A method for detecting defoamer 204

Abstract

This invention relates to a method for detecting defoamer 204, belonging to the field of bioproduct quality testing. This invention employs HPLC-ELSD to detect defoamer 204, providing a solution for determining the content of defoamer 204 in bioproducts by optimizing sample pretreatment conditions, HPLC parameters (mobile phase selection, elution gradient), and ELSD parameters (carrier gas flow rate, drift tube temperature, detector gain). Furthermore, this invention exhibits a wide linear range (R04 at 2.5~50 μg / mL) when used for defoamer 204 detection. 2 With advantages such as >0.99, high accuracy (recovery rate of 91.4~98.5%), high sensitivity (limit of detection of 1.0 μg / mL, limit of quantitation of 2.5 μg / mL), good specificity, and simple operation, it can accurately detect the presence and content of defoamer 204 in biological products, filling a gap in the field and having great significance for the quality monitoring and control of biological products.

Description

Technical Field

[0001] This invention relates to a method for detecting defoamer 204, which belongs to the field of biological product quality testing. Background Technology

[0002] In recent years, with the rapid growth of the global biopharmaceutical industry and the swift progress of biopharmaceutical product registration, the safety of biopharmaceuticals has become a key focus for national drug regulatory authorities and biopharmaceutical companies. During the microbial fermentation production of biopharmaceuticals, a large amount of foam is generated due to aeration, stirring, the presence of sugars and proteins in the culture medium, and the accumulation of microbial metabolites. Improper defoaming can lead to loss of effective volume, the risk of liquid escape and contamination, and can also cause inhibition of dissolved oxygen transfer and abnormal microbial metabolism. Therefore, foam control is a crucial step in ensuring production efficiency and product quality. Adding an appropriate amount of defoamer in microbial fermentation production can inhibit foam generation; however, defoamer residue can pose safety risks.

[0003] Antifoam 204 (CAS: 172964-47-5) is a 100% active organic non-silicone defoamer. It is a mixture composed of polypropylene polyether (PPG) dispersions, free of silicone, mineral oil, and animal / plant-derived components, and belongs to a fully synthetic, silicone-free pure polyether structure. Its mechanism of action is as follows: the PPG structure has surface activity, disrupting foam stability by reducing gas-liquid interfacial tension. Because it does not contain silicone oil, it avoids the risk of silicone oil encapsulating bacteria or cells, thus avoiding the inhibition of cell metabolism caused by silicone oil-based defoamers. It is particularly suitable for the production of high-value-added bioproducts with strict residue control. As mentioned above, if antifoam 204 is used in the production of bioproducts, it is necessary to test its residue level to meet safety requirements. However, a search of patent databases and CNKI (China National Knowledge Infrastructure) reveals that existing technologies do not disclose how to detect the content of antifoam 204 in bioproducts. Furthermore, existing methods for detecting other defoamers are not applicable to the determination of antifoam 204 content.

[0004] (a) CN114720573A is used for qualitative detection of organosilicon defoamers (whose main component is dimethylsiloxane), while defoamer 204 belongs to the structure of silicone-free pure polyether. Therefore, the method disclosed in this patent is not applicable to the determination of the content of defoamer 204.

[0005] (ii) Existing technologies for determining the content of polyether defoamers mainly focus on polyoxyethylene-polyoxypropylene (PEO-PPO) copolymers (e.g., Defoamer) and polyhydroxy polyethers (e.g., Dowfax DF103). Although defoamer 204 belongs to polyether defoamers, its structure and properties differ significantly from Defoamer and Dowfax DF103: (1) Defoamer contains hydrophilic segments (PEO) and is highly water-soluble, while defoamer 204 is polypropylene-based polyether (PPG), which does not contain PEO segments and is highly hydrophobic. Therefore, CN117825596A, which is used to detect Defoamer, is not applicable to the detection of defoamer 204; (2) Dowfax DF103 has a polyhydroxy polyether structure, while defoamer 204 does not have a polyhydroxy structure. Therefore, CN117310044A, which is used to detect Dowfax DF103, is also not applicable to the content determination of defoamer 204. Furthermore, the aforementioned two patents use an electrospray detector (CAD) to determine the content of foaming agent or other substances. However, the purchase and maintenance costs of CAD equipment are relatively high, and its operation is complex. It is a proprietary technology of Thermo Fisher Scientific, which has a monopolistic nature and is not conducive to popularization.

[0006] In summary, the existing technology is not publicly available and cannot be applied to the determination of the content of defoamer 204. Therefore, there is an urgent need to develop a dedicated method for detecting the content of defoamer 204 in biological products. Summary of the Invention

[0007] To address the limitation of existing technologies in determining the content of defoamer 204, this invention provides a method based on high-performance liquid chromatography combined with evaporative light scattering detector (HPLC-ELSD) for determining the content of defoamer 204 in biological products. This method optimizes the sample pretreatment conditions (selection of organic solvent I for extracting defoamer 204), HPLC parameters (mobile phase selection, elution gradient), and ELSD parameters (carrier gas flow rate, drift tube temperature, detector gain), achieving qualitative detection of the presence of defoamer 204 in biological products and quantitative determination of its content.

[0008] (I) To achieve the above objectives, the present invention provides a method for detecting defoamer 204 in a test sample based on HPLC-ELSD, as follows:

[0009] (1) HPLC-ELSD method:

[0010] HPLC (High Performance Liquid Chromatography): A reversed-phase column (C4 or C8 column, preferably C4) is used, with mobile phase A being ultrapure water and mobile phase B being methanol or acetonitrile (preferably methanol); gradient elution is employed.

[0011] The gradient elution can employ either elution gradient 1 or elution gradient 2 (elution gradient 1 is preferred):

[0012] Elution gradient 1: 0.00~3.00 min, 50% A; 3.00~5.00 min, 50% A~0% A; 5.00~11.00 min, 0% A; 11.00~12.00 min, 0% A~50% A; 12.00~18.00 min, 50% A, where percentages are volume ratios.

[0013] Elution gradient 2: 0.00~3.00 min, 80% A; 3.00~5.00 min, 80% A~0% A; 5.00~11.00 min, 0% A; 11.00~12.00 min, 0% A~80% A; 12.00~18.00 min, 80% A, where percentages are volume ratios.

[0014] ELSD (Evaporative Light Scattering Detection): Nitrogen or air is used as the carrier gas, with a flow rate of 2.0~3.2 L / min (nitrogen is preferred, with a flow rate of 3.2 L / min); the drift tube temperature is 80~110℃ (preferably 110℃); the detector gain is 1~2 (preferably gain 1); the impactor is in mode 1 (i.e., shunt mode).

[0015] As a preferred embodiment, the parameters of the above HPLC-ELSD detection method are set as follows: ①HPLC: ChromCore 300 C4-T column (column length 4.6×150mm, particle size 5μm); column temperature 45℃; flow rate 1.0mL / min; sample chamber temperature 6℃; injection volume 40μL; mobile phase A is ultrapure water, mobile phase B is methanol, and the elution gradient is the above-mentioned elution gradient 1. ②ELSD: carrier gas is nitrogen, flow rate 3.2L / min; drift tube temperature 110℃; detector gain value 1; impactor mode 1.

[0016] (2) Analysis of test results: If the chromatogram of the test sample solution contains the chromatographic peak of defoamer 204 (consistent with the positive control), the test result is judged to be positive, that is, the test sample contains defoamer 204.

[0017] (ii) Further, the sample to be tested is the upstream fermentation broth in the bioproduct preparation process, or the downstream process sample after the chromatography step (including but not limited to process intermediates, semi-finished bioproducts or finished bioproducts).

[0018] a) When the sample to be tested is fermentation broth, the sample to be tested shall be pretreated before HPLC-ELSD detection: take an appropriate amount of the sample to be tested, centrifuge at 10000~14000 r / min for 3~8 min (preferably centrifuge at 12000 r / min for 5 min), discard the precipitate, take an appropriate amount of the supernatant and mix it with organic solvent I in an equal proportion, vortex for 2~8 min (preferably 5 min), then centrifuge at 10000~14000 r / min for 3~8 min (preferably centrifuge at 12000 r / min for 5 min), and take the organic layer as the test solution.

[0019] b) When the sample to be tested is a process sample after chromatography, the sample to be tested shall be pretreated before HPLC-ELSD detection: take an appropriate amount of the sample to be tested, mix it with organic solvent I in an equal proportion and vortex for 2-8 min (preferably 5 min), then centrifuge at 10000-14000 r / min for 3-8 min (preferably 12000 r / min for 5 min), take the organic layer, dry it by nitrogen blowing or vacuum drying and concentration, take an appropriate amount of organic solvent II to redissolve, let it stand and take the supernatant as the test solution;

[0020] When the sample to be tested is a fermentation broth or a process sample after a chromatography step, preferably, the organic solvent I is selected from one of the following: chloroform, n-hexane, ethylene glycol, methanol, toluene, a mixture of chloroform and methanol in equal proportions, or a mixture of toluene and methanol in equal proportions; wherein, chloroform, methanol, n-hexane or ethylene glycol are preferred, and chloroform is even more preferred.

[0021] When the sample to be tested is a process sample after a chromatography step, preferably, the organic solvent II is selected from chloroform or methanol, with methanol being more preferred.

[0022] (iii) Furthermore, if the test result in step (2) is positive, the defoamer 204 can be quantitatively detected using the standard curve method:

[0023] s1) The standard curve is plotted with the natural logarithm of the concentration of defoamer 204 standard as the abscissa and the natural logarithm of the corresponding chromatographic peak area as the ordinate. Linear regression is then performed to obtain the regression equation.

[0024] s2) Quantitative determination: Based on the natural logarithm of the peak area of ​​defoamer 204 in the measured sample, i.e. ln (peak area), the concentration of defoamer 204 in the measured sample is calculated by referring to the regression equation of the standard curve.

[0025] Preferably, the process for preparing the standard curve regression equation is as follows: Defoamer 204 standard is weighed, dissolved in chloroform or methanol, and prepared into a series of standard solutions with concentrations in the range of 2.5~50 μg / mL (e.g., 2.5, 5.0, 10.0, 20.0, 50.0 μg / mL). HPLC-ELSD detection is performed on these solutions using the same method as for the test sample. Then, a linear regression is performed with the natural logarithm of the concentration of defoamer 204 standard as the abscissa and the natural logarithm of the corresponding chromatographic peak area as the ordinate to obtain the regression equation.

[0026] Preferably, the regression equation is: y = 2.1391x + 0.0165 (R0). 2 =0.9975).

[0027] Preferably, step s2) involves quantitative calculation:

[0028] i) When ln (peak area) is lower than the linear minimum point, it is considered that the sample tested did not detect defoamer 204;

[0029] ii) When ln (peak area) is in the linear range, calculate the content of defoamer 204 using the standard curve regression equation;

[0030] iii) When ln (peak area) exceeds the linear range, dilute the test sample and perform HPLC-ELSD detection again until ln (peak area) falls into the linear range. Then calculate the content of defoamer 204 through the standard curve regression equation.

[0031] The beneficial effects of this invention are:

[0032] (1) By optimizing the pretreatment conditions of the sample to be tested (organic solvent I used to extract defoamer 204), HPLC parameters (selection of organic solvent in mobile phase B, elution gradient), and ELSD parameters (carrier gas flow rate, drift tube temperature, detector gain value), this invention has successfully developed an HPLC-ELSD method for detecting the content of defoamer 204, providing a solution for the determination of the content of defoamer 204.

[0033] (2) When this method is used to detect defoamer 204, the chromatographic peak shape is good and the analysis time is short. A good linear relationship exists between the peak area and the defoamer 204 content when it is between 2.5 and 50 μg / mL. 2 The concentration of the antifoaming agent was >0.99; the recovery rate was 91.4-98.5%, which is good; the limit of quantitation was determined to be 2.5 μg / mL and the limit of detection was 1.0 μg / mL by the signal-to-noise ratio. Therefore, this method has the advantages of simple operation, wide linear range, high sensitivity, and good specificity, and is suitable for detecting the presence and content of antifoaming agent 204 in biological products.

[0034] (3) This invention enables the determination of the content of defoamer 204 in process samples of microbial fermentation production and fermentation product purification of biological products, filling the gap in the determination of the content of this substance. It can be used to accurately evaluate the quality of biological products and is of great significance in the quality control of biological products and the safety of clinical drug use. Attached Figure Description

[0035] Figure 1 : Overlay spectrum of various dilutions of defoamer 204 reference standard in Example 1;

[0036] Figure 2 Standard curves were plotted for each dilution of the defoamer 204 reference standard from Example 1.

[0037] Figure 3 : Overlay spectrum of the test sample (fermentation broth) in Example 1;

[0038] Figure 4 : The overlay spectrum of the test sample (chromatographic process sample) in Example 1;

[0039] Figure 5 Comparative Example 1: Column-to-column superimposed chromatograms;

[0040] Figure 6 Comparative Example 3: Flow Relative Overlay Pattern;

[0041] Figure 7 Comparative Example 4: Elution gradient comparison overlay map;

[0042] Figure 8 Comparative Example 5: Overlay graphs showing the comparison of carrier gas velocities;

[0043] Figure 9 Comparative Example 6: Temperature comparison and overlay of drift tubes;

[0044] Figure 10 Comparative Example 7: Gain Value Comparison and Overlay Spectra. Detailed Implementation

[0045] To facilitate understanding of the present invention, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] Example 1

[0047] In this embodiment, the parameter settings and operation steps for detecting defoamer 204 by HPLC-ELSD method are provided, and the feasibility, accuracy and sensitivity of the method for detecting defoamer 204 in biological products (fermentation broth, chromatographic process samples) are verified.

[0048] 1.1 HPLC-ELSD method for detecting defoamer 204:

[0049] (1) HPLC: An Agilent 1260 high performance liquid chromatograph was used; the chromatographic parameters were as follows: the column was ChromCore300 C4-T (4.6×150mm, 5μm); the column temperature was 45℃; the flow rate was 1.0mL / min; the sample chamber temperature was 6℃; mobile phase A was ultrapure water, mobile phase B was methanol, and the ratio of mobile phase A and B was set according to Table 1. The injection volume was 40μL.

[0050] Table 1. Elution gradient settings for mobile phases A and B

[0051]

[0052] (2) ELSD: The ELSD6100 evaporative light scattering detector of Aote is used; the ELSD parameters are as follows: the carrier gas is air or nitrogen (nitrogen is an inert gas and is theoretically superior to air; in this embodiment, air is used as the carrier gas, and nitrogen is also feasible when air is feasible), the flow rate is 3.2 L / min; the drift tube temperature is 110℃; the detector gain value is 1; the impactor is mode 1 (split mode).

[0053] 1.2 Standard Curve Construction:

[0054] (1) Preparation of antifoaming agent 204 reference solution: Accurately weigh 100 mg of antifoaming agent 204 reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with methanol, mix well, and the resulting antifoaming agent 204 reference standard stock solution (1.0 mg / mL). Take an appropriate amount of the above stock solution and dilute it with methanol to a series of reference standard solutions with concentrations of 2.5, 5.0, 10.0, 20.0, and 50.0 μg / mL.

[0055] (2) Defoamer 204 reference standard detection: Defoamer 204 was detected according to the method described in Section 1.1. Linear regression was performed on the natural logarithm of the peak area of ​​defoamer 204 against the natural logarithm of the solution concentration to obtain the linear equation between the two, and R was calculated. 2 .

[0056] See test results Figure 1 , Figure 2 : Figure 1 The graph shows the superimposed spectra of various dilutions of defoamer 204 reference standard. Figure 2 Standard curves were plotted for each dilution of defoamer 204 reference standard. The results showed that the retention time of the defoamer 204 reference standard solution was 9.028 min, with a concentration ranging from 2.5 to 50 μg / mL. The natural logarithm of the defoamer 204 reference standard concentration showed a good linear relationship with the natural logarithm of its peak area, with the regression equation y = 2.1391x + 0.0165, R0.2 =0.9975.

[0057] 1.3 Detection of the sample (fermentation broth):

[0058] (1) Preparation of test solution: Take an appropriate amount of fermentation broth containing defoamer 204, centrifuge at 12000 r / min for 5 min, discard the precipitate, take an appropriate amount of supernatant and mix it with organic solution I (trichloromethane is used in this example) in an equal proportion, vortex for 5 min, centrifuge at 12000 r / min for 5 min after completion, and take the organic layer as the test solution. For the negative control, take fermentation broth without defoamer 204 and operate in the same way.

[0059] (2) Detection of test solution: Defoamer 204 was detected according to the method described in Section 1.1. i) When ln (peak area) is below the minimum linear point, it is considered not detected; ii) When ln (peak area) is in the linear range, the content of defoamer 204 was calculated by the standard curve equation; iii) When ln (peak area) exceeds the linear range, the test sample was diluted and then HPLC-ELSD was performed until ln (peak area) fell into the linear range. Then the content of defoamer 204 was calculated by the standard curve regression equation.

[0060] See test results Figure 3 Substituting the natural logarithm of the measured peak area into the regression equation, the results showed that the fermentation broth containing defoamer 204 had a concentration of 16.5 μg / mL.

[0061] 1.4 Detection of the sample to be tested (process sample for chromatography step):

[0062] (1) Preparation of test solution: Take an appropriate amount of the sample to be tested (process sample of chromatography step), mix it with organic solution I (trichloromethane in this example) in equal proportion and vortex for 5 min. After completion, centrifuge at 12000 r / min for 5 min, take out the organic layer, dry it by nitrogen blowing, take an appropriate amount of organic solution II (methanol in this example) to redissolve, let it stand and take the supernatant as the test solution.

[0063] (2) Detection of test solution: Defoamer 204 was detected according to the method described in Section 1.1. i) When ln (peak area) is below the minimum linear point, it is considered undetectable; ii) When ln (peak area) is in the linear range, the content of defoamer 204 was calculated by the standard curve equation; iii) When ln (peak area) exceeds the linear range, the test sample was diluted and then HPLC-ELSD was performed until ln (peak area) fell into the linear range. Then the content of defoamer 204 was calculated by the standard curve regression equation.

[0064] See test results Figure 4The results showed that the chromatographic sample analysis data were not detected.

[0065] It is evident that the present invention has the advantages of simple operation and wide linear range, and can be used for qualitative detection and quantitative determination of defoamer 204 in biological products.

[0066] Example 2

[0067] This embodiment aims to study the accuracy, sensitivity, and specificity of the detection method for defoamer 204 provided by the present invention.

[0068] 2.1 Accuracy Testing

[0069] Preparation of accuracy test solution: Take an appropriate amount of the sample to be tested (process sample of the chromatography step, where defoamer 204 was not detected), and add three concentrations of defoamer 204 reference solution (high, medium, and low, final concentrations: 40, 10, and 3 μg / mL), respectively, and mix well. Take an appropriate amount and mix with organic solution I (chloroform) in an equal proportion and vortex for 5 min. After completion, centrifuge at 12000 r / min for 5 min, collect the organic layer, and dry it using a nitrogen blower. Take an appropriate amount of organic solution II (methanol) to reconstitute the solution, let it stand, and take the supernatant as the accuracy solution.

[0070] Defoamer 204 detection: Defoamer 204 was detected by HPLC-ELSD as described in Section 1.1 of Example 1, and the recovery rate was recorded. Generally, a recovery rate between 80% and 120% is considered to meet the accuracy requirements.

[0071] The results showed that the high, medium, and low recovery rates of defoamer 204 were 98.5%, 95.3%, and 91.4%, respectively, proving that the detection method for defoamer 204 provided by this invention has good accuracy.

[0072] 2.2 Sensitivity Detection

[0073] Preparation of sensitivity detection solutions: Take an appropriate amount of the stock solution of the 204 standard (without foaming agent) and dilute it with methanol to prepare sensitivity solutions of concentrations of 0.5, 1.0, and 2.5 μg / mL.

[0074] Defoamer 204 detection: Defoamer 204 was detected by HPLC-ELSD method as described in Section 1.1 of Example 1. The signal-to-noise ratio (S / N) was recorded. The limit of detection (S / N) should be ≥3 and the limit of quantitation (S / N) should be ≥10.

[0075] The results showed that when the content of defoamer 204 was 1.0 μg / mL, the S / N ratio was 9, which was the limit of detection for this method; when the content was 2.5 μg / mL, the S / N ratio was 27, which was the limit of quantitation for this method.

[0076] 2.3 Specificity Detection

[0077] Preparation of specific detection solution: When the sample to be tested is fermentation broth, select fermentation broth without defoamer 204 as the negative control solution; for example, when the sample to be tested is a sample from a chromatography process step, select its corresponding buffer solution as the negative control solution. The negative control solution is processed according to Sections 1.3 and 1.4 of Example 1, and the defoamer 204 is detected by HPLC-ELSD method as described in Section 1.1. The negative control solution should have no interfering peaks at the elution point of the positive control.

[0078] The negative control chromatogram is shown below. Figure 3 , Figure 4 The corresponding spectral lines of the "negative control" were obtained. The results showed that no interfering peaks appeared at the 204 elution point of the above negative control solution, indicating good specificity (judgment criterion: the chromatogram of the negative control solution should have no interfering peaks).

[0079] In summary, the present invention is used for the detection of defoamer 204 and has the advantages of high accuracy, high sensitivity and good specificity.

[0080] Comparative Example 1

[0081] This comparative example aims to compare the effect of column selection in the HPLC process on the detection of defoamer 204 by HPLC-ELSD: the HPLC-ELSD parameters were set according to Section 1.1 of Example 1, with the only difference being that column 1 was C8 (AdvanceBioRP-mAb SB-C8, 2.1×100mm); and column 2 was C4 (ChromCore 300 C4-T 4.6×150mm).

[0082] Preparation of antifoaming agent 204 reference solution: Accurately weigh 100 mg of antifoaming agent 204 reference standard into a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and this is the antifoaming agent 204 reference standard stock solution. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for comparative studies of the above chromatographic columns (chromatographic column 1 and chromatographic column 2).

[0083] See test results Figure 5 The results showed that the peak shape of defoamer 204 on column 2 (C4) was better than that on column 1 (C8) (narrower peak shape and higher peak height), which could achieve higher sensitivity and a more stable baseline. Therefore, column 2 (C4) was preferred.

[0084] Comparative Example 2

[0085] This comparative example aims to compare the effect of the selection of extraction reagent, i.e., organic solvent I, in the sample pretreatment stage on the detection of defoamer 204 by HPLC-ELSD: the sample pretreatment conditions are as described in Sections 1.3 and 1.4 of Example 1, except for the selection of organic solvent I (chloroform, n-hexane, ethylene glycol, methanol, toluene, a mixture of chloroform and methanol in equal proportions, a mixture of toluene and methanol in equal proportions, acetone); the HPLC-ELSD parameter settings are as described in Section 1.1 of Example 1.

[0086] Take an appropriate amount of the process sample after the chromatography step, and add an appropriate concentration of defoamer 204 reference standard (final concentration of 50 μg / ml). Mix it with the above organic solvent I in equal proportion and vortex for 5 min. Then centrifuge at 12000 r / min for 5 min, collect the organic layer, and dry it by nitrogen blowing or vacuum drying and concentration. Take an appropriate amount of organic solvent II (methanol) to redissolve it, let it stand, and take the supernatant as the test solution. Refer to the spiking recovery test carried out in the accuracy test in Example 2, and select the better organic solvent I according to the spiking recovery rate.

[0087] Table 2. Comparison of different organic solvents I

[0088]

[0089] Note: Acetone was miscible with the sample (without separation), so 204 could not be extracted by liquid-liquid extraction, and therefore no further operations were performed.

[0090] The results showed that: (1) Except for acetone, all other organic reagents I could be used to extract defoamer 204. According to the recovery rate of the spiked test sample, the recovery rates were as follows: chloroform (98.6%) > methanol (94.1%) > n-hexane (90.0%) > ethylene glycol (85.2%) > chloroform + methanol (1:1) (83.9%) > toluene (81.2%) > toluene + methanol (1:1) (76.9%), where ">" indicates "better than". (2) Therefore, among the above organic solvents I, chloroform, methanol, n-hexane or ethylene glycol are preferred as extraction reagents in the sample pretreatment process, and chloroform is the most preferred organic reagent I for sample pretreatment.

[0091] Comparative Example 3

[0092] This comparative example aims to compare the effect of the choice of organic solvent in mobile phase B on the detection of defoamer 204 by HPLC-ELSD: the HPLC-ELSD parameters were set according to Section 1.1 of Example 1, except that mobile phase B was methanol or acetonitrile.

[0093] Preparation of defoamer 204 reference solution: Accurately weigh 100 mg of defoamer 204 reference standard into a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and this is the defoamer 204 reference standard stock solution. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for the comparative study of the above mobile phase B (methanol vs. acetonitrile).

[0094] The results show that methanol, as mobile phase B, has lower baseline noise, a more stable baseline, and a larger peak area than acetonitrile, resulting in higher sensitivity. Therefore, methanol is the preferred mobile phase B. See attached spectral data. Figure 6 .

[0095] Comparative Example 4

[0096] This comparative example aims to compare the effects of different elution gradient settings in the HPLC process on the detection of defoamer 204 by HPLC-ELSD: the HPLC-ELSD parameter settings are as described in Section 1.1 of Example 1, except that the elution gradients of mobile phases A and B are set differently (elution gradient 1 vs. elution gradient 2: see Table 3 below).

[0097] Table 3: Gradient elution settings

[0098]

[0099] Preparation of antifoaming agent 204 reference solution: Accurately weigh 100 mg of antifoaming agent 204 reference standard into a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and this is the antifoaming agent 204 reference standard stock solution. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for comparative studies of the above elution gradient.

[0100] The results show that elution gradient 1 has lower baseline noise and a more stable baseline than elution gradient 2. Therefore, elution gradient 1 is preferred. (See graph below.) Figure 7 .

[0101] Comparative Example 5

[0102] This comparative example aims to compare the effect of carrier gas flow rate on the detection of defoamer 204 by HPLC-ELSD in the ELSD process: the HPLC-ELSD parameters were set according to Section 1.1 of Example 1, except that the carrier gas flow rate was set differently (2.0 L / min, 3.2 L / min).

[0103] Preparation of defoamer 204 reference solution: Accurately weigh 100 mg of defoamer 204 reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with methanol, mix well, and this is the defoamer 204 reference standard stock solution. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for the comparative study of the above carrier gas flow rates of 2.0 and 3.2 L / min.

[0104] The results show that a carrier gas flow rate of 3.2 L / min results in a more stable baseline and lower noise, making 3.2 L / min the preferred carrier gas flow rate. See attached graph. Figure 8 .

[0105] Comparative Example 6

[0106] This comparative example aims to compare the effect of drift tube temperature on the detection of defoamer 204 by HPLC-ELSD in the ELSD process: the HPLC-ELSD parameters were set according to Section 1.1 of Example 1, except that the drift tube temperature was set differently (80 / 100 / 110℃).

[0107] Preparation of defoamer 204 reference solution: Accurately weigh 100 mg of defoamer 204 reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with methanol, and mix well. This is the stock solution of defoamer 204 reference standard. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for comparative studies of drift tube temperatures of 80 / 100 / 110℃.

[0108] The results show that as the drift tube temperature increases, the baseline becomes more stable and the noise decreases; the optimal drift tube temperature is 110℃. (See attached graph.) Figure 9 .

[0109] Comparative Example 7

[0110] This comparative example aims to compare the effect of detector gain value in the ELSD process on the detection of defoamer 204 by HPLC-ELSD: the HPLC-ELSD parameter settings are the same as in Section 1.1 of Example 1, except that the detector gain value is set differently (1 or 2).

[0111] Preparation of defoamer 204 reference solution: Accurately weigh 100 mg of defoamer 204 reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with methanol, and mix well. This is the defoamer 204 reference standard stock solution. Take an appropriate amount of the above stock solution and dilute it with methanol to a suitable concentration for the comparative study of gain values ​​1 and 2 mentioned above.

[0112] The results showed that when the gain value was set to 2, the peak height response value exceeded the maximum peak height range when the concentration of the reference defoamer 204 solution was 50 μg / mL, and the baseline noise was high. Therefore, a gain value of 1 is preferred. See attached graph. Figure 10 .

[0113] As demonstrated by the above embodiments, the present invention is effective and feasible for determining the content of defoamer 204 in biological products. This method has advantages such as simple operation, speed, high accuracy, high sensitivity, and low cost, and is suitable for determining the content of defoamer 204 in biological products, filling a gap in its content determination.

Claims

1. A method for detecting defoamer 204 in a test sample based on HPLC-ELSD, characterized in that: (1) HPLC-ELSD method: HPLC: A reversed-phase column was used; mobile phase A was ultrapure water, and mobile phase B was methanol or acetonitrile; gradient elution was used, with either elution gradient 1 or elution gradient 2 as follows: a) Elution gradient 1: 0.00~3.00 min, 50% A; 3.00~5.00 min, 50% A~0% A; 5.00~11.00 min, 0% A; 11.00~12.00 min, 0% A~50% A; 12.00~18.00 min, 50% A, where percentages are volume ratios. b) Elution gradient 2: 0.00~3.00 min, 80% A; 3.00~5.00 min, 80% A~0% A; 5.00~11.00 min, 0% A; 11.00~12.00 min, 0% A~80% A; 12.00~18.00 min, 80% A, where percentages are volume ratios. ELSD: Nitrogen or air is used as the carrier gas, with a flow rate of 2.0~3.2 L / min; the drift tube temperature is 80~110℃; the detector gain is 1~2; the impactor adopts a shunt mode; (2) Analysis of test results: According to the chromatogram obtained in step (1), if there is a chromatographic peak that is consistent with the positive control of defoamer 204, the test result is initially judged to be positive, that is, defoamer 204 is present in the sample.

2. The method according to claim 1, characterized in that, In HPLC, the reversed-phase column is a C4 or C8 column.

3. The method according to claim 2, characterized in that, In HPLC, the reversed-phase column used is a C4 column with a length of 4.6 × 150 mm and a particle size of 5 μm.

4. The method according to claim 1, characterized in that, In HPLC, the mobile phase B is methanol.

5. The method according to claim 1, characterized in that, HPLC: column temperature 45℃; flow rate 1.0 mL / min; sample chamber temperature 2~8℃; injection volume 40 μL.

6. The method according to claim 1, characterized in that, ELSD: Nitrogen gas is used as the carrier gas, and the flow rate is 3.2 L / min.

7. The method according to claim 1, characterized in that, ELSD: Select 110℃ for the drift tube temperature and 1 for the gain value.

8. The method according to claim 1, characterized in that, The sample to be tested is the upstream fermentation broth of a bioproduct or the downstream process sample after a chromatography step.

9. The method according to claim 8, characterized in that, The sample to be tested is a fermentation broth. Before performing HPLC-ELSD detection, the sample to be tested is pretreated as follows: Take an appropriate amount of the sample to be tested, centrifuge at 10000~14000 r / min for 3~8 min, discard the precipitate, take an appropriate amount of the supernatant and mix it with organic solvent I in an equal proportion, vortex for 2~8 min, then centrifuge at 10000~14000 r / min for 3~8 min, and take the organic layer as the test solution; wherein, the organic solvent I is selected from one of the following: chloroform, n-hexane, ethylene glycol, methanol, toluene, a mixture of chloroform and methanol in an equal proportion, or a mixture of toluene and methanol in an equal proportion.

10. The method according to claim 8, characterized in that, The sample to be tested is the process sample after the chromatography step. Before performing HPLC-ELSD detection, the sample to be tested is pretreated as follows: Take an appropriate amount of the sample to be tested, mix it with organic solvent I in an equal proportion and vortex for 2-8 min, then centrifuge at 10000-14000 r / min for 3-8 min, aspirate the organic layer, and dry it by nitrogen blowing or vacuum drying and concentration. Redissolve the sample with organic solvent II, let it stand, and take the supernatant as the test solution. The organic solvent I is selected from one of the following: chloroform, n-hexane, ethylene glycol, methanol, toluene, a mixture of chloroform and methanol in an equal proportion, or a mixture of toluene and methanol in an equal proportion; the organic solvent II is selected from chloroform or methanol.

11. The method according to any one of claims 1-10, characterized in that, If the test result in step (2) is positive, the defoamer 204 can be further quantitatively determined using the standard curve method: s1) The standard curve is plotted with the natural logarithm of the concentration of defoamer 204 standard as the abscissa and the natural logarithm of the corresponding chromatographic peak area as the ordinate. Linear regression is then performed to obtain the regression equation. s2) Quantitative determination: Based on the natural logarithm of the peak area of ​​defoamer 204 in the measured sample, i.e. ln (peak area), the concentration of defoamer 204 in the measured sample is calculated by referring to the regression equation of the standard curve.

12. The method according to claim 11, characterized in that, The process for preparing the regression equation is as follows: Defoamer 204 standard is weighed, dissolved in chloroform or methanol, and prepared into a series of standard solutions with concentrations in the range of 2.5~50μg / mL. HPLC-ELSD detection is performed on each solution using the same method as the test sample. A graph is plotted with the natural logarithm of the concentration of defoamer 204 standard as the abscissa and the natural logarithm of the corresponding chromatographic peak area as the ordinate. Linear regression is then performed to obtain the regression equation.

13. The method according to claim 11, characterized in that, The regression equation is: y = 2.1391x + 0.0165.

14. The method according to claim 11, characterized in that, In step s2) quantitative determination: i) When ln (peak area) is lower than the linear minimum point, it is considered that the sample tested did not detect defoamer 204; ii) When ln (peak area) is in the linear range, calculate the content of defoamer 204 using the standard curve regression equation; iii) When ln (peak area) exceeds the linear range, dilute the test sample and perform HPLC-ELSD detection until ln (peak area) falls into the linear range. Then calculate the content of defoamer 204 through the standard curve regression equation.

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