be gradient eluted. The gel chromatography has low sensitivity and is not suitable for residual detection (LI N, ZIEGEMEIER D, BASS L, WANG W. Quantitation of free polyethylene glycol in PEGylated protein conjugate by size exclusion HPLC with refractive index (RI) detection [J]. J Pharm Biomed Anal. 2008; 48 (5): 1332-1338. WANG J, TANG S, ZHANG K. Quantitation of polyethylene glycol by size exclusion chromatography with charged aerosol, differential refractive index, and multi-angle lightscattering detectors [J]. J Pharm Biomed Anal. 2024; 238: 115854.). Studies have shown that C4, C8, C18, and HILIC columns all produce multiple peaks for PEG6000, and are difficult to separate from the main component (CHENG TL, CHUANG KH, CHEN BM, et al. Analytical measurement of PEGylated molecules [J]. Bioconjug Chem. 2012; 23(5): 881-899.).There are two methods for determining Tween 80: direct method and indirect method. The direct method includes spectrophotometry and SEC-ELSD method. The accuracy and repeatability of spectrophotometry are poor. The accuracy of SEC-ELSD method is greatly challenged because Tween 80 is not a single chemical component, and the composition of different manufacturers varies greatly, and the chromatographic behavior is complex (ZHANGS, XIAO H, LI N. The Characterization of Polysorbate [J]. Curr Protoc. 2022; 2 (7): e489. Nayak VS, Tan Z, Ihnat PM, Russell RJ, Grace MJ. Evaporative light scattering detection based HPLC method for the determination of polysorbate 80 in therapeutic protein formulations. J Chromatogr Sci. 2012; 50 (1): 21-25.). Indirect methods include HPLC-UV and gas chromatography. The HPLC-UV method requires hydrolysis of Tween 80 into oleic acid, which is a complex and time-consuming process, and the oleic acid formed by hydrolysis has poor stability. The gas chromatography method requires hydrolysis of Tween 80 into oleic acid, and then immediately methylates it to obtain methyl oleate for detection. This method has high sensitivity, but the increase in processing steps has an adverse effect on accuracy and precision (DEGTEREV MB, BADAUKAYTE RA, SHUKUROV RR. A development of high-throughput HPLC method for the polysorbate 80 quantitation in protein therapeutic products [J]. J Chromatogr B Anallyt Technol Biomed Life Sci. 2019; 1133: 121847. SHENDE N, KARALE A, BHAGADE S, et al. Evaluation of a sensitive GC-MS method to detect polysorbate 80 in vaccine preparation [J]. JPharm Biomed Anal.2020;183:113126.).

[0116] Considering that both PEG6000 and Tween 80 are non-ionic surfactants, PEG6000 has only ether structure and hydroxyl structure, no ultraviolet absorption, and Tween 80 has no strong chromophore. The present invention uses a reverse / ion exchange mixed mode ChromCore SAA surfactant-specific chromatographic column, and according to the difference in sample hydrophobicity, optimizes the gradient elution method to achieve baseline separation of antigen protein, PEG6000 and Tween80 in the sample. The separation eluent is introduced into a universal detector ELSD that does not rely on the optical properties of the analyte. The temperature and airflow controlled ELSD atomizer atomizes the eluent into a droplet plume of uniform size, and then the solvent component is removed through an independently temperature-controlled evaporation tube, leaving solute particles with low volatility. Finally, the solute particles enter the optical detection cell for detection, and finally achieve simultaneous and efficient analysis of PEG6000 and Tween 80. This method was comprehensively methodologically validated in accordance with the guidelines for validation of analytical methods for drug quality standards in Part III of the 2020 edition of the Chinese Pharmacopoeia 9101. The method of the present invention has good specificity, linearity and durability, high accuracy and precision, and can be used to detect the residual amounts of PEG6000 and Tween 80 in the recombinant norovirus bivalent vaccine stock solution.

[0117] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in a recombinant norovirus vaccine stock solution, characterized in that: The following steps are involved: (A) Setting the test conditions HPLC conditions: the mobile phase was a 0.1% acetic acid aqueous solution and a 0.1% acetic acid isopropanol solution in a volume ratio of (0-90): (10-100), gradient elution; the flow rate was 1.2 mL / min, the column temperature was 25°C, the injection volume was 5-100 μL; the detection time was 20 min, and the post-run time was 5 min; ELSD conditions: evaporation temperature 60-75°C, atomization temperature 55-70°C, gas flow rate 1.60SLM; (B) Draw a standard curve Prepare 100, 50, 25, 12.5 and 10 μg / mL of PEG6000 and Tween80 reference substances, respectively; mix the PEG6000 and Tween80 reference substances in an equal volume ratio of 1:1 to prepare 50, 25, 12.5, 6.25 and 5 μg / mL of PEG6000+Tween80 mixed reference substances; inject and test under the conditions of step (A) to obtain the peak areas of PEG6000 and Tween80; plot standard curves with the logarithms of the concentrations of PEG6000 and Tween80 as the abscissa and the logarithms of the elution peak areas of the elution peaks as the ordinate, and obtain regression equations for PEG6000 and Tween80; (C) The vaccine stock solution is tested under the conditions of step (A), and the contents of PEG6000 and Tween80 in the vaccine stock solution are calculated based on the obtained peak area and the standard curve and regression equation obtained in step (B).

2. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: A NanoChrom Ghost-Remover column with an inner diameter of 4.6 mm and a length of 50 mm was installed at the front end of the injector according to the direction of liquid flow.

3. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: The chromatographic column is a ChromCore SAA column with an inner diameter of 4.6 mm, a column length of 150 mm, and a particle size of 5 μm.

4. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: The gradient elution method is as follows, recording the elution peak at 4.1-14.7 min in the chromatogram:

5. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: The injection volume was 100 μL.

6. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: The evaporation temperature of the evaporative light scattering detector was 75 °C and the nebulization temperature was 65 °C.

7. The method for simultaneously determining the residual amounts of polyethylene glycol 6000 and Tween 80 in the recombinant norovirus vaccine stock solution according to claim 1, characterized in that: The limits of quantification of PEG6000 and Tween 80 were both 3.125 μg / mL.

Images