HPLC-ELSD determination method for residues in human blood coagulation factor VIII

Abstract

The invention belongs to the technical field of blood product detection methods, and particularly relates to an HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector) determination method for residues in a human blood coagulation factor VIII. According to the scheme, an analysis method for detecting polysorbate 80 and polyethylene glycol 3350 in the human blood coagulation factor VIII by adopting a reversed-phase high-performance liquid chromatography in combination with an evaporative light-scattering detector is established. After the sample reconstitution fluid is subjected to high-speed centrifugation or filtration, supernate is taken and analyzed on a machine. Quantitative analysis results show that for polysorbate 80, the correlation coefficient is greater than 0.999 in the range of 10-500 mg / L; for polyethylene glycol 3350, the correlation coefficient is greater than 0.998 in the range of 50-2000mg / L. According to the method, the two residues can be rapidly and accurately detected, the technical problem that the accuracy of the detection result of the two residues is not ideal in the detection method of the human blood coagulation factor VIII in the prior art can be solved, and the method has wide popularization and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of blood product detection methods, and in particular relates to a HPLC-ELSD determination method for residues in human coagulation factor VIII. Background Art

[0002] Human coagulation factor VIII is a plasma glycoprotein that has a corrective effect on coagulation dysfunction caused by a deficiency of this substance. It is mainly used to prevent and treat bleeding symptoms caused by hemophilia A and acquired coagulation factor VIII deficiency, as well as to treat surgical bleeding in such patients.

[0003] Polysorbate 80 (also known as Tween 80) is a non-ionic surfactant and emulsifier, widely used as a pharmaceutical excipient. In the production of human coagulation factor VIII, polysorbate 80 is usually used as a cosolvent, or combined with tributyl phosphate as a virus inactivator. Polyethylene glycol (PEG) is a hydrophilic polymer that is widely used in the separation and purification of biological macromolecules such as proteins and nucleic acids. PEG3350 is a polyethylene glycol with an average molecular weight of 3350 and is a commonly used reagent in the separation and preparation of human coagulation factor VIII. Relevant studies have shown that polyethylene glycol may be a hapten and should be quality controlled. The above two substances are reagents used in the separation and preparation of human coagulation factor VIII, and both will form residues in the finished product. The above substances need to be controlled within a certain range to ensure product quality and safety. Therefore, it is particularly important to develop accurate and reliable detection methods for the two substances.

[0004] The determination of polysorbate 80 residues under the human coagulation factor VIII test item in Part III of the "Chinese Pharmacopoeia (2020 Edition)" adopts UV-visible spectrophotometry in accordance with General Rule 3203, and the determination of polyethylene glycol residues adopts UV spectrophotometry in accordance with General Rule 3202. The ingredients of human coagulation factor VIII products are complex. In addition to human coagulation factor VIII, they also contain a variety of plasma proteins, heparin sodium, amino acids, tributyl phosphate and many other ingredients. The use of ultraviolet spectrophotometry to determine the polysorbate 80 residues and polyethylene glycol residues has low selectivity and the measurement results are easily interfered. Another commonly used method for polymer analysis is size exclusion chromatography. Since the molecular weight distributions of polymers such as polysorbate 80, polyethylene glycol 3350, and heparin sodium overlap, they are difficult to separate, affecting the quantitative accuracy. Therefore, further research is urgently needed on the detection methods of the two human coagulation factor VIII residues to ensure the accuracy of the detection of the residues and to obtain a pretreatment method that is simple, linear, repeatable, accurate and reliable to meet relevant detection needs. Summary of the invention

[0005] The present invention aims to provide an HPLC-ELSD determination method for polymer residues in human coagulation factor VIII, so as to solve the technical problem that the detection results of polysorbate 80 and polyethylene glycol 3350 in the prior art human coagulation factor VIII are not accurate enough.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] The invention discloses an HPLC-ELSD determination method for polymer residues in human coagulation factor VIII, wherein the residues include polysorbate 80 and polyethylene glycol 3350; the HPLC-ELSD determination method includes detection for polysorbate 80 and detection for polyethylene glycol 3350.

[0008] Further, the parameter conditions adopted for HPLC-ELSD determination are:

[0009] Chromatographic column: Shimadzu surfactant special analytical column ShimNex S-C18-SAA (150mm×4.6mm, 5μm);

[0010] Mobile phase: Mobile phase A is 0.1% acetic acid in water, and mobile phase B is 0.1% acetic acid in isopropanol;

[0011] Injection volume: 10 μL;

[0012] Flow rate: 1.0 mL / min;

[0013] Column temperature: 30°C;

[0014] Dynamic gain: wide;

[0015] Drift tube temperature: 80°C;

[0016] Atomizing gas pressure: 350KPa;

[0017] Elution mode: gradient elution.

[0018] Further, in the detection of polysorbate 80, the elution procedure of the mobile phase is shown in the following table:

[0019] Time (min) Mobile phase A percentage (volume) Mobile phase B percentage (volume) 0.00 90％ 10％ 1.00 80％ 20％ 3.40 80％ 20％ 3.50 0％ 100％ 6.50 0％ 100％ 6.60 90％ 10％ 10.00 90％ 10％

[0020] The initial concentration of mobile phase B was 10%.

[0021] Further, in the detection of polyethylene glycol 3350, the elution procedure of the mobile phase is shown in the following table:

[0022]

[0023]

[0024] The initial concentration of mobile phase B was 0%.

[0025] Furthermore, in the detection of polysorbate 80, the sample to be tested is taken and redissolved, and then centrifuged to obtain the supernatant; the supernatant is subjected to HPLC-ELSD detection.

[0026] Furthermore, in the detection of polysorbate 80, the centrifugation parameter was 14000 rpm for 10 min.

[0027] Furthermore, in the detection of polyethylene glycol 3350, the sample to be tested is taken and redissolved, the sample is filtered and the filtrate is taken for HPLC-ELSD detection.

[0028] Furthermore, in the detection of polyethylene glycol 3350, an acetate fiber filter membrane with a pore size of 0.45 um was used for the filtration process.

[0029] Furthermore, in the detection of polysorbate 80, the linear range was 10-500 mg / L; in the detection of polyethylene glycol 3350, the linear range was 50-2000 mg / L.

[0030] Furthermore, the product of human coagulation factor VIII contains plasma proteins and excipients; the plasma proteins include human coagulation factor VIII, VWF factor, fibronectin, fibrinogen, IgA protein, IgM protein, IgG protein, and fibrinogen; the excipients include polyethylene glycol 3350, polysorbate 80, tributyl phosphate, glycine, histidine, heparin, and citric acid.

[0031] The principle and beneficial effects of this technical solution are:

[0032] The present technical solution provides an analytical method for determining the residual amount of polysorbate 80 and polyethylene glycol 3350 (PEG3350) in human coagulation factor VIII by combining Shimadzu ShimNex S-C18-SAA surfactant-specific column and Shimadzu's new ELSD-LT III detector with high sensitivity and wide linear range. After screening and trying a large number of chromatographic columns, the inventor found that the ShimNex S-C18-SAA surfactant-specific column can achieve good separation of PEG3350 and polysorbate 80 from other substances such as macromolecular proteins, amino acids and heparin sodium in human coagulation factor VIII products. In addition, the chromatographic column of the present solution can be directly injected (after very simple centrifugation or filtration treatment), without the need for pre-treatment by methods such as protein precipitation or solid phase extraction, and without the need to worry about macromolecular proteins clogging the chromatographic column. This scheme uses Shimadzu's new ELSD-LT III detector, which has the characteristics of high sensitivity and dynamic gain function, and can meet the high-sensitivity test requirements of PEG3350 and polysorbate 80 in human coagulation factor VIII. The wide linear range means that high-concentration samples can be accurately measured without dilution, which saves time and effort. This scheme adopts a reasonable mobile phase composition and elution procedure, which saves reagents and costs while achieving good separation of the chromatographic peaks of PEG3350 and polysorbate 80. In summary, this method has high sensitivity, a wide linear range, and simple pretreatment, and is suitable for the determination of the residual amount of PEG3350 and polysorbate 80 in human coagulation factor VIII with complex composition.

[0033] In this technical solution, the key technical points for determining the residual amounts of PEG3350 and polysorbate 80 in human coagulation factor VIII are:

[0034] (1) The use of ShimNex S-C18-SAA surfactant-specific column can avoid the generation of PEG3350 double peaks compared to other types of chromatographic columns, ensuring the accuracy of quantitative detection (see Comparative Example 2, Figure 11-13 ). And the use of this chromatographic column can reduce the need for complex sample pretreatment. Due to the coexistence of large molecular proteins, amino acids and heparin sodium in human coagulation factor VIII products, the use of other chromatographic columns requires more complex sample pretreatment. Moreover, even if the corresponding pretreatment is performed, it is difficult to effectively ensure the chromatographic peak shape of PEG3350 using other types of chromatographic columns.

[0035] (2) The concentration of the acetic acid aqueous solution in the mobile phase (A and B) is low, which is one tenth of the conventional method in the prior art. This can meet the analysis requirements and save reagent costs. The mild conditions are also conducive to extending the service life of the chromatographic column (see Fig.15 ).

[0036] (3) Different starting ratios of mobile phase B were used for PEG3350 and polysorbate 80 to ensure ideal peak shapes for the two target substances (see Figure 8 , Fig.16 ). In particular, for PEG3350, 0% of the initial mobile phase B had a very significant effect on the peak shape, which was not expected by the inventors before the experiment, and an unexpected technical effect was obtained (see Fig.16 ).

[0037] (4) When the concentration of polysorbate 80 in the sample is high, two polysorbate 80 peaks will appear in the chromatogram, one of which has a retention time close to that of PEG3350, affecting quantitative detection. Using the detection conditions of this scheme, PEG3350 and polysorbate 80 can be fully separated to ensure detection accuracy (see Fig.14 ). To ensure that the polysorbate 80 peak and the PEG3350 peak are fully separated, the setting of the elution gradient is very critical. Using other elution gradient forms will result in unsatisfactory separation of the two peaks (see Fig. 9 ).

[0038] Due to the setting of the above key technical points, the accurate determination of the residual amounts of PEG3350 and polysorbate 80 in human coagulation factor VIII is guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the chemical information of polysorbate 80.

[0040] Figure 2 Chemical information for polyethylene glycol 3350.

[0041] Figure 3 The chromatogram of the polysorbate 80 standard and the specificity of the PEG3350 standard of Example 1 are shown.

[0042] Figure 4 This is the calibration curve of the polysorbate 80 and PEG3350 standards of Example 1.

[0043] Figure 5 The chromatograms of the human coagulation factor VIII sample and the spiked sample in Example 1 are overlapped (for polysorbate 80).

[0044] Figure 6 The overlapping chromatograms of the human coagulation factor VIII sample, standard and spiked sample of Example 1 (for PEG3350).

[0045] Figure 7 The overlapping chromatograms of Comparative Example 1 (top: mixed standard of polysorbate 80 and PEG 3350; bottom: human coagulation factor VIII sample).

[0046] Figure 8 Overlapping chromatograms of polysorbate 80 at different initial concentrations of phase B in Comparative Example 1 (50 μg / mL).

[0047] Fig. 9 This is the effect of the gradient change amplitude of Comparative Example 1 on the analysis of polysorbate 80.

[0048] Fig.10 This is the influence of the blank interference peak of polysorbate 80 in comparative example 1.

[0049] Fig.11 This is the separation chromatogram of the target substance PEG3350 in human coagulation factor VIII and other substances in the sample in comparative example 2 (overlapping chromatogram of the separation effect of ShimNex S-C18-SAA column).

[0050] Fig.12 The SEC chromatographic column separation effect overlay chromatogram of Comparative Example 2.

[0051] Fig.13 The above is an overlapping chromatogram of the separation effect of the C18 chromatographic column of Comparative Example 2.

[0052] Fig.14 The chromatogram is a diagram showing the separation of PEG3350 and polysorbate 80 in Comparative Example 2.

[0053] Fig.15 This is the overlapping chromatogram of PEG3350 and polysorbate 80 standards under the conditions of 0.1% and 1% acetic acid ratio in the mobile phase of Comparative Example 2 (both concentrations are 10 mg / mL, injection volume is 1 μL).

[0054] Fig.16 The overlapping chromatograms of PEG3350 single label of comparative example 2 at initial concentrations of 10% and 0% in phase B (both concentrations were 1 mg / mL, and the injection volume was 1 μL). DETAILED DESCRIPTION

[0055] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are conventional methods; the materials, reagents, etc. used can all be obtained from commercial sources.

[0056] Example 1

[0057] (1) The equipment used for HPLC-ELSD analysis and detection is:

[0058] Analytical instrument: integrated high performance liquid chromatograph LC-2040C 3D;

[0059] Detector: ELSD-LT III;

[0060] Chromatography workstation: LabSolutions Ver.5.120.

[0061] (2) Analysis conditions (HPLC-ELSD analysis parameters)

[0062] Chromatographic column: Shimadzu surfactant special analytical column ShimNex S-C18-SAA (150mm×4.6mm, 5μm);

[0063] Mobile phase: Mobile phase A is 0.1% acetic acid in water; Mobile phase B is 0.1% acetic acid in isopropanol;

[0064] Injection volume: 10 μL;

[0065] Flow rate: 1.0 mL / min;

[0066] Column temperature: 30°C;

[0067] Dynamic gain: wide;

[0068] Drift tube temperature: 80°C;

[0069] Atomizing gas pressure: 350KPa;

[0070] Different elution methods were used for polyethylene glycol 3350 (PEG3350) and polysorbate 80.

[0071] The elution mode of PEG3350 was: gradient elution, and the initial concentration of mobile phase B was 0% (see Table 1 for details).

[0072] The elution mode of polysorbate 80 was: gradient elution, and the initial concentration of mobile phase B was 10% (see Table 2 for details).

[0073] Table 1: Elution gradient for PEG3350

[0074] Time (min) Flow rate Mobile phase A percentage (volume) Mobile phase B percentage (volume) 0.00 1.0mL / min 100％ 0％ 1.00 1.0mL / min 90％ 10％ 3.40 1.0mL / min 80％ 20％ 3.50 1.0mL / min 0％ 100％ 6.50 1.0mL / min 0％ 100％ 6.60 1.0mL / min 100％ 0％ 10.00 1.0mL / min 100％ 0％

[0075] Table 2: Elution gradient for polysorbate 80

[0076] Time (min) Flow rate Mobile phase A percentage (volume) Mobile phase B percentage (volume) 0.00 1.0mL / min 90％ 10％ 1.00 1.0mL / min 80％ 20％ 3.40 1.0mL / min 80％ 20％ 3.50 1.0mL / min 0％ 100％ 6.50 1.0mL / min 0％ 100％ 6.60 1.0mL / min 90％ 10％ 10.00 1.0mL / min 90％ 10％

[0077] (3) Preparation of standard solution

[0078] Standard stock solution: Weigh an appropriate amount of polysorbate 80 standard, dissolve it in ultrapure water, mix it well, and prepare a 10 mg / mL solution. Weigh an appropriate amount of PEG3350 standard, dissolve it in ultrapure water, mix it well, and prepare a 10 mg / mL solution.

[0079] Standard working solution: For polysorbate 80, dilute the standard stock solution with ultrapure water to prepare standard solutions with concentrations of 10, 20, 50, 100, 200, and 500 mg / L. For PEG3350, dilute the standard stock solution with ultrapure water to prepare standard solutions with concentrations of 50, 100, 200, 500, 1000, and 2000 mg / L.

[0080] (4) Sample pretreatment

[0081] Take the human coagulation factor sample reconstituted solution, centrifuge at 14000rpm for 10min, take the supernatant for analysis (if the detection object is polysorbate 80, this method is preferred); or take the human coagulation factor sample reconstituted solution, filter with a 0.45um acetate fiber filter membrane, and take the filtrate for analysis (if the detection object is PEG3350, this method is preferred).

[0082] (5) Test results

[0083] (5.1) Specificity and linear relationship

[0084] The chromatogram of polysorbate 80 standard solution (10 mg / L) is as follows: Figure 3 The calibration curve is shown in Figure 3 . Six standard working solutions were prepared as described above, and the determination was carried out according to the above analytical conditions. The calibration curve was drawn by the external standard method with the logarithm of the peak area as the ordinate and the logarithm of the concentration as the abscissa. Polysorbate 80 had good linearity in the concentration range of 10-500 mg / L, with a correlation coefficient of 0.9997 and a readback value accuracy of 92.5-103.3%.

[0085] The overlapping chromatogram of PEG3350 standard solution (50 mg / L) and solvent blank showed that there was no obvious interference at the retention time of the target peak, indicating good method specificity. For the specificity and calibration curve of PEG3350, see Figure 4 . Six standard working solutions were prepared as described above, and the determination was performed according to the above analytical conditions. The calibration curve was drawn by the external standard method with the logarithm of the peak area as the ordinate and the logarithm of the concentration as the abscissa. PEG3350 had good linearity in the concentration range of 50-2000 mg / L, with a correlation coefficient of 0.9983 and a readback value accuracy of 90.9-109.3%.

[0086] (5.2) Precision test

[0087] The standard solutions of three different concentrations of polysorbate 80 were injected six times in succession, and the precision results of retention time and peak area are shown in Table 3. The RSDs of retention time and peak area were between 0.04-0.20% and 1.49-2.84%, respectively, and the instrument precision was good.

[0088] Table 3: Polysorbate 80 precision results (n=6)

[0089]

[0090] The PEG3350 standard solution with a concentration of 100 mg / L was injected 6 times continuously, and the precision results of retention time and peak area are shown in Table 4. The RSDs of retention time and peak area were between 0.10% and 2.72%, respectively, and the instrument precision was good.

[0091] Table 4: PEG3350 precision results (n=6)

[0092]

[0093]

[0094] (5.3) Spike recovery experiment

[0095] The residual amount of polysorbate 80 in a sample reconstituted solution of human coagulation factor VIII was 38.9 mg / L. Polysorbate 80 standard solution was added to the reconstituted solution of human coagulation factor VIII sample to prepare spiked samples with spiked concentrations of 10, 50 and 200 mg / L. The samples were processed according to the above method and measured on the instrument. The overlapping chromatograms of the sample and the spiked sample (10 mg / L) are shown in Figure 5 The spike recovery results are shown in Table 5. The spike recovery rates of three different concentrations were between 93.2% and 98.2%.

[0096] Table 5: Polysorbate 80 spike recovery results

[0097]

[0098] No PEG3350 was detected in a sample of human coagulation factor VIII. Spiked samples with concentrations of 100 and 1000 mg / L were prepared and measured on the machine. The overlapping chromatograms of the sample, standard (50 mg / L) and spiked sample (100 mg / L) are shown in Figure 6 The spike recovery results are shown in Table 6. At 100 and 1000 mg / L spike concentrations, the spike recoveries were 103.6% and 112.3%, respectively.

[0099] Table 6: PEG3350 spike recovery results (ND means not detected)

[0100]

[0101] In summary, this protocol establishes an analytical method for detecting polysorbate 80 and PEG3350 residues in human coagulation factor VIII using reversed-phase high performance liquid chromatography combined with evaporative light scattering detector. ELSD-LT III has an original dynamic range extension function, a wide linear range, and simple parameter settings. Under the detection conditions of this protocol, polysorbate 80 and PEG3350 are well separated from other components in the sample. The results show that the method has simple pretreatment, good linearity, good repeatability, accuracy and reliability, and is suitable for the detection of polysorbate 80 and PEG3350 residues in human coagulation factor VIII.

[0102] Comparative Example 1: Study on the detection conditions of polysorbate 80

[0103] The ingredients of human coagulation factor VIII products are complex, as shown in Table 7. In addition to human coagulation factor VIII, it also contains a variety of plasma proteins, heparin sodium, PEG3350, polysorbate 80, amino acids, tributyl phosphate and many other ingredients. Therefore, the detection of target residues (whether polysorbate 80 or PEG3350) has caused certain difficulties. The polymer impurities in protein drug products are usually determined by size exclusion chromatography (SEC), which can be separated according to the difference in molecular weight. However, PEG3350, polysorbate 80 and heparin sodium in human coagulation factor VIII are all polymers, and there are overlapping parts in the molecular weight distribution, which makes it difficult to use SEC to separate them according to the difference in molecular size. Because the sample contains a large amount of large molecular plasma proteins, it cannot be directly injected using a reversed phase C8 or C18 column, which increases the difficulty of separation and is easy to clog the chromatographic column. It is usually necessary to use protein precipitation or solid phase extraction and other methods for pretreatment before the sample can be loaded for analysis. The pretreatment is complicated and time-consuming. Because the control limit of polysorbate 80 in human coagulation factor VIII is low and the limit of quantification is required to reach 10 ng / mL, it is necessary to establish a highly sensitive analytical method while avoiding interference from coexisting substances.

[0104] Table 7: Analysis of components of human coagulation factor VIII products

[0105]

[0106] This technical solution provides an analytical method for determining the residual amount of polysorbate 80 in human coagulation factor VIII by combining Shimadzu ShimNex S-C18-SAA surfactant column and Shimadzu's new ELSD-LT III detector with high sensitivity and wide linear range. Using ShimNex S-C18-SAA surfactant column, PEG3350 and polysorbate 80 can be well separated from macromolecular proteins, amino acids, and heparin sodium in human coagulation factor VIII products without complicated sample pretreatment. There is no need to worry about other coexisting substances in the sample interfering with the chromatographic peak of the target substance, nor is there any need to worry about macromolecular proteins clogging the chromatographic column. Shimadzu's new ELSD-LT III detector has the characteristics of high sensitivity and dynamic gain function, which can meet the high-sensitivity test requirements of polysorbate 80 in human coagulation factor VIII. The wide linear range means that high-concentration samples can be accurately measured without dilution, which saves time and effort. The method has high sensitivity, wide linear range and simple pretreatment, and is suitable for the determination of polysorbate 80 residues in human coagulation factor VIII with complex composition.

[0107] In the process of studying the detection method of polysorbate 80, the mobile phase was optimized as follows:

[0108] (1) Concentration of mobile phase A: acetic acid aqueous solution

[0109] This technical solution uses an aqueous acetic acid solution and an isopropanol solution as the mobile phase. The acetic acid content added to the mobile phase is low (0.1%), which can meet the analysis requirements and save reagent costs. In addition, mild conditions are also conducive to extending the service life of the chromatographic column. This solution uses Shimadzu's new ELSD-LT III detector, which has the characteristics of high sensitivity and dynamic gain function. The detection method of this solution has a wider linear range (10-500mg / L) and a higher method sensitivity (10mg / L), which can meet the quantitative analysis requirements of polysorbate 80 in human coagulation factor VIII. See the separation chromatogram of polysorbate 80 and other substances in the sample. Figure 7 Polysorbate is well separated from the main components of PEG3350 and human coagulation factor VIII and their auxiliary materials and can be accurately quantified.

[0110] (2) Effect of initial concentration of mobile phase B on analysis

[0111] The effect of initial concentration of mobile phase B on the analysis of polysorbate 80 was investigated. Figure 8 It can be seen that when the initial concentration of phase B is 35%, polysorbate is divided into multiple peaks, the peaks are broadened, and the sensitivity decreases; when the initial concentration of phase B is 10% compared with 0%, the retention time is slightly advanced, the peak shape is better, and the baseline noise is smaller, so the initial concentration is finally determined to be 10%.

[0112] (3) Impact of gradient variation on analysis

[0113] Referring to the aforementioned detection method of polysorbate 80, the gradient elution procedure was studied, and a comparative experiment was conducted using different elution gradient procedures. When a steep gradient (Phase B: 0%-100%, 0-3.5min; 3.5-6.5min to maintain 100% of Phase B) was used for analysis, the polysorbate 80 standard formed two peaks (including polysorbate 80 peak 1 with a relatively early retention time and polysorbate 80 peak 2 with a relatively late retention time, the latter being used for the quantitative calculation of polysorbate 80). By comparing with the chromatogram of the PEG3350 standard, it was found that the first peak of polysorbate 80 (polysorbate 80 peak 1) could not be separated from PEG3350, and the peak shape of polysorbate 80 peak 2 was not ideal (comparable Fig. 9 The chromatograms of the steep gradient mixed standard and two steep gradient single standards in the above table are shown in Table 1). However, when using a slow gradient (Phase B: 0%-10%, 0-1min; 10%-20%, 1-3.4min; 20%-100%, 3.4-3.5min; the same as the steep gradient after 3.5min; the slow gradient is basically consistent with the gradient elution method shown in Table 1 as a whole), the two chromatographic peaks of polysorbate can be well separated from PEG3350, and the separation degree is greater than 1.5. Therefore, combined with the experimental study on the gradient elution conditions and the study on the effect of the concentration of the initial mobile phase B on polysorbate 80 and PEG3350, this scheme adopts a slow gradient with a starting concentration of 10% for the detection of polysorbate 80; this scheme adopts a slow gradient with a starting concentration of 0% for the detection of PEG3350. In addition, it should be noted that when the injection volume is small or the concentration of polysorbate 80 in the sample is low (for example, the concentration of polysorbate 80 is below 50ppm), due to the problem of setting the ratio of the visualized chromatogram, the polysorbate 80 peak 1 may not be clearly visible on some chromatograms, but its interference with the PEG3350 chromatographic peak does exist. The poor separation between the polysorbate 80 peak 1 and the PEG3350 chromatographic peak will affect the quantitative accuracy of PEG3350.

[0114] (3) Solution to the polysorbate 80 blank interference peak

[0115] Depend on Fig.10 It can be seen that the blank has an interference peak at the retention time of polysorbate 80, which affects the quantification of low-concentration samples. The blank was injected twice in succession, and the repeatability was good. Therefore, the background subtraction function in the software was used, and the standard and sample were analyzed after the background was subtracted, and good sensitivity, linearity, repeatability and spike recovery results were obtained.

[0116] Comparative Example 2: Study on the detection conditions for PEG3350

[0117] The residual level of PEG3350 in human coagulation factor VIII is low, so it is necessary to establish a highly sensitive analytical method while avoiding interference from coexisting substances.

[0118] (1) Comparison of separation effects of different chromatographic columns

[0119] This technical solution uses Shimadzu ShimNex S-C18-SAA surfactant-specific column. Compared with other solutions that use reversed-phase C8 or C18 analytical columns, samples containing large molecular proteins can be directly injected, PEG3350 can be well separated from other coexisting substances, and the difficulty of sample processing is significantly reduced. The separation chromatogram of the target substance PEG 3350 in human coagulation factor VIII and other substances in the sample is detailed in Fig.11 . The ShimNex S-C18-SAA surfactant-specific column has a strong retention effect on PEG3350 and polysorbate 80, and PEG3350 is well separated from the main components of human coagulation factor VIII. In addition, conventional C8 or C18 columns usually do not support direct loading of samples containing high concentrations of plasma proteins, and require protein precipitation or solid phase extraction before loading and analysis, which requires complex pretreatment. Using traditional chromatographic columns, direct loading will cause chromatographic column blockage, and the chromatographic peaks of other components in human coagulation factor VIII are difficult to separate from the chromatographic peak of PEG3350, resulting in low separation and inability to guarantee accurate quantification. Using this technical solution, human coagulation factor VIII containing large molecular plasma proteins is directly sampled and analyzed after filtration through a filter membrane, and pretreatment is simpler.

[0120] The samples were tested using different chromatographic columns, as described in Example 1. The selected chromatographic columns included: the chromatographic column of this scheme (ShimNex S-C18-SAA surfactant-specific column), SEC column (TOSOH TSK-GELG2500PWXL, 7.8 mm × 300 mm, 7 μm) and C18 column (Shim-pack Scepter C18-120, 5 μm 4.6 × 250 mm). The separation effect overlap chromatograms obtained are detailed in Fig.11 , Fig.12 and Fig.13 .from Fig.11 It can be seen that PEG3350 is a single peak on the selected chromatographic column ShimNex S-C18-SAA with good peak shape, and is well separated from the complex coexisting substances in the human coagulation factor VIII sample, and can be accurately quantified. Fig.12 It can be seen that PEG3350 is a double peak on the SEC column. Even if the human coagulation factor VIII sample is treated with common protein precipitants such as sulfosalicylic acid, trichloroacetic acid, perchloric acid and saturated sodium chloride ethanol, the interference cannot be removed and accurate quantification cannot be achieved. Fig.13It can be seen that PEG3350 on the C18 column is also a double peak. We tried to use sulfosalicylic acid protein precipitation for sample treatment, but still could not remove the interference and could not accurately quantify. Process optimization research on SEC column and C18 column did not find a solution to the PEG3350 double peak problem. It can be seen that in order to ensure the accurate quantification of the residual level of PEG3350 in human coagulation factor VIII, the selection of ShimNex S-C18-SAA surfactant column is very necessary. This column has achieved good technical effects in avoiding the generation of PEG3350 double peaks, reducing sample pretreatment procedures and increasing quantitative accuracy.

[0121] (2) Mobile phase

[0122] The detection method of PEG3350 of this scheme has achieved an ideal quantitative detection effect. The specific conditions of the mobile phase used in this scheme are: mobile phase A is 0.1% acetic acid aqueous solution, mobile phase B is 0.1% acetic acid isopropanol solution, the initial concentration is 0% mobile phase B, and the optimized gradient elution program (see Table 1). The existing technology usually uses isocratic elution, or gradient elution with an initial organic phase concentration greater than 10% to detect PEG3350, without considering the separation degree of PEG 3350 and other coexisting substances in human coagulation factor VIII. The mobile phase composition and gradient time program of this scheme can separate PEG3350 and other coexisting substances to achieve accurate quantification. In particular, polysorbate 80 has two chromatographic peaks, one of which is very close to the retention time of the PEG 3350 chromatographic peak. This method can achieve good separation of PEG 3350 and polysorbate 80, with a separation degree greater than 1.5, and can be accurately quantified. The chromatogram of the separation of PEG3350 and polysorbate 80 can be seen in Fig.14 . The polysorbate 80 standard has two chromatographic peaks, at retention times 3.889 min and 5.789 respectively. The chromatographic peak at retention time 3.889 min is very close to PEG3350 (retention time 4.383 min). This method can achieve effective separation of PEG3350 and polysorbate, with a separation degree greater than 1.5, and can be accurately quantified. Specifically, in the figure, the separation degree of PEG3350 (4.383 min) and polysorbate 80 (3.889 min) is 1.910; the separation degree of polysorbate 80 (5.789 min) and PEG 3350 (4.383 min) is 8.150.

[0123] (2.1) Optimization of acetic acid ratio in mobile phase

[0124] Referring to the aforementioned detection method of PEG 3350, the concentration of acetic acid in the mobile phase was studied, and a comparative experiment was conducted using different concentrations of acetic acid. When using ShimNex S-C18-SAA chromatographic column for polymer analysis, the commonly used mobile phase acetic acid ratio is 1% (mobile phase A and B), and the present invention reduces it to 0.1% (mobile phase A and B). It is found that the PEG 3350 peak shape is good, and the peaks 1 and 2 formed with the polysorbate 80 standard are well separated, and the separation degree is greater than 1.5, which can meet the quantitative analysis requirements, such as Fig.15 The proportion of acetic acid used in the present invention is only one tenth of the conventional amount, which can not only meet the analysis requirements and save reagent costs, but also the mild analysis conditions are conducive to extending the service life of the chromatographic column and the instrument.

[0125] (2.2) Optimization of initial mobile phase concentration

[0126] Referring to the aforementioned detection method of PEG 3350, the initial concentration of mobile phase B was studied, and a comparative experiment was conducted using different initial concentrations of mobile phase B. The present invention investigated the effect of the initial concentration of PEG 3350 mobile phase on the analysis, and found that when the initial concentration of phase B was 10%, the PEG 3350 chromatographic peak was broadened and bifurcated, and the chromatographic peak lasted for about 2 minutes from the starting point to the end point, with low sensitivity. When the initial concentration of phase B was adjusted to 0%, the PEG 3350 peak was sharp and symmetrical, with high sensitivity (see Fig.16 ). It can be seen that the detection of PEG 3350 is different from that of polysorbate 80. The concentration of the initial mobile phase B needs to be adjusted to 0%. The peak shape of the obtained chromatographic peak is significantly optimized, and an unexpected technical effect is obtained.

[0127] This protocol uses Shimadzu's new ELSD-LT III detector, which has high sensitivity and dynamic gain function. This makes the linear range of this detection method wider (50-2000mg / L), and high-concentration samples can be accurately measured without dilution, saving time and effort. The method has higher sensitivity (as low as 50mg / L), meeting the needs of quantitative analysis of PEG3350 in human coagulation factor VIII.

[0128] In summary, this method has high sensitivity, wide linear range and simple pretreatment, and is suitable for the determination of residual PEG3350 in complex human coagulation factor VIII.

[0129] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A HPLC-ELSD method for determining polymer residues in human coagulation factor VIII, characterized in that: The polymer residues include polysorbate 80 and polyethylene glycol 3350; the HPLC-ELSD determination method includes detection for polysorbate 80 and detection for polyethylene glycol 3350.

2. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 1, characterized in that: The parameters used in the HPLC-ELSD determination are: Chromatographic column: Shimadzu surfactant special analytical column ShimNex S-C18-SAA (150mm×4.6mm, 5μm); Mobile phase: Mobile phase A is 0.1% acetic acid in water, and mobile phase B is 0.1% acetic acid in isopropanol; Injection volume: 10 μL; Flow rate: 1.0 mL / min; Column temperature: 30°C; Dynamic gain: wide; Drift tube temperature: 80°C; Atomizing gas pressure: 350KPa; Elution mode: gradient elution.

3. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 2, characterized in that: In the detection of polysorbate 80, the elution procedure of the mobile phase is shown in the following table: The initial concentration of mobile phase B was 10%.

4. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 2, characterized in that: In the detection of polyethylene glycol 3350, the elution program of the mobile phase is shown in the following table: The initial concentration of mobile phase B was 0%.

5. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to any one of claims 1 to 4, characterized in that: In the detection of polysorbate 80, the sample to be tested is taken and redissolved, and then centrifuged to obtain the supernatant; the supernatant is subjected to HPLC-ELSD detection.

6. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 5, characterized in that: In the test for polysorbate 80, the centrifugation parameter was 14000 rpm for 10 min.

7. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 6, characterized in that: In the detection of polyethylene glycol 3350, the sample to be tested is taken and redissolved, and then the sample is filtered and processed, and the filtrate is taken for HPLC-ELSD detection.

8. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 7, characterized in that: In the detection of polyethylene glycol 3350, the filtration process uses an acetate fiber filter membrane with a pore size of 0.45um.

9. The HPLC-ELSD determination method of residues in human coagulation factor VIII according to claim 8, characterized in that: In the detection of polysorbate 80, the linear range is 10-500 mg / L; in the detection of polyethylene glycol 3350, the linear range is 50-2000 mg / L.

10. The HPLC-ELSD determination method of polymer residues in human coagulation factor VIII according to claim 9, characterized in that: The product of human coagulation factor VIII contains plasma proteins and excipients; the plasma proteins include human coagulation factor VIII, VWF factor, fibronectin, fibrinogen, IgA protein, IgM protein, IgG protein, and fibrinogen; the excipients include polyethylene glycol 3350, polysorbate 80, tributyl phosphate, glycine, histidine, heparin, and citric acid.

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