Content detection method of recombinant human erythropoietin

Abstract

The present invention relates to a method for detecting the content of recombinant human erythropoietin, comprising the following steps: mixing a physical and chemical reference substance of recombinant human erythropoietin with a solvent to prepare reference substances with different concentrations; taking a sample of recombinant human erythropoietin to prepare a test sample; performing high performance liquid chromatography on the reference substances with different concentrations to establish the corresponding relationship between the concentration and the peak area; performing high performance liquid chromatography on the test sample, substituting the obtained peak area into the corresponding relationship between the concentration and the peak area, and calculating the content of recombinant human erythropoietin in the test sample. This detection method has high detection accuracy and is simple to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a method for detecting the content of recombinant human erythropoietin. Background Art

[0002] Erythropoietin (abbreviated as EPO), first discovered in 1906, is a glycoprotein mainly synthesized and secreted by cells near the proximal convoluted tubules of the kidney. It belongs to the sialylglycoprotein hormone, and the protein part consists of 166 amino acids with a molecular weight of 34KD. Erythropoietin can be divided into two types, α and β, according to the difference in its glycan structure. The α type contains 34% carbohydrates, and the β type contains 26% carbohydrates. The two types are identical in biological characteristics, antigenicity, and other effects.

[0003] In 1985, people expressed recombinant human erythropoietin (rhEPO) using genetic recombination technology. The gene of EPO was constructed into Chinese hamster ovary cells (CHO cells) using DNA recombination technology to form CHO cells that can express the rhEPO gene. rhEPO has the same physiological activity as endogenous EPO in the human body. In June 1989, the US FDA officially approved the listing of recombinant human erythropoietin developed by Amgen. rhEPO is mainly used clinically to treat anemia such as anemia associated with chronic renal failure, anemia in chronic renal failure (CRF), anemia in myelodysplastic syndrome (MDS), anemia in AIDS patients. Currently, there are also reports on its application in treating anemia caused by chemotherapy in cancer patients, correcting anemia related to multiple myeloma (MM), anemia in premature infants, and anemia during pregnancy and after childbirth in pregnant women, chronic anemia in patients with rheumatoid arthritis, lupus erythematosus, and severe parasitic diseases, sickle cell anemia and thalassemia in hemoglobinopathy, and anemia associated with inflammatory bowel disease. At the same time, rhEPO can also be used to enhance exercise endurance, reduce altitude sickness, and in patients with angina pectoris who require blood transfusion. In short, rhEPO is one of the earliest recombinant protein drugs globally and is also a genetically engineered drug with mature technology and definite efficacy. However, in traditional technologies, the efficiency of CHO cells expressing rhEPO is not high, resulting in the overall problems of low production rate and high cost of rhEPO, which affects its promotion in clinical practice.

[0004] In order to improve the production rate of rhEPO, it is necessary to optimize the CHO cell culture conditions and downstream purification processes. By quantitatively detecting the content of rhEPO in cell supernatants and intermediates, the advantages and disadvantages of various preparation process parameters can be quickly and accurately evaluated, and the purpose of improving the production rate of rhEPO can be achieved.

[0005] At present, the methods for detecting the content of rhEPO are mainly divided into two categories: one is the biological method, including in vivo determination and in vitro determination. Since the biological method detects the biological activity of cytokines, it has high affinity, but has the disadvantages of poor specificity, cumbersome operation, and susceptibility to interference. The other is the immunological method, which is a detection method developed along with immunolabeling technology and is currently the most widely used method for detecting rhEPO. The current immunological methods can be further divided into: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), chemiluminescence immunoassay (CLTA), etc.

[0006] Initially, the detection of rhEPO content basically used the RIA method. This method has mature technology and high sensitivity. However, its disadvantages are manual operation, slow result output, short reagent half-life, environmental pollution, etc., which limit the application of this method. ELISA is a sensitive technology that can quantify the measured substance at the microgram or even nanogram level and has the advantages of simplicity, low cost, and easy standardization. The disadvantages are large sample consumption, about 200 μL of sample is required for each detection, long detection time, and large human error. FIA is the most sensitive in non-radioimmunoassays. It is often used to detect bioactive substances with very low content and has high specificity and sensitivity. The disadvantage is that the reagent kit is expensive and not suitable for use in the process exploration stage. CLTA is an analytical technique that uses chemiluminescence signals to trace and detect antigens or antibodies. Its sensitivity is generally higher than that of ELISA and has the advantages of strong specificity, stable label, wide linear range, and fast detection speed. Currently, CLIA is developing rapidly and has the trend of replacing RIA as the mainstream. However, there are also deficiencies such as the high price of the detection system, which is difficult to popularize, or the low price but the sensitivity and reliability of detection cannot be guaranteed.

[0007] There is a method involving an erythropoietin detection method based on magnetic beads as a carrier. The EPO in serum is captured by coupling a capture antibody (anti-EPO antibody) to the magnetic beads. Subsequently, a biotinylated detection antibody is added to form an immune complex of "capture antibody, EPO, detection antibody" with the magnetic beads as the carrier. Then, streptavidin-conjugated enzyme is added to bind to the biotin labeled on the detection antibody to label the immune complex. This immunoassay method has extremely high sensitivity (19 pg / mL), which is 13 times higher than that of traditional immunoassay methods. The sample volume (15 μL) is 13 times less than that of traditional immunoassay methods, and the sample detection experimental period is shortened to 34 min, which is much shorter than that of traditional immunoassay methods (6 h - 8 h). However, although this method has high sensitivity, low sample volume, and short detection period, the preparation of the "reagent" with magnetic beads as the carrier is complex. There are problems such as the need to prepare anti-rhEPO antibodies, difficulty in forming commercial products, and possible quality differences between batches of "magnetic bead reagents". Moreover, the detection process is too cumbersome, there is a risk of anti-rhEPO antibody shedding, which in turn affects the accuracy of rhEPO content detection. Summary of the Invention

[0008] Based on this, the present invention provides a method for detecting the content of recombinant human erythropoietin with high detection accuracy and simple operation.

[0009] The specific technical solution is as follows:

[0010] A method for detecting the content of recombinant human erythropoietin, comprising the following steps:

[0011] Mix the recombinant human erythropoietin (rhEPO) physical and chemical reference substance with a solvent to prepare reference substances with different concentrations;

[0012] Take a sample of recombinant human erythropoietin to prepare a sample to be tested;

[0013] Detect the reference substances with different concentrations by reversed-phase high performance liquid chromatography to establish the corresponding relationship between concentration and peak area;

[0014] Detect the sample to be tested by reversed-phase high performance liquid chromatography, substitute the obtained peak area into the corresponding relationship between concentration and peak area, and calculate the content of recombinant human erythropoietin in the sample to be tested;

[0015] Wherein, the mobile phase used in the reversed-phase high performance liquid chromatography includes mobile phase A and mobile phase B;

[0016] By volume percentage, mobile phase A includes: 65% - 75% water (H2O) and 35% - 25% acetonitrile (ACN); and 0.05% - 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile;

[0017] The mobile phase B comprises, by volume percentage: 35% to 45% water (H2O) and 65% to 55% acetonitrile (ACN); and 0.05% to 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile.

[0018] In one embodiment, the elution program used in the reversed-phase high performance liquid chromatography includes:

[0019] From 0 to 1 minute, the volume fraction of the mobile phase A is maintained at 100% and the volume fraction of the mobile phase B is 0%;

[0020] From 1 minute to 25 minutes, the volume fraction of the mobile phase A changes from 100% to 0% and the volume fraction of the mobile phase B changes from 0% to 100%;

[0021] From 25 minutes to 38 minutes, the volume fraction of the mobile phase A is maintained at 0% and the volume fraction of the mobile phase B is 100%.

[0022] In one embodiment, the elution program used in the reversed-phase high performance liquid chromatography further includes:

[0023] From 38 minutes to 43 minutes, the volume fraction of the mobile phase A changes from 0% to 100% and the volume fraction of the mobile phase B changes from 100% to 0%;

[0024] From 43 minutes to 55 minutes, the volume fraction of the mobile phase A is maintained at 100% and the volume fraction of the mobile phase B is 0%.

[0025] In one embodiment, the chromatographic column used in the reversed-phase high performance liquid chromatography is packed with one of C4 - C18 alkylsilyl-bonded silica gels.

[0026] In one embodiment, the chromatographic column used in the reversed-phase high performance liquid chromatography is packed with C4, C6, C8 or C18 alkylsilyl-bonded silica gel.

[0027] In one embodiment, the conditions of the reversed-phase high performance liquid chromatography further include: the flow rate is 0.9 - 1.1 mL / min, the temperature is 25°C - 35°C, and the wavelength of the ultraviolet detector is 209 nm - 211 nm.

[0028] In one embodiment, the conditions of the reversed-phase high performance liquid chromatography further include: the flow rate is 1 mL / min, the temperature is 28°C - 32°C, and the wavelength of the ultraviolet detector is 210 nm.

[0029] In one embodiment, the integration conditions for the detection results are: the peak width is 50, the threshold is 200, and the integration interval is 7 minutes - 30 minutes.

[0030] In one embodiment, the retention time of the peak corresponding to recombinant human erythropoietin is 19.3 ± 0.2 min.

[0031] In one embodiment, the corresponding relationship between the concentration and the peak area is y = 74056.85x - 108767.04, where x represents the concentration and y represents the peak area of recombinant human erythropoietin at the said concentration.

[0032] The above method for detecting the content of recombinant human erythropoietin can accurately determine the recombinant human erythropoietin in the sample to be tested by using reversed-phase high performance liquid chromatography and reasonably controlling the detection conditions of high performance liquid chromatography. The operation is simple and does not require complex operations such as antibody conjugation in traditional methods. Compared with traditional biological detection methods and immunological detection methods, it has the advantages of less sample requirement, low detection cost, less time consumption, high accuracy, and can also detect samples with complex components such as cell culture supernatant, which has very high guiding significance for the process optimization and production of recombinant human erythropoietin.

[0033] At the same time, the above method for detecting the content of recombinant human erythropoietin has been comprehensively verified, including verification items such as system suitability, specificity, linearity, accuracy, precision and intermediate precision, quantitation limit and detection limit, durability, etc. It has high detection accuracy, the spiked recovery rate is between 95.05% and 100.66%, and the detection precision is high. This also makes the method have good repeatability, the quantitation limit is as low as 30 μg / mL rhEPO, and makes the determination results of the content of recombinant human erythropoietin in cell culture and downstream purification processes more accurate and reliable. Description of the Drawings

[0034] Figure 1 It is the typical HPLC chromatogram of the blank solution for linearity verification;

[0035] Figure 2 It is the typical HPLC chromatogram of the test solution at 20 μg / mL for linearity verification;

[0036] Figure 3 It is the typical HPLC chromatogram of the test solution at 40 μg / mL for linearity verification;

[0037] Figure 4 It is the typical HPLC chromatogram of the test solution at 80 μg / mL for linearity verification;

[0038] Figure 5 It is the typical HPLC chromatogram of the test solution at 160 μg / mL for linearity verification;

[0039] Figure 6 It is the typical HPLC chromatogram of the test solution at 320 μg / mL for linearity verification;

[0040] Figure 7 It is the HPLC typical chromatogram of the test solution at 640 μg / mL for linearity verification;

[0041] Figure 8 It is the HPLC typical chromatogram of the blank solution for system suitability verification;

[0042] Figure 9 It is the HPLC typical chromatogram of the system suitability solution for system suitability verification;

[0043] Figure 10 It is the HPLC chromatogram of the blank solution for specificity verification;

[0044] Figure 11 It is the HPLC chromatogram of the reference substance for specificity verification;

[0045] Figure 12 It is the HPLC chromatogram of the test solution for specificity verification;

[0046] Figure 13 It is the HPLC typical chromatogram of the blank solution for precision and intermediate precision verification;

[0047] Figure 14 It is the HPLC typical chromatogram of the test solution for precision and intermediate precision verification;

[0048] Figure 15 It is the HPLC typical chromatogram of the blank solution for accuracy verification;

[0049] Figure 16 It is the HPLC typical chromatogram of the cell supernatant for accuracy verification;

[0050] Figure 17 It is the HPLC typical chromatogram of the spiked sample at 30 μg / mL for accuracy verification;

[0051] Figure 18 It is the HPLC typical chromatogram of the spiked sample at 330 μg / mL for accuracy verification;

[0052] Figure 19 It is the HPLC typical chromatogram of the spiked sample at 630 μg / mL for accuracy verification;

[0053] Figure 20 It is the HPLC typical chromatogram of the blank solution for limit of quantitation and limit of detection verification;

[0054] Figure 21 It is the HPLC typical chromatogram at the limit of detection of 20 μg / mL;

[0055] Figure 22 It is the HPLC typical chromatogram at the limit of quantitation of 30 μg / mL;

[0056] Figure 23 HPLC chromatogram of the reference substance at 200 μg / mL (28 °C);

[0057] Figure 24 HPLC chromatogram of the reference substance at 200 μg / mL (30 °C);

[0058] Figure 25 HPLC chromatogram of the reference substance at 200 μg / mL (32 °C);

[0059] Figure 26 HPLC chromatogram of the reference substance at 200 μg / mL (208 nm);

[0060] Figure 27 HPLC chromatogram of the reference substance at 200 μg / mL (210 nm);

[0061] Figure 28 HPLC chromatogram of the reference substance at 200 μg / mL (212 nm);

[0062] Figure 29 HPLC chromatogram of the reference substance at 200 μg / mL (0.8 ml / min);

[0063] Figure 30 HPLC chromatogram of the reference substance at 200 μg / mL (1.0 mL / min);

[0064] Figure 31 HPLC chromatogram of the reference substance at 200 μg / mL (1.2 mL / min). Detailed implementation mode

[0065] The following further elaborates in detail on the content detection method of recombinant human erythropoietin of the present invention in combination with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0067] In the present invention, among the technically characterized described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0068] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0069] In the present invention, the percentage content involved, unless otherwise specified, refers to the mass percentage for solid-liquid mixtures and solid-solid mixtures, and refers to the volume percentage for liquid-liquid mixtures. Unless otherwise specified, the solvent of the solution sample is water.

[0070] In the present invention, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0071] In the present invention, the temperature parameter, unless otherwise specifically limited, allows both isothermal treatment and treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0072] The present invention provides a method for detecting the content of recombinant human erythropoietin, comprising the following steps:

[0073] Mix the physical and chemical reference substance of recombinant human erythropoietin (rhEPO) with a solvent to prepare reference substances with different concentrations;

[0074] Take a sample of recombinant human erythropoietin and prepare a sample to be tested;

[0075] Detect the reference substances with different concentrations by reversed-phase high performance liquid chromatography to establish the corresponding relationship between the concentration and the peak area;

[0076] Detect the sample to be tested by reversed-phase high performance liquid chromatography, substitute the obtained peak area into the corresponding relationship between the concentration and the peak area, and calculate the content of recombinant human erythropoietin in the sample to be tested;

[0077] Wherein, the mobile phase used in the reversed-phase high performance liquid chromatography includes mobile phase A and mobile phase B;

[0078] By volume percentage, mobile phase A includes: 65% - 75% water (H2O) and 35% - 25% acetonitrile (ACN); and 0.05% - 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile;

[0079] By volume percentage, mobile phase B includes: 35% to 45% water (H2O) and 65% to 55% acetonitrile (ACN); and 0.05% to 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile.

[0080] Understandably, the sample to be tested can be CHO cell culture supernatant and intermediates in the downstream purification process.

[0081] In some specific examples, the solvent is Tris-HCl buffer solution.

[0082] In some specific examples, by volume percentage, mobile phase A includes: 68% to 72% water (H2O) and 32% to 28% acetonitrile (ACN); and 0.05% to 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile. Specifically, by volume percentage, mobile phase A includes: 70% water (H2O) and 30% acetonitrile (ACN); and 0.1% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile.

[0083] In some specific examples, by volume percentage, mobile phase B includes: 38% to 42% water (H2O) and 62% to 58% acetonitrile (ACN); and 0.05% to 0.15% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile. Specifically, by volume percentage, mobile phase B includes: 40% water (H2O) and 60% acetonitrile (ACN); and 0.1% trifluoroacetic acid (TFA) based on the total volume of water and acetonitrile.

[0084] In some specific examples, the elution program used in the reversed-phase high-performance liquid chromatography includes:

[0085] From 0 to 1 minute, keep the volume fraction of mobile phase A at 100% and the volume fraction of mobile phase B at 0%;

[0086] From 1 minute to 25 minutes, the volume fraction of mobile phase A changes from 100% to 0%, and the volume fraction of mobile phase B changes from 0% to 100%;

[0087] From 25 minutes to 38 minutes, keep the volume fraction of mobile phase A at 0% and the volume fraction of mobile phase B at 100%.

[0088] Furthermore, the elution program used in the reversed-phase high-performance liquid chromatography further includes:

[0089] From 38 minutes to 43 minutes, the volume fraction of mobile phase A changes from 0% to 100%, and the volume fraction of mobile phase B changes from 100% to 0%.

[0090] From 43 min to 55 min, maintain the volume fraction of mobile phase A at 100% and the volume fraction of mobile phase B at 0%.

[0091] In some specific examples, the integration conditions for the detection results are as follows: peak width 50, threshold 200, and integration interval from 7 min to 30 min.

[0092] In some specific examples, the peak area of recombinant human erythropoietin is calculated by the area normalization method.

[0093] In some specific examples, the chromatographic column used in the reversed-phase high-performance liquid chromatography is packed with one of C4 - C18 alkylsilyl-bonded silica gels. Further, the chromatographic column used in the reversed-phase high-performance liquid chromatography is packed with C4, C6, C8 or C18 alkylsilyl-bonded silica gel. Specifically, the chromatographic column used in the reversed-phase high-performance liquid chromatography is phenomenex C18 column.

[0094] In some specific examples, the parameters of the chromatographic column used in the reversed-phase high-performance liquid chromatography include: 4.6 mm × (150 - 250) mm, pore size 12 - 30 nm, particle size 3.5 - 10 μm.

[0095] In some specific examples, the conditions of the reversed-phase high-performance liquid chromatography further include: flow rate 0.9 - 1.1 mL / min, temperature 25°C - 35°C, and wavelength of the ultraviolet detector 209 nm - 211 nm. Further, the conditions of the reversed-phase high-performance liquid chromatography further include: flow rate 1 mL / min, temperature 28°C - 32°C, and wavelength of the ultraviolet detector 210 nm.

[0096] In some specific examples, the retention time of the peak corresponding to recombinant human erythropoietin is 19.3 ± 0.2 min.

[0097] In some specific examples, the correspondence between the concentration and the peak area refers to the standard curve. Specifically, the standard curve is y = 74056.85x - 108767.04, where x represents the concentration and y represents the peak area of recombinant human erythropoietin at the said concentration.

[0098] The following are specific examples.

[0099] Example 1

[0100] This example is a method for detecting the content of recombinant human erythropoietin, and the steps are as follows:

[0101] (1) Chromatographic column: phenomenex C18 column, using octadecylsilyl silica gel as the packing material, 4.6 mm × 250 mm, CV = 4.15 ml, pore size 30 nm, particle size 5 μm, column efficiency ≥ 25411.

[0102] (2) Mobile phase:

[0103] Mobile phase A (volume percentage): 70% H2O + 30% ACN, 0.1% TFA;

[0104] Mobile phase B (volume percentage): 40% H2O + 60% ACN, 0.1% TFA.

[0105] (3) Blank solution: 10 mM Tris-HCl buffer, pH 7.00.

[0106] (4) Physicochemical reference solution of rhEPO: Purchased from Sinovac Biotech Co., Ltd., calibrated by the Lowry method, protein concentration 3.77 g / L, electrophoresis purity ≥ 99%.

[0107] (5) Sample to be tested: Culture supernatant containing rhEPO obtained from CHO cell culture, filtered through a 0.45 μm membrane.

[0108] (6) High performance liquid chromatography:

[0109] The sample was detected using a high performance liquid chromatography system (Agilent 110 Series), flow rate 1 mL / min, temperature 30 °C, ultraviolet detector wavelength 210 nm, injection volume 100 μL. The mobile phase elution gradient is shown in Table 1 below:

[0110] Table 1 Detection method solution gradient table

[0111] Time (min) A(％) B(％) 0 100 0 1 100 0 25 0 100 38 0 100 43 100 0 55 100 0

[0112] The integration conditions for the liquid chromatogram are: peak width 50, threshold 200, integration interval 7 min - 30 min. The peak area of rhEPO was calculated by the area normalization method, and the retention time of the target protein peak was 19.3 ± 0.2 min.

[0113] (7) Standard curve construction: Take the physicochemical reference solution of rhEPO and dilute it with 10 mM Tris-HCl buffer into six different concentrations of test solutions: 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, and 640 μg / mL. Inject the blank solution and the test solutions in sequence twice for detection. Using the concentration as the abscissa and the average peak area after subtracting the noise as the ordinate, establish a standard curve, with the requirement that the linear equation R 2 ≥ 0.995. The verification results are shown in Table 2 and Figures 1 - 7As shown below:

[0114] Table 2 Linear Verification Results

[0115]

[0116] Based on the above table data, a standard curve was established with the equation y = 74056.85x - 108767.04, and R 2 = 0.99971. It can be seen from Table 2 that with the concentration as the abscissa and the average peak area after noise deduction as the ordinate, a linear relationship between concentration and peak area was obtained, with an R 2 = 0.99971, which is greater than 0.995, indicating that there is a certain linear relationship between concentration and peak area, and the linearity is good, and the linear verification is qualified.

[0117] (8) Test Results: The concentration of rhEPO cell culture supernatant is 43.68 μg / mL.

[0118] Example 2

[0119] This example conducts a methodological verification of the content detection method of the recombinant human erythropoietin in Example 1.

[0120] (1) System Suitability Verification

[0121] System suitability verification is to check whether the high-performance liquid chromatography system and the chromatographic column meet the detection requirements. By checking the retention time (Rt) of the target protein peak and the relative standard deviation (RSD) of the peak area, it is determined whether the detection results are accurate and reliable and whether they are applicable to the detection of rhEPO content.

[0122] Take the rhEPO physical and chemical reference solution and dilute it to 200 μg / mL with 10 mM Tris-HCl buffer as the system suitability solution. Inject the blank solution twice and the system suitability solution continuously four times, and record the chromatogram. It is required that the blank solution has no interference, and for the system suitability solution injected continuously four times, the RSD of the main peak retention time ≤ 1.0%, and the RSD of the main peak area ≤ 2.0%. The verification results are shown in Table 3 below and Figures 8 - 9 as follows:

[0123] Table 3 System Suitability Verification Results

[0124]

[0125]

[0126] It can be seen from Table 3 and Figures 8 - 9It can be seen that there is no ultraviolet absorption in the retention time of the main peak after the blank solution is injected, indicating that the blank solution has no interference. The RSD of the retention time of the main peak and the RSD of the main peak area of the system suitability solution are both within the specified ranges. Therefore, the system suitability verification is qualified, and the high-performance liquid chromatography system and chromatographic column used meet the detection requirements and are applicable to the detection of the rhEPO content.

[0127] (2) Specificity verification

[0128] Specificity verification refers to the ability of the analytical method used to correctly determine the analyte in the presence of other components (such as impurities, degradation products, excipients, etc.). For rhEPO cell supernatant, the components are relatively complex. Tris-HCl buffer is used in the rhEPO purification process. Therefore, 10 mM Tris-HCl buffer is used to dilute the reference substance, and 10 mM Tris-HCl buffer is used as the blank to verify whether it has interference on the determination of the rhEPO content.

[0129] Take the rhEPO physical and chemical reference substance solution, dilute it to 40 μg / mL with 10 mM Tris-HCl buffer as the reference substance solution. Take the rhEPO cell supernatant as the test sample solution. Inject the blank solution, reference substance solution, and test sample solution once each, and record the chromatogram. It is required that the blank solution has no interference, and the deviation of the retention time of the main peak of the test sample solution from the retention time of the main peak of the reference substance solution should be <1%. The verification results are shown in Table 4 and Figures 10 - 12 as follows:

[0130] Table 4 Specificity verification results

[0131]

[0132] It can be seen from Table 4 and Figures 10 - 12 that: the blank solution has no interference, and the deviation of the retention time of the main peak of the test sample from the retention time of the main peak of the reference substance solution is 0.7%, and the retention times are basically the same. Therefore, in the presence of other components (such as impurities, degradation products, excipients, etc.) and the blank solution, this detection method also has the ability to correctly determine the analyte, and the specificity verification is qualified.

[0133] (3) Precision and intermediate precision verification

[0134] Precision and intermediate precision verification are used for the performance inspection of the instrument, and also to confirm the analysis ability of the analyst to ensure the accuracy, reliability, and good repeatability of the test results.

[0135] The first person takes the rhEPO physical and chemical reference substance solution, dilutes it to 200 μg / mL with 10 mM Tris-HCl buffer solution as the test sample solution, and prepares three portions in parallel. Inject the blank solution once and each portion of the test sample solution once, and record the chromatogram. It is required that the blank solution has no interference. In the three portions of the test sample solution of the first person, the RSD of the main peak retention time ≤ 1.0%, and the RSD of the main peak area ≤ 5.0%.

[0136] The second person takes the rhEPO physical and chemical reference substance solution, dilutes it to 200 μg / mL with 10 mM Tris-HCl buffer solution as the test sample solution, and prepares three portions in parallel. Replace with another piece of equipment, inject the blank solution once and each portion of the test sample solution once, and record the chromatogram. It is required that the blank solution has no interference. In the three portions of the test sample solution of the second person, the RSD of the main peak retention time ≤ 1.0%, and the RSD of the main peak area ≤ 5.0%; in the six portions of the test sample solution of the two persons, the RSD of the main peak retention time ≤ 1.0%, and the RSD of the main peak area ≤ 5.0%. The verification results are shown in Table 5 and Figures 13 - 14 as follows:

[0137] Table 5 Verification Results of Precision and Intermediate Precision

[0138]

[0139]

[0140] From Table 5 and Figures 13 - 14 it can be seen that: in the three portions of the test sample solution of the first person, the blank solution has no interference, the RSD of the main peak retention time = 0.032%, and the RSD of the main peak area = 1.59%. In the three portions of the test sample solution of the second person, the blank solution has no interference, the RSD of the main peak retention time = 0.083%, and the RSD of the main peak area = 0.66%. In the six samples of the two persons, the RSD of the main peak retention time = 0.11%, and the RSD of the main peak area = 1.82%. For the three samples of the first person, the three samples of the second person, and the six samples of the two persons, the RSD of the main peak retention time and the peak area are all less than the specified qualified range. Therefore, there are no problems with the performance of the instrument and the analysis ability of the analysts, and the verification of precision and intermediate precision is qualified.

[0141] (5) Accuracy Verification

[0142] Accuracy verification refers to the degree of closeness between the measured value obtained by this detection method and the true value, which is expressed by the recovery rate of the target protein.

[0143] Take the rhEPO physical and chemical reference substance solution, and dilute it with 10 mM Tris-HCl buffer into reference substance dilutions at three concentrations of 16 μg / mL, 616 μg / mL, and 1216 μg / mL. Take one sample solution (rhEPO cell supernatant) and mix it with the reference substance dilutions at a volume ratio of 1:1 to form three spiked samples with different concentrations. Inject the above three spiked samples three times each and the sample solution twice, and calculate the concentration and spiked recovery rate. It is required that the blank solution has no interference, the average spiked recovery rate of the main peak at each concentration is between 90% and 110%, the RSD of the spiked recovery rate of each group of samples is ≤ 5%, and the RSD of the spiked recovery rate of the 9 samples is ≤ 5%. The verification results are shown in Table 6 and Figures 15 - 19 as follows:

[0144] Table 6 Accuracy Verification Results

[0145]

[0146] From Table 6 and Figures 15 - 19 it can be seen that: the average spiked recovery rate at each concentration is 95.05% - 100.66%, the RSD of the spiked recovery rate of each group of samples is 0.10% - 2.24%, the RSD of the spiked recovery rate of the 9 samples is 2.79%. The obtained spiked recovery rate and RSD are both within the specified qualified range. The degree of approximation between the measured value and the true value obtained by this detection method is high, and the relative standard deviation is small. Therefore, the accuracy verification is qualified.

[0147] (6) Verification of Quantitation Limit and Detection Limit

[0148] The quantitation limit refers to the lowest amount of the analyte in the sample that can be quantitatively determined with appropriate accuracy and precision. The detection limit refers to the lowest amount of the analyte in the sample that can be detected, but it does not necessarily need to be accurately quantified.

[0149] Take the rhEPO physical and chemical reference substance solution, and dilute it with the 10 mM Tris-HCl buffer into 20 μg / mL and 30 μg / mL, which are used as the detection limit sample and the quantitation limit sample respectively. Inject the blank solution twice, the detection limit sample twice, and the quantitation limit sample three times. It is required that the detection limit sample can detect the target protein, and the ratio of the average peak area to the average noise peak area (signal-to-noise ratio) ≥ 3, the ratio of the average peak area of the quantitation limit sample to the average noise peak area ≥ 5, the RSD of the peak area of the quantitation limit sample ≤ 10%, and the RSD of the retention time of the main peak ≤ 2%. The verification results are shown in Table 7 and Figures 20 - 22 as follows:

[0150] Table 7 Verification Results of Quantitation Limit and Detection Limit

[0151]

[0152] From Table 7 andFigures 20 - 21 It can be seen that when the sample concentration is 20 μg / mL, since the blank solution has no ultraviolet absorption within the retention time of the main peak, the signal-to-noise ratio is 41, which is much greater than 3. From Figure 21 it can be seen that the response value of the main peak is already relatively low at this time. If the concentration is further decreased, the main peak can still be detected, but the peak shape will be poor. Moreover, the detection limit of 20 μg / mL can meet the general process requirements of the industry. Therefore, the detection limit of this rhEPO content detection method is set at 20 μg / mL.

[0153] When the sample concentration is 30 μg / mL, the signal-to-noise ratio is 73, which is much greater than 5. The main peak peak area RSD = 0.44% and the main peak retention time RSD = 0.06% after injecting samples continuously three times, both of which are less than the specified qualified range. Coupled with the fact that good linearity and accuracy have been demonstrated for 30 μg / mL in the previous linearity and accuracy verifications, setting the quantification limit of this rhEPO content detection method at 30 μg / mL can meet the general process requirements of the industry.

[0154] (7) Durability verification

[0155] 7.1 Comparison of detection results at different column temperatures

[0156] Take the rhEPO physical and chemical reference substance solution, dilute it to 200 μg / mL with 10 mM Tris-HCl buffer solution, keep other conditions unchanged, and detect it at 28 °C, 30 °C, and 32 °C respectively. Observe the detection results as shown in Table 8 below and Figures 23 - 25 as follows:

[0157] Table 8 Influence of different column temperatures on the detection of rhEPO content

[0158]

[0159] From Table 8 and Figures 23 - 25 it can be seen that when other conditions remain unchanged and the column temperature fluctuates within the range of 30 °C ± 2 °C, this detection method has a certain stability and will not affect the detection of rhEPO content.

[0160] 7.2 Comparison of detection results at different wavelengths

[0161] Take the rhEPO physical and chemical reference substance solution, dilute it to 200 μg / mL with 10 mM Tris-HCl buffer solution, keep other conditions unchanged, and detect it at 208 nm, 210 nm, and 210 nm respectively. Observe the detection results as shown in Table 9 below and Figures 26 - 28 as follows:

[0162] Table 9 Influence of different wavelengths on the detection of rhEPO content

[0163]

[0164] As can be seen from Table 9: with other conditions remaining unchanged, when the detection wavelength is 208 nm, the measured rhEPO content deviates by 16.74% compared to the normal conditions. The reason for the deviation may be that the organic solvent used in this detection method is acetonitrile, and the ultraviolet absorption cut-off wavelength of acetonitrile is 190 - 210 nm. When the wavelength < 210 nm, some acetonitrile may be absorbed, resulting in a higher measured value of the target protein concentration. When the detection wavelength is 212 nm, the measured rhEPO content deviates by -12.54% compared to the normal conditions. The reason for the deviation may be that some of the target protein may not be absorbed by ultraviolet light, leading to a lower measured value of the target protein concentration.

[0165] In summary, this rhEPO content detection method is sensitive to the detection wavelength, and wavelength changes will have a greater impact on the detection results. Therefore, during the detection process, it should be carried out according to a detection wavelength of 210 nm.

[0166] 7.3 Comparison of Detection Results at Different Flow Rates

[0167] Take the rhEPO physical and chemical reference solution, dilute it to 200 μg / mL with 10 mM Tris-HCl buffer solution, keep other conditions unchanged, and detect it under the conditions of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively, and observe the detection results as shown in Table 10 and Figures 29 - 31 as follows:

[0168] Table 10 Influence of Different Flow Rates on the Detection of rhEPO Content

[0169]

[0170] As can be seen from Table 10: with other conditions remaining unchanged, when the detection flow rate is 0.8 mL / min, the measured rhEPO content deviates by 25.20% compared to the normal conditions. The reason for the deviation may be that after the flow rate decreases, the mass transfer rate of the mobile phase becomes relatively slower, the retention time of the main peak lags, and the peak shape is wider (large volume, low concentration). In addition, the detector of the HPLC used in this verification is a concentration-type detector, and the peak area is inversely proportional to the flow rate of the mobile phase. Therefore, the peak area is higher compared to the detection value under normal conditions, resulting in a higher measured concentration. When the detection flow rate is 1.2 mL / min, the mass transfer rate of the mobile phase becomes relatively faster, the retention time of the main peak advances, and the peak shape is sharp and narrow (small volume, high concentration), which may lead to a smaller integrated peak area and a lower measured concentration compared to the normal conditions.

[0171] In summary, the rhEPO content detection method is sensitive to the detection flow rate, and changes in the flow rate will have a greater impact on the detection results. Therefore, during the detection process, it should be strictly implemented according to the detection flow rate of 1.0 mL / min.

[0172] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0173] The above-described embodiments merely represent several implementation manners of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as limiting the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention patent should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for detecting the content of recombinant human erythropoietin, characterized in that It includes the following steps: Mix the recombinant human erythropoietin physical and chemical reference substance with a solvent to prepare reference substances with different concentrations; Take a sample of recombinant human erythropoietin and prepare the test sample; Detect the reference substances with different concentrations by reverse-phase high performance liquid chromatography to establish the corresponding relationship between concentration and peak area; Detect the test sample by reverse-phase high performance liquid chromatography, substitute the obtained peak area into the corresponding relationship between concentration and peak area, and calculate the content of recombinant human erythropoietin in the test sample; Among them, the mobile phase used in the reverse-phase high performance liquid chromatography includes mobile phase A and mobile phase B; By volume percentage, mobile phase A includes: 70% water and 30% acetonitrile; and 0.1% trifluoroacetic acid based on the total volume of water and acetonitrile; By volume percentage, mobile phase B includes: 40% water and 60% acetonitrile; and 0.1% trifluoroacetic acid based on the total volume of water and acetonitrile; The test sample is the supernatant of CHO cell culture; The conditions of the reverse-phase high performance liquid chromatography include: the wavelength of the ultraviolet detector is 210 nm; The elution program used in the reverse-phase high performance liquid chromatography includes: From 0 to 1 min, keep the volume fraction of mobile phase A at 100% and the volume fraction of mobile phase B at 0%; From 1 min to 25 min, the volume fraction of mobile phase A changes from 100% to 0%, and the volume fraction of mobile phase B changes from 0% to 100%; From 25 min to 38 min, keep the volume fraction of mobile phase A at 0% and the volume fraction of mobile phase B at 100%; The chromatographic column used in the reverse-phase high performance liquid chromatography is filled with C18 alkylsilyl-bonded silica gel.

2. The method for detecting the content of recombinant human erythropoietin according to claim 1, wherein The elution program used in the reverse-phase high performance liquid chromatography also includes: From 38 min to 43 min, the volume fraction of mobile phase A changes from 0% to 100%, and the volume fraction of mobile phase B changes from 100% to 0%; From 43 min to 55 min, keep the volume fraction of mobile phase A at 100% and the volume fraction of mobile phase B at 0%.

3. The method for detecting the content of recombinant human erythropoietin according to claim 1, wherein The conditions of the reverse-phase high performance liquid chromatography also include: the flow rate is 0.9 - 1.1 mL / min.

4. The method for detecting the content of recombinant human erythropoietin according to claim 3, wherein, The conditions of the reverse-phase high performance liquid chromatography also include: the flow rate is 1 mL / min.

5. The method for detecting the content of recombinant human erythropoietin according to claim 1, characterized in that, The conditions of the reverse-phase high performance liquid chromatography also include: the temperature is 25°C - 35°C.

6. The method for detecting the content of recombinant human erythropoietin according to claim 5, characterized in that, The conditions of the reverse-phase high performance liquid chromatography also include: the temperature is 28°C - 32°C.

7. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1 to 6, characterized in that, The integration conditions for the test results are: peak width 50, threshold 200, and the integration interval is 7 min - 30 min.

8. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1 to 6, characterized in that, The retention time of the peak corresponding to recombinant human erythropoietin is 19.3 ± 0.2 min.

9. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1 to 6, characterized in that, The corresponding relationship between concentration and peak area is y = 74056.85x - 108767.04, where x represents the concentration and y represents the peak area of recombinant human erythropoietin at the said concentration.

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