CN115015415A - Content detection method of recombinant human erythropoietin - Google Patents
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- G—PHYSICS
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Abstract
The invention relates to a content detection method of recombinant human erythropoietin, which comprises the following steps: mixing a physicochemical reference substance of the recombinant human erythropoietin with a solvent to prepare reference substances with different concentrations; taking a sample of the recombinant human erythropoietin to prepare a sample to be detected; detecting the reference substances with different concentrations by high performance liquid chromatography, and establishing a corresponding relation between the concentration and the peak area; and detecting the product to be detected by high performance liquid chromatography, substituting the obtained peak area into the corresponding relation between the concentration and the peak area, and calculating the content of the recombinant human erythropoietin in the product to be detected. The detection method has high detection accuracy and simple operation.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a content detection method of recombinant human erythropoietin.
Background
Erythropoietin (EPO), first discovered in 1906, is a glycoprotein synthesized and secreted mainly by cells in the vicinity of the proximal convoluted tubule of the kidney, belonging to salivary glycoprotein hormone, and consists of 166 amino acids with a molecular weight of 34 KD. Erythropoietin can be classified into alpha type and beta type according to the difference of the glycoform structures, wherein the alpha type contains 34% of carbohydrate, the beta type contains 26% of carbohydrate, and the two types have the same biological characteristics, antigenicity and other effects.
In 1985, recombinant human erythropoietin (rhEPO) is expressed by utilizing a gene recombination technology, and the gene of the EPO is constructed into Chinese hamster ovary cells (CHO cells) by utilizing a DNA recombination technology to form the CHO cells capable of expressing the rhEPO gene, wherein the rhEPO has the same physiological activity as human endogenous EPO. In 6 months 1989, the U.S. FDA officially approved the introduction of recombinant human erythropoietin, developed by the company Ann. rhEPO is mainly clinically used for treating anemia such as anemia caused by chronic renal failure combined anemia, Chronic Renal Failure (CRF) anemia, myelodysplastic syndrome (MDS) anemia, and AIDS associated anemia of patients, and is also reported to be applied to treating anemia caused by cancer patients receiving chemotherapy, correcting anemia related to Multiple Myeloma (MM), anemia of premature infants, gestational anemia and postpartum anemia of pregnant women, chronic anemia of patients suffering from rheumatoid arthritis, lupus erythematosus and severe parasites, sickle cell anemia of hemoglobinopathy, thalassemia and anemia related to inflammatory bowel diseases. Meanwhile, the rhEPO can also be used for enhancing exercise endurance and reducing patients with altitude sickness and angina pectoris needing blood transfusion. In conclusion, rhEPO is one of the earliest recombinant protein medicines in the world and is also a gene engineering medicine with mature technology and definite curative effect. However, in the conventional technology, the efficiency of expressing rhEPO by CHO cells is not high, so that the problems of low yield and high cost of the rhEPO exist integrally, and the clinical popularization of the rhEPO is influenced.
In order to improve the yield of the rhEPO, CHO cell culture conditions and downstream purification processes need to be optimized, and the advantages and disadvantages of various preparation process parameters can be quickly and accurately evaluated by quantitatively detecting the rhEPO content of cell supernatants and intermediate products, so that the aim of improving the yield of the rhEPO is fulfilled.
Currently, methods for detecting the content of rhEPO are mainly divided into two categories: the method has the advantages that the biological method detection comprises an in vivo detection method and an in vitro detection method, the biological method detection is the biological activity of the cell factor, so the biological method has high affinity, but the biological method detection has the defects of poor specificity, complex operation, easy interference and the like; and the second is immunological detection, which is a detection method developed along with an immunological labeling technology and is the most widely applied method for detecting rhEPO at present. The current immunological methods for detection can be further classified as: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLTA), and the like.
The initial assay for rhEPO content essentially employed the RIA method. The method has mature technology and high sensitivity, has the defects of manual operation, slow result, short half-life period of the reagent, environmental pollution and the like, and limits the application of the method. ELISA is a sensitive technology, can quantify the measured substance at microgram or even nanogram level, has the advantages of simplicity, low cost, easy standardization and the like, and has the defects of large sample dosage, about 200 mu L of sample required for each detection, long detection time and large human error. FIA has the highest sensitivity in a non-radioimmunoassay, is usually used for detecting bioactive substances with very low content, has high specificity and sensitivity, and has the defect that a kit is expensive and is not suitable for being used in a process exploration stage. CLTA is an analytical technique for detecting antigen or antibody by tracing chemiluminescent signals, has sensitivity generally higher than that of ELISA, and has the advantages of strong specificity, stable marker, wide linear range, high detection speed and the like. Although CLIA is currently rapidly developed and tends to become the mainstream of CLIA instead of RIA, it also has disadvantages that a detection system is expensive and difficult to popularize, or the price is low and sensitivity and detection reliability are not guaranteed.
Some methods involve an erythropoietin detection method based on magnetic beads as carriers, EPO in serum is captured by coupling capture antibodies (anti-EPO antibodies) on the magnetic beads, then biotinylated detection antibodies are added to form immune complexes of capture antibodies, EPO and detection antibodies using the magnetic beads as carriers, and then streptavidin coupled enzyme is added to bind with biotin labeled on the detection antibodies to label the immune complexes. The immunoassay method has ultrahigh sensitivity (19pg/mL), is improved by 13 times compared with the traditional immunoassay method, has 13 times less sample dosage (15 mu L) compared with the traditional immunoassay method, shortens the sample detection experiment period to 34min, and is far shorter than the traditional immunoassay method (6 h-8 h). However, although the method has high sensitivity, small sample usage amount and short detection period, the preparation of the 'reagent' using magnetic beads as carriers is complicated, the problems of quality difference and the like possibly existing between batches of the 'magnetic bead reagent' and the like due to the fact that the anti-rhEPO antibody needs to be prepared, commercial products are difficult to form, and the detection process is too complicated, so that the risk of falling of the anti-rhEPO antibody exists, and the accuracy of detecting the rhEPO content is further influenced.
Disclosure of Invention
Based on the content, the invention provides the content detection method of the recombinant human erythropoietin, which has high detection accuracy and simple operation.
The specific technical scheme is as follows:
a method for detecting the content of recombinant human erythropoietin comprises the following steps:
mixing a physicochemical reference substance of recombinant human erythropoietin (rhEPO) with a solvent to prepare reference substances with different concentrations;
taking a sample of the recombinant human erythropoietin, and preparing a to-be-detected product;
carrying out reversed-phase high performance liquid chromatography detection on the reference substances with different concentrations, and establishing a corresponding relation between the concentration and the peak area;
carrying out reversed-phase high performance liquid chromatography detection on the to-be-detected product, substituting the obtained peak area into the corresponding relation between the concentration and the peak area, and calculating the content of the recombinant human erythropoietin in the to-be-detected product;
wherein the mobile phase adopted by the reversed-phase high performance liquid chromatography comprises a mobile phase A and a mobile phase B;
the mobile phase A comprises the following components in percentage by volume: 65% -75% of water (H) 2 O) and 35 to 25 percent of Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile;
the mobile phase B comprises the following components in percentage by volume: 35% -45% of water (H) 2 O) and 65% to 55% Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile.
In one embodiment, the reversed-phase high performance liquid chromatography employs an elution procedure comprising:
keeping the volume fraction of the mobile phase A to be 100% and the volume fraction of the mobile phase B to be 0% for 0-1 min;
1-25 min, wherein the volume fraction of the mobile phase A is changed from 100% to 0%, and the volume fraction of the mobile phase B is changed from 0% to 100%;
25 min-38 min, keeping the volume fraction of the mobile phase A at 0 percent and the volume fraction of the mobile phase B at 100 percent.
In one embodiment, the elution procedure employed by the reverse phase high performance liquid chromatography further comprises:
38 min-43 min, the volume fraction of the mobile phase A is changed from 0% to 100%, and the volume fraction of the mobile phase B is changed from 100% to 0%;
and (3) 43-55 min, keeping the volume fraction of the mobile phase A at 100% and the volume fraction of the mobile phase B at 0%.
In one embodiment, the reversed-phase high performance liquid chromatography adopts a chromatographic column which takes one of C4-C18 alkyl silane bonded silica gel as a filler.
In one embodiment, the reversed-phase high performance liquid chromatography uses a column packed with C4, C6, C8, or C18 alkylsilane chemically bonded silica.
In one embodiment, the conditions of the reverse phase high performance liquid chromatography further comprise: the flow rate is 0.9-1.1 mL/min, the temperature is 25-35 ℃, and the wavelength of the ultraviolet detector is 209-211 nm.
In one embodiment, the conditions of the reverse phase high performance liquid chromatography further comprise: the flow rate is 1mL/min, the temperature is 28-32 ℃, and the wavelength of the ultraviolet detector is 210 nm.
In one embodiment, the integration condition of the detection result is: the peak width is 50, the threshold value is 200, and the integration interval is 7 min-30 min.
In one embodiment, the peak retention time for recombinant human erythropoietin is 19.3 ± 0.2 min.
In one embodiment, the relationship between the concentration and the peak area is y-74056.85 x-108767.04, wherein x represents the concentration and y represents the peak area of recombinant human erythropoietin at that concentration.
According to the content detection method of the recombinant human erythropoietin, the reversed-phase high-performance liquid chromatography is adopted, the detection conditions of the high-performance liquid chromatography are reasonably controlled, the recombinant human erythropoietin in a sample to be detected can be accurately detected, the operation is simple and convenient, complex operations such as antibody coupling in the traditional method are not needed, and compared with the traditional biological detection method and the immunological detection method, the method has the advantages of small sample demand, low detection cost, less time consumption and high accuracy, can also detect samples with complex components such as cell culture supernatant, and has very high guiding significance for process optimization and production development of the recombinant human erythropoietin.
Meanwhile, the content detection method of the recombinant human erythropoietin is comprehensively verified, and comprises verification items such as system applicability, specificity, linearity, accuracy, precision, intermediate precision, quantitative limit, detection limit and durability, the detection accuracy is high, the standard addition recovery rate is 95.05% -100.66%, the detection precision is high, the repeatability of the method is good, the quantitative limit is as low as 30 mu g/mL rhEPO, and the content measurement result of the recombinant human erythropoietin in cell culture and downstream purification processes is more accurate and reliable.
Drawings
FIG. 1 is a typical HPLC profile of a linearly validated blank solution;
FIG. 2 is a typical HPLC chromatogram for linearly verifying a 20. mu.g/mL test solution;
FIG. 3 is a typical HPLC chromatogram of a sample solution with linear validation of 40 μ g/mL;
FIG. 4 is a typical HPLC chromatogram for linearly verifying a sample solution of 80. mu.g/mL;
FIG. 5 is a typical HPLC chromatogram for linearly verifying 160 μ g/mL test solution;
FIG. 6 is a typical HPLC chromatogram of a test solution with a linear validation rate of 320. mu.g/mL;
FIG. 7 is a typical HPLC chromatogram of a 640. mu.g/mL sample solution for linear validation;
FIG. 8 is a typical HPLC profile of a blank solution for verifying system suitability;
FIG. 9 is a typical HPLC profile of a system suitability verification system suitability solution;
FIG. 10 is a specific validation blank solution HPLC profile;
FIG. 11 is a HPLC profile of a proprietary validation control;
FIG. 12 is a HPLC chromatogram of a specific verification sample;
FIG. 13 is a typical HPLC profile of precision and intermediate precision validation blank solution;
FIG. 14 is a typical HPLC chromatogram of a sample solution for precision and intermediate precision verification;
FIG. 15 is a typical HPLC profile of a blank solution for accuracy verification;
FIG. 16 is a typical HPLC profile of the cell supernatant for accuracy verification;
FIG. 17 is a typical HPLC profile for verifying accuracy of a 30 μ g/mL spiked sample;
FIG. 18 is a typical HPLC profile for verifying accuracy of 330. mu.g/mL labeled samples;
FIG. 19 is a typical HPLC profile for accuracy verification of 630 μ g/mL spiked samples;
FIG. 20 is a typical HPLC profile of a blank solution for validation and verification of quantitation limit and detection limit;
FIG. 21 is a typical HPLC profile with a detection limit of 20. mu.g/mL;
FIG. 22 is a typical HPLC profile with a limit of quantitation of 30. mu.g/mL;
FIG. 23 is a 200. mu.g/mL control (28 ℃) HPLC profile;
FIG. 24 is a 200. mu.g/mL control (30 ℃) HPLC profile;
FIG. 25 is a 200. mu.g/mL control (32 ℃) HPLC profile;
FIG. 26 is a 200. mu.g/mL control (208nm) HPLC profile;
FIG. 27 is a 200. mu.g/mL control (210nm) HPLC profile;
FIG. 28 is a 200. mu.g/mL control (212nm) HPLC profile;
FIG. 29 is an HPLC chromatogram of a 200. mu.g/mL control (0.8 mL/min);
FIG. 30 is an HPLC chromatogram of a 200. mu.g/mL control (1.0 mL/min);
FIG. 31 is an HPLC chromatogram of a 200. mu.g/mL control (1.2 mL/min).
Detailed Description
The method for detecting the content of recombinant human erythropoietin according to the present invention will be described in further detail with reference to the following specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, the technical features described in the open type include a closed technical solution including the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical intervals are regarded as continuous, and include the minimum and maximum values of the range and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid mixing, and volume percentages for liquid-liquid mixing. The solvent of the solution sample is water unless otherwise specified.
The percentage concentrations referred to in the present invention are, unless otherwise specified, the final concentrations. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
The invention provides a content detection method of recombinant human erythropoietin, which comprises the following steps:
mixing a physicochemical reference substance of recombinant human erythropoietin (rhEPO) with a solvent to prepare reference substances with different concentrations;
taking a sample of the recombinant human erythropoietin to prepare a sample to be detected;
carrying out reversed-phase high performance liquid chromatography detection on the reference substances with different concentrations, and establishing a corresponding relation between the concentration and the peak area;
carrying out reversed-phase high performance liquid chromatography detection on the to-be-detected product, substituting the obtained peak area into the corresponding relation between the concentration and the peak area, and calculating the content of the recombinant human erythropoietin in the to-be-detected product;
wherein the mobile phase adopted by the reversed-phase high performance liquid chromatography comprises a mobile phase A and a mobile phase B;
the mobile phase A comprises the following components in percentage by volume: 65% -75% of water (H) 2 O) and 35 to 25 percent of Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile;
the mobile phase B comprises the following components in percentage by volume: 35% -45% of water (H) 2 O) and 65% to 55% Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile.
It is understood that the sample to be tested may be an intermediate product in the CHO cell culture supernatant and the downstream purification process.
In some specific examples, the solvent is Tris-HCl buffer.
In some specific examples, the mobile phase a comprises, in volume percent: 68-72% of water (H) 2 O) and 32 to 28 percent of Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile. Specifically, the mobile phase a comprises, in volume percent: 70% of water (H) 2 O) and 30% Acetonitrile (ACN); and trifluoroacetic acid (TFA) at 0.1% of the total volume of water and acetonitrile.
In some specific examples, the mobile phase B comprises, in volume percent: 38% -42% of water (H) 2 O) and 62 to 58 percent of Acetonitrile (ACN); and trifluoroacetic acid (TFA) accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile. Specifically, the mobile phase B comprises, in volume percent: 40% of water (H) 2 O) and 60% Acetonitrile (ACN); and trifluoroacetic acid (TFA) at 0.1% of the total volume of water and acetonitrile.
In some specific examples, the reversed-phase high performance liquid chromatography employs an elution procedure comprising:
keeping the volume fraction of the mobile phase A to be 100% and the volume fraction of the mobile phase B to be 0% for 0-1 min;
1-25 min, wherein the volume fraction of the mobile phase A is changed from 100% to 0%, and the volume fraction of the mobile phase B is changed from 0% to 100%;
25 min-38 min, keeping the volume fraction of the mobile phase A at 0 percent and the volume fraction of the mobile phase B at 100 percent.
Further, the elution procedure adopted by the reversed-phase high performance liquid chromatography further comprises the following steps:
38 min-43 min, the volume fraction of the mobile phase A is changed from 0% to 100%, and the volume fraction of the mobile phase B is changed from 100% to 0%;
and (3) 43-55 min, and keeping the volume fraction of the mobile phase A to be 100% and the volume fraction of the mobile phase B to be 0%.
In some specific examples, the integration condition of the detection result is: the peak width is 50, the threshold value is 200, and the integration interval is 7 min-30 min.
In some specific examples, the peak area of recombinant human erythropoietin is calculated by area normalization.
In some specific examples, the reversed-phase high performance liquid chromatography uses a column packed with one of C4-C18 alkylsilane bonded silica gel. Furthermore, the chromatographic column adopted by the reversed-phase high performance liquid chromatography uses C4, C6, C8 or C18 alkylsilane bonded silica gel as a filler. In particular to a chromatographic column phenomenex adopted by the reversed phase high performance liquid chromatography
C18 column.
In some specific examples, the reversed-phase high performance liquid chromatography employs column parameters including: 4.6mm x (150-250) mm, a pore diameter of 12-30 nm, and a particle diameter of 3.5-10 μm.
In some specific examples, the conditions of the reverse phase high performance liquid chromatography further comprise: the flow rate is 0.9-1.1 mL/min, the temperature is 25-35 ℃, and the wavelength of the ultraviolet detector is 209-211 nm. Further, the conditions of the reversed-phase high performance liquid chromatography further comprise: the flow rate is 1mL/min, the temperature is 28-32 ℃, and the wavelength of the ultraviolet detector is 210 nm.
In some specific examples, the peak retention time for recombinant human erythropoietin is 19.3 ± 0.2 min.
In some specific examples, the correspondence between the concentration and the peak area refers to a standard curve. Specifically, the standard curve is y-74056.85 x-108767.04, wherein x represents the concentration and y represents the peak area of recombinant human erythropoietin at that concentration. .
Specific examples are as follows.
Example 1
This embodiment is a method for detecting the content of recombinant human erythropoietin, comprising the following steps:
(1) a chromatographic column: phenomenex
C18 column, using octadecyl silane bonded silica gel as filler, 4.6mm × 250mm, CV ═ 4.15ml, pore diameter 30nm, particle diameter 5 μm, column efficiency not less than 25411.
(2) Mobile phase:
mobile phase a (volume percent): 70% H 2 O+30％ACN，0.1％TFA；
Mobile phase B (volume percent): 40% H 2 O+60％ACN，0.1％TFA。
(3) Blank solution: 10mM Tris-HCl buffer, pH 7.00.
(4) rhEPO physicochemical control solution: purchased from Koxing biological pharmacy Co., Ltd, and calibrated by Lowry method, the protein concentration is 3.77g/L, and the electrophoresis purity is more than or equal to 99%.
(5) And (3) a to-be-detected product: the culture supernatant containing rhEPO obtained from the CHO cell culture was filtered through a 0.45 μm membrane.
(6) High performance liquid chromatography:
the sample detection is carried out by adopting a high performance liquid chromatography system (Agilent 110Series), the flow rate is 1mL/min, the temperature is 30 ℃, the wavelength of an ultraviolet detector is 210nm, the sample injection volume is 100 mu L, and the mobile phase elution gradient table is shown in the following table 1:
TABLE 1 detection method gradient meter for solution
| Time (min) | A(％) | B(％) |
| 0 | 100 | 0 |
| 1 | 100 | 0 |
| 25 | 0 | 100 |
| 38 | 0 | 100 |
| 43 | 100 | 0 |
| 55 | 100 | 0 |
The integration conditions of the liquid phase map are as follows: the peak width is 50, the threshold value is 200, the integration interval is 7 min-30 min, the peak area of the rhEPO is calculated according to an area normalization method, and the retention time of the peak of the target protein is 19.3 +/-0.2 min.
(7) Construction of a standard curve: taking the physical and chemical control rhEPO solution, and diluting the solution into six test sample solutions with different concentrations by using 10mM Tris-HCl buffer solution, wherein the six test sample solutions have different concentrations, and the concentration is 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 160 mu g/mL, 320 mu g/mL and 640 mu g/mL. Sequentially sampling blank solution and sample for twice detection, taking concentration as abscissa and average value of peak area after noise subtraction as ordinate, establishing standard curve, and requiring linear equation R 2 Not less than 0.995. The verification results are shown in the following table 2 and fig. 1 to 7:
TABLE 2 Linear verification results
The data in the table are used for establishing a standard curve, and the equation is that y is 74056.85x-108767.04, R 2 0.99971. As can be seen from Table 2: taking the concentration as the abscissa and the average value of the peak area after noise deduction as the ordinate to obtain a linear relation between the concentration and the peak area, wherein R is 2 When the concentration is 0.99971 and is more than 0.995, the concentration has a certain linear relation with the peak area, and the linearity is good and passes the linear verification.
(8) And (3) detection results: the rhEPO cell culture supernatant concentration is 43.68 mu g/mL.
Example 2
This example demonstrates the methodology of the method for determining the amount of recombinant human erythropoietin of example 1.
(1) System suitability verification
The system suitability verification is to check whether the high performance liquid chromatography system and the chromatographic column meet the detection requirements, and determine whether the detection result is accurate and reliable or not and whether the detection result is suitable for detecting the content of the rhEPO or not by checking the retention time (Rt) of a target protein peak and the Relative Standard Deviation (RSD) of a peak area.
The rhEPO physicochemical control solution is diluted to 200 mug/mL by using 10mM Tris-HCl buffer solution to be used as a system applicability solution. And (4) carrying out sample injection twice on the blank solution, carrying out continuous sample injection four times on the system applicability solution, and recording a chromatogram. The blank solution is required to be free of interference, the system applicability solution is continuously fed for four times, the retention time RSD of the main peak is less than or equal to 1.0 percent, and the area RSD of the main peak is less than or equal to 2.0 percent. The verification results are shown in the following table 3 and fig. 8 to 9:
TABLE 3 System suitability verification results
As can be seen from Table 3 and FIGS. 8 to 9: after the blank solution is injected, no ultraviolet absorption exists in the retention time of the main peak, which indicates that the blank solution has no interference. System applicability the solution major peak retention time RSD and major peak area RSD are within specified ranges. Therefore, the system is qualified in suitability verification, and the used high performance liquid chromatography system and chromatographic column meet the detection requirements and are suitable for detecting the content of the rhEPO.
(2) Attribute validation
The specificity verification means that the analysis method can correctly determine the capability of the detected object in the possible presence of other components (such as impurities, degradation products, auxiliary materials and the like), and the components of the rhEPO cell supernatant are relatively complex. Since each rhEPO purification process used Tris-HCl buffer, the control was diluted with 10mM Tris-HCl buffer and the reference was checked to have no interference with rhEPO content measurement using 10mM Tris-HCl buffer as a blank.
The rhEPO physicochemical control solution was diluted to 40. mu.g/mL with 10mM Tris-HCl buffer as a control solution. Taking the supernatant of the rhEPO cell as a test solution. The blank solution, the reference solution and the sample solution are added once respectively, and the chromatogram is recorded. The blank solution is required to be free of interference, and the deviation of the main peak retention time of the test solution and the main peak retention time of the reference solution is less than 1%. The verification results are shown in the following table 4 and fig. 10 to 12:
TABLE 4 results of the Attribute verification
As can be seen from Table 4 and FIGS. 10 to 12: the blank solution has no interference, the retention time of the main peak of the test sample and the retention time of the main peak of the reference solution deviate by 0.7 percent, and the retention times are basically consistent. Therefore, under the condition that other components (such as impurities, degradation products, auxiliary materials and the like) and a blank solution exist, the detection method also has the capability of correctly detecting the detected object, and the specificity is verified to be qualified.
(3) Verification of precision and intermediate precision
The precision and intermediate precision verification is used for testing the performance of the instrument and confirming the analysis capability of an analyst, thereby ensuring the accuracy and reliability of the test result and good repeatability.
The first person takes a physical and chemical control rhEPO solution, and uses 10mM Tris-HCl buffer solution to dilute to 200 mug/mL as a test solution, and prepares three parts in parallel. And (4) carrying out sample injection once on the blank solution and each part of the sample solution, and recording the chromatogram. The blank solution is required to be free of interference, the retention time RSD of the main peak is less than or equal to 1.0 percent, and the peak area RSD of the main peak is less than or equal to 5.0 percent in the first person three test sample solutions.
The second person takes the rhEPO physicochemical reference substance solution, uses 10mM Tris-HCl buffer solution to dilute to 200 mug/mL as the test substance solution, and prepares three parts in parallel. And replacing one device, feeding the blank solution once, feeding the sample solution once for each part of the sample solution, and recording the chromatogram. Requiring the blank solution to be free of interference, wherein the retention time RSD of the main peak is less than or equal to 1.0 percent and the peak area RSD of the main peak is less than or equal to 5.0 percent in the second human three test sample solutions; the retention time RSD of the main peak in the six test sample solutions of the two persons is less than or equal to 1.0 percent, and the peak area RSD of the main peak is less than or equal to 5.0 percent. The verification results are shown in the following table 5 and FIGS. 13 to 14:
TABLE 5 results of precision and intermediate precision verification
As can be seen from Table 5 and FIGS. 13 to 14: in the first three test solutions, the blank solution is not interfered, the retention time RSD of the main peak is 0.032%, and the peak area RSD of the main peak is 1.59%. In the second three test solutions, the blank solution is free of interference, the main peak retention time RSD is 0.083%, the main peak area RSD is 0.66%, in the six samples of the two persons, the main peak retention time RSD is 0.11%, and the main peak area RSD is 1.82%. The retention time of the main peak and the RSD of the peak area of the first person sample, the second person sample and the second person sample are all smaller than the specified qualified range. Therefore, the performance of the instrument and the analysis capability of an analyst are not problematic, and the precision and the intermediate precision are qualified.
(5) Accuracy verification
The accuracy verification means that the degree of similarity between a measured value and a true value obtained by the detection method is expressed by the recovery rate of the target protein.
Taking an rhEPO physicochemical reference substance solution, diluting the rhEPO physicochemical reference substance solution into reference substance diluent with three concentrations of 16 mu g/mL, 616 mu g/mL and 1216 mu g/mL by using 10mM Tris-HCl buffer solution, mixing a part of test substance solution (rhEPO cell supernatant) and the reference substance diluent according to the volume ratio of 1:1 to obtain three standard adding samples with different concentrations, respectively injecting the three standard adding samples for three times, injecting the test substance for two times, and calculating the concentration and the standard adding recovery rate. The blank solution is required to be free of interference, the average standard adding recovery rate of the main peak under each concentration is between 90 and 110 percent, the standard adding recovery rate RSD of each of the three groups of samples is less than or equal to 5 percent, and the standard adding recovery rate RSD of 9 samples is less than or equal to 5 percent. The verification results are shown in the following table 6 and fig. 15 to 19:
table 6 accuracy verification results
As can be seen from Table 6 and FIGS. 15 to 19: the average standard adding recovery rate under each concentration is 95.05-100.66%, the standard adding recovery rate RSD of each of three groups of samples is 0.10-2.24%, the standard adding recovery rate RSD of 9 samples is 2.79%, the obtained standard adding recovery rate and RSD are all in the specified qualified range, the similarity degree between the measured value and the actual value obtained by the detection method is high, the relative standard deviation is small, and therefore the accuracy verification is qualified.
(6) Quantitative limit and detection limit verification
The limit of quantitation refers to the lowest amount of analyte that can be quantitatively determined in a sample with suitable accuracy and precision. The limit of detection is the minimum amount of analyte in the sample that can be detected, but does not necessarily need to be accurately quantified.
Taking the rhEPO physicochemical control solution, diluting the rhEPO physicochemical control solution into 20 mu g/mL and 30 mu g/mL by using 10mM Tris-HCl buffer solution, and respectively using the rhEPO physicochemical control solution as a detection limit sample and a quantification limit sample. Taking blank solution for sample injection twice, detecting limit sample for sample injection twice, and quantifying limit sample for sample injection three times. The detection limit sample is required to detect the target protein, the ratio (signal to noise ratio) of the peak area mean value to the noise peak area mean value is more than or equal to 3, the ratio of the quantitative limit sample peak area mean value to the noise peak area mean value is more than or equal to 5, the quantitative limit sample peak area RSD is less than or equal to 10%, and the main peak retention time RSD is less than or equal to 2%. The verification results are shown in the following table 7 and fig. 20 to 22:
TABLE 7 verification results of quantitation limit and detection limit
As can be seen from Table 7 and FIGS. 20 to 21: when the sample concentration is 20 mug/mL, the detection limit of the rhEPO content detection method is 20 mug/mL because the blank solution has no ultraviolet absorption in the retention time of the main peak, the signal-to-noise ratio is 41, which is much greater than 3, and it can be seen from fig. 21 that the response value of the main peak is low at this time, the main peak can be detected if the concentration is continuously reduced, but the peak type is poor, and the detection limit of 20 mug/mL can meet the general process requirements of the industry.
When the concentration of the sample is 30 mug/mL, the signal-to-noise ratio is 73, which is far more than 5, three times of continuous sample introduction are carried out, the peak area RSD of the main peak is 0.44%, the retention time RSD of the main peak is 0.06%, which are all smaller than the specified qualified range, and the 30 mug/mL has been proved to have good linearity and accuracy in the linearity and accuracy verification in the early stage, so that the limit of the quantitation of the rhEPO content detection method to 30 mug/mL can meet the general process requirements of the industry.
(7) Durability verification
7.1 comparison of test results at different column temperatures
Taking the rhEPO physicochemical reference substance solution, diluting the rhEPO physicochemical reference substance solution to 200 mu g/mL by using 10mM Tris-HCl buffer solution, keeping other conditions unchanged, detecting the rhEPO physicochemical reference substance solution at the conditions of 28 ℃, 30 ℃ and 32 ℃, and observing the detection results, wherein the detection results are shown in the following table 8 and figures 23-25:
TABLE 8 influence of different column temperatures on the determination of the content of rhEPO
As can be seen from Table 8 and FIGS. 23-25: other conditions are unchanged, and when the column temperature fluctuates within the range of 30 +/-2 ℃, the detection method has certain stability and cannot influence the detection of the content of the rhEPO.
7.2 comparison of detection results at different wavelengths
Taking the rhEPO physicochemical control solution, diluting the rhEPO physicochemical control solution into 200 mu g/mL by using 10mM Tris-HCl buffer solution, keeping other conditions unchanged, detecting the rhEPO physicochemical control solution under the conditions of 208nm, 210nm and 210nm respectively, and observing the detection results as shown in the following table 9 and figures 26-28:
TABLE 9 Effect of different wavelengths on rhEPO content determination
As can be seen from table 9: other conditions were unchanged, and when the detection wavelength was 208nm, the rhEPO content measured at this time deviated from that under normal conditions by 16.74%, which may be caused by: the organic solvent used in the detection method is acetonitrile, the ultraviolet absorption cut-off wavelength of the acetonitrile is 190-210 nm, and when the wavelength is less than 210nm, part of the acetonitrile can be absorbed, so that the measured value of the concentration of the target protein is higher. When the detection wavelength is 212nm, the rhEPO content measured at this time deviates by-12.54% from that under normal conditions, and the reason for the deviation may be: there may be a portion of the protein of interest that is not absorbed by the ultraviolet light, resulting in a lower measured concentration of the protein of interest.
In summary, the rhEPO content detection method is sensitive to the detection wavelength, and the wavelength variation has a large influence on the detection result, so that the detection process should be performed according to the detection wavelength of 210 nm.
7.3 comparison of the measurement results at different flow rates
Taking the rhEPO physicochemical control solution, diluting the rhEPO physicochemical control solution into 200 mu g/mL by using 10mM Tris-HCl buffer solution, keeping other conditions unchanged, detecting the rhEPO physicochemical control solution under the conditions of 0.8mL/min, 1.0mL/min and 1.2mL/min respectively, and observing the detection results, wherein the detection results are shown in the following table 10 and figures 29-31:
TABLE 10 Effect of different flow rates on rhEPO content determination
As can be seen from table 10: other conditions were unchanged, and when the detected flow rate was 0.8mL/min, the rhEPO content measured at this time deviated by 25.20% from that under normal conditions, and the reason for the deviation may be: after the flow rate is reduced, the mass transfer speed of the mobile phase is relatively reduced, the retention time of a main peak is delayed, the peak type is wide (large in volume and low in concentration), and the HPLC detector used in the verification is a concentration type detector, and the peak area is inversely proportional to the flow rate of the mobile phase, so that the peak area is higher than the detection value under the normal condition, and the detected concentration is larger. When the detection flow rate is 1.2mL/min, the mass transfer speed of the mobile phase is relatively high, the retention time of a main peak is advanced, the peak shape is sharp and narrow (the volume is small and the concentration is high), the peak area possibly integrated is small, and the measured concentration is relatively low compared with the normal condition.
In summary, the rhEPO content detection method is sensitive to the detection flow rate, and the flow rate variation can cause great influence on the detection result, so that the rhEPO content detection method is strictly executed according to the detection flow rate of 1.0mL/min in the detection process.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Claims (10)
1. A method for detecting the content of recombinant human erythropoietin is characterized by comprising the following steps:
mixing a physicochemical reference substance of the recombinant human erythropoietin with a solvent to prepare reference substances with different concentrations;
taking a sample of the recombinant human erythropoietin, and preparing a to-be-detected product;
carrying out reversed-phase high performance liquid chromatography detection on the reference substances with different concentrations, and establishing a corresponding relation between the concentration and the peak area;
carrying out reversed-phase high performance liquid chromatography detection on the to-be-detected product, substituting the obtained peak area into the corresponding relation between the concentration and the peak area, and calculating the content of the recombinant human erythropoietin in the to-be-detected product;
wherein the mobile phase adopted by the reversed-phase high performance liquid chromatography comprises a mobile phase A and a mobile phase B;
the mobile phase A comprises the following components in percentage by volume: 65 to 75 percent of water and 35 to 25 percent of acetonitrile; and trifluoroacetic acid accounting for 0.05-0.15% of the total volume of water and acetonitrile;
the mobile phase B comprises the following components in percentage by volume: 35 to 45 percent of water and 65 to 55 percent of acetonitrile; and trifluoroacetic acid accounting for 0.05-0.15 percent of the total volume of the water and the acetonitrile.
2. The method for detecting the content of recombinant human erythropoietin according to claim 1, wherein the reversed-phase high-performance liquid chromatography uses an elution program comprising:
keeping the volume fraction of the mobile phase A to be 100% and the volume fraction of the mobile phase B to be 0% for 0-1 min;
1-25 min, wherein the volume fraction of the mobile phase A is changed from 100% to 0%, and the volume fraction of the mobile phase B is changed from 0% to 100%;
25 min-38 min, keeping the volume fraction of the mobile phase A at 0 percent and the volume fraction of the mobile phase B at 100 percent.
3. The method for detecting the content of recombinant human erythropoietin according to claim 2, wherein the elution procedure used in the reversed-phase high-performance liquid chromatography further comprises:
38 min-43 min, the volume fraction of the mobile phase A is changed from 0% to 100%, and the volume fraction of the mobile phase B is changed from 100% to 0%;
and (3) 43-55 min, keeping the volume fraction of the mobile phase A at 100% and the volume fraction of the mobile phase B at 0%.
4. The method for detecting the content of recombinant human erythropoietin according to claim 1, wherein the reversed-phase high performance liquid chromatography uses one of C4-C18 alkylsilane chemically bonded silica as a filler.
5. The method for detecting the content of recombinant human erythropoietin according to claim 4, wherein the reversed-phase high performance liquid chromatography uses C4, C6, C8 or C18 alkylsilane chemically bonded silica as a filler.
6. The method for detecting the content of recombinant human erythropoietin according to claim 1, wherein the conditions of the reversed-phase high performance liquid chromatography further comprise: the flow rate is 0.9-1.1 mL/min, the temperature is 25-35 ℃, and the wavelength of the ultraviolet detector is 209-211 nm.
7. The method for detecting the content of recombinant human erythropoietin according to claim 6, wherein the conditions of the reversed-phase high performance liquid chromatography further comprise: the flow rate is 1mL/min, the temperature is 28-32 ℃, and the wavelength of the ultraviolet detector is 210 nm.
8. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1 to 7, wherein the integration conditions of the detection results are as follows: the peak width is 50, the threshold value is 200, and the integration interval is 7 min-30 min.
9. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1 to 7, wherein the retention time of the peak corresponding to recombinant human erythropoietin is 19.3 ± 0.2 min.
10. The method for detecting the content of recombinant human erythropoietin according to any one of claims 1-7, wherein the correlation between the concentration and the peak area is y-74056.85 x-108767.04, wherein x represents the concentration and y represents the peak area of recombinant human erythropoietin at the concentration.
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