CN107941983B - Detection method and purity analysis method of rhPTH protein or rhPTH protein preparation - Google Patents
Detection method and purity analysis method of rhPTH protein or rhPTH protein preparation Download PDFInfo
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Abstract
The invention provides a detection method and a purity analysis method of rhPTH protein or a rhPTH protein preparation, belonging to the field of medical inspection and drug analysis. The detection method of the rhPTH protein or the rhPTH protein preparation further improves the specificity, detection limit and durability of detection by optimizing the conditions of the proportion of the mobile phase, the concentration of a sample, the detection time, the detection wavelength, the column temperature and the like; the purity analysis by adopting the detection method also has accurate detection data with good and reliable detection results, and has great practical application value.
Description
Technical Field
The invention relates to the field of medical inspection and drug analysis, in particular to a detection method and a purity analysis method of rhPTH protein or a rhPTH protein preparation.
Background
In the process of drug development, the detection method of protein content and purity is an important evaluation parameter for drug screening and quality control, and the method needs to pass method verification to meet the requirements of method verification aspects such as specificity, precision, accuracy and the like, so that the reliability of the detection result can be ensured and the method can be used for guiding drug screening and product quality control.
Human parathyroid hormone is one of the important hormones for maintaining the blood calcium balance of the body, the basic physiological effect of which is to promote bone resorption and growth by increasing the number of osteoblasts, and rhPTH, an active fragment of human endogenous parathyroid hormone, increases the accumulation of new bone on the surface of cancellous and cortical bone by stimulating the activity of osteoblasts by once daily injection.
Because the rhPTH product is in the development stage, the specificity, detection limit, durability and precision of the existing detection method aiming at the content and purity of the rhPTH are not enough, and further improvement is needed.
Disclosure of Invention
The first purpose of the invention is to provide a detection method of rhPTH protein or rhPTH protein preparation, which can better improve the specificity, detection limit and precision of detection and provide accurate and reliable detection data.
The second objective of the invention is to provide an analysis method for purity of rhPTH protein or rhPTH protein preparation, by which purity and content information of different samples can be better analyzed, and better information guarantee is provided for quality control and the like.
In order to achieve the above purpose of the invention, the following technical scheme is adopted:
a detection method of rhPTH protein or a rhPTH protein preparation comprises the following steps:
preparing rhPTH protein or a rhPTH protein preparation into a solution to be detected, injecting the solution to be detected into a high performance liquid chromatograph, and detecting under the following conditions:
chromatographic conditions are as follows: the chromatographic column is a reversed phase chromatographic column, and the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 8-15:85-92, and the mobile phase B is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 45-55:45: 55;
the pH values of the mobile phase A and the mobile phase B are 1.9-2.5;
elution conditions: elution time and volume content of mobile phase B during elution time were as follows: 0-5min, 0-25%; 25 to 35 percent of 5 to 15 min; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%;
the detection wavelength is 210-220 nm.
A method for analyzing purity of rhPTH protein or rhPTH protein preparation comprises the steps of preparing a gradient concentration solution of a rhPTH protein standard product and a solution of a to-be-detected product of the rhPTH protein or the rhPTH protein preparation, carrying out chromatographic detection according to the detection method of the rhPTH protein or the rhPTH protein preparation, and calculating the purity result of the to-be-detected product of the rhPTH protein or the rhPTH protein preparation according to the result of the chromatographic detection.
Compared with the prior art, the invention has the beneficial effects that: the detection method of the rhPTH protein or the rhPTH protein preparation further improves the specificity, detection limit and durability of detection by optimizing the conditions of the proportion of the mobile phase, the concentration of a sample, the detection time, the detection wavelength, the column temperature and the like; the purity analysis by adopting the detection method also has accurate detection data with good and reliable detection results, and has great practical application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a chromatogram of the current assay provided in Experimental example 1 of the present invention;
FIG. 2 is a chromatogram of the original drug of the present invention provided in Experimental example 1;
FIG. 3 is a chromatogram for adjustment of elution conditions provided in Experimental example 1 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following is a detailed description of a method for detecting rhPTH protein or a rhPTH protein preparation and a method for analyzing purity according to an embodiment of the present invention.
A detection method of rhPTH protein or a rhPTH protein preparation comprises the following steps:
preparing rhPTH protein or a rhPTH protein preparation into a solution to be detected, injecting the solution to be detected into a high performance liquid chromatograph, and detecting under the following conditions:
chromatographic conditions are as follows: the chromatographic column is a reversed phase chromatographic column, and the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 8-15:85-92, and the mobile phase B is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 45-55:45: 55;
the pH values of the mobile phase A and the mobile phase B are 1.9-2.5;
elution conditions: elution time and volume content of mobile phase B during elution time were as follows: 0-5min, 0-25%; 25 to 35 percent of 5 to 15 min; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%;
the detection wavelength is 210-220 nm.
Further, in the preferred embodiment of the present invention, the flow rates of mobile phase A and mobile phase B are 0.8mL/min to 1.3 mL/min.
Further, in the preferred embodiment of the present invention, the reverse phase chromatography column is a C18 column or a C8 column.
Furthermore, in the preferred embodiment of the present invention, the sample loading amount of the solution to be tested is 45-60 μ L.
Further, in the preferred embodiment of the present invention, the column temperature of the column is 38 ℃ to 44 ℃.
Further, in the preferred embodiment of the present invention, the concentration of the solution to be tested is 0.2mg/mL-0.3 mg/mL.
Further, in a preferred embodiment of the present invention, the test solution is prepared from a test mother liquor prepared from rhPTH protein or rhPTH protein.
Further, in a preferred embodiment of the invention, a stock solution of 2mg/mL to 3mg/mL is tested.
A method for analyzing purity of rhPTH protein or rhPTH protein preparation comprises the steps of preparing a gradient concentration solution of a rhPTH protein standard product and a solution of a to-be-detected product of the rhPTH protein or the rhPTH protein preparation, carrying out chromatographic detection according to the detection method of the rhPTH protein or the rhPTH protein preparation, and calculating the purity result of the to-be-detected product of the rhPTH protein or the rhPTH protein preparation according to the result of the chromatographic detection.
Further, in a preferred embodiment of the present invention, obtaining a standard curve of the rhPTH protein standard is also included.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a detection method of rhPTH protein or a rhPTH protein preparation, which comprises the following specific detection steps:
1.1 preparing rhPTH protein or rhPTH protein preparation to be detected into test mother liquor, wherein the concentration of the test mother liquor is 2 mg/mL;
1.2 diluting the test mother solution into rhPTH protein or rhPTH protein preparation solution to be tested; the concentration of the solution to be detected is 0.2 mg/mL;
1.3 the mobile phase comprises mobile phase a and mobile phase B, mobile phase a consisting essentially of acetonitrile solution and sodium sulfate solution in a ratio of 8: 92, the pH value of the mobile phase A is 1.9, the mobile phase B is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 45:55, and the pH value of the mobile phase B is 2.5;
1.4 selecting a C18 column as a reversed-phase chromatographic column, adding 45 mu L of solution to be detected into the chromatographic column, and detecting at the detection wavelength of 210nm and the column temperature of 38 ℃;
1.5 flow rate of mobile phase was 0.8 mL/min.
The elution conditions were: elution time and volume content of mobile phase B during elution time were as follows: 0-5min, 0-25%; 25 to 35 percent of 5 to 15 min; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%.
Example 2
The embodiment provides a detection method of rhPTH protein or a rhPTH protein preparation, which comprises the following specific detection steps:
1.1 preparing rhPTH protein or rhPTH protein preparation to be detected into test mother liquor, wherein the concentration of the test mother liquor is 3 mg/mL;
1.2 diluting the test mother solution into rhPTH protein or rhPTH protein preparation solution to be tested; the concentration of the solution to be detected is 0.3 mg/mL;
1.3 the mobile phase comprises mobile phase a and mobile phase B, the mobile phase a consisting essentially of acetonitrile solution and sodium sulfate solution in a ratio of 15:85, the pH value of the mobile phase A is 2.5, the mobile phase B is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 55:45, and the pH value of the mobile phase B is 1.9;
1.4 selecting a C8 column as a reversed-phase chromatographic column, adding 60 mu L of solution to be detected into the chromatographic column, and detecting at the detection wavelength of 220nm and the column temperature of 44 ℃;
1.5 flow rate of mobile phase 1.3 mL/min.
The elution conditions were: elution time and volume content of mobile phase B during elution time were as follows: 0-5min, 0-25%; 25 to 35 percent of 5 to 15 min; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%.
Example 3
The embodiment provides a detection method of rhPTH protein or a rhPTH protein preparation, which comprises the following specific detection steps:
1.1 preparing rhPTH protein or rhPTH protein preparation to be detected into test mother liquor, wherein the concentration of the test mother liquor is 2.5 mg/mL;
1.2 diluting the test mother solution into rhPTH protein or rhPTH protein preparation solution to be tested; the concentration of the solution to be detected is 0.25 mg/mL;
1.3 the mobile phase comprises mobile phase a and mobile phase B, the mobile phase a consisting essentially of acetonitrile solution and sodium sulfate solution in a ratio of 10: 90, the pH value of the mobile phase A is 2.3, the mobile phase B is mainly prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 50:50, and the pH value of the mobile phase B is 2.3;
1.4 selecting a C18 column as a reversed-phase chromatographic column, adding 50 mu L of solution to be detected into the chromatographic column, and detecting at the detection wavelength of 210nm and the column temperature of 40 ℃;
1.5 flow rate of mobile phase was 1.0 mL/min.
The elution conditions were: elution time and volume content of mobile phase B during elution time were as follows: 0-5min, 0-25%; 25 to 35 percent of 5 to 15 min; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%. .
Example 4
The embodiment provides an analysis method for purity of rhPTH protein or a rhPTH protein preparation, which comprises the following specific steps:
1.1 preparing a gradient concentration solution of a rhPTH protein standard product and a solution of a rhPTH protein or a rhPTH protein preparation to-be-detected product;
1.2 carrying out chromatographic detection according to the detection method provided in example 3;
1.3 calculating a standard curve of the rhPTH protein standard according to the result of chromatographic detection;
1.4 calculating the purity of the rhPTH protein or the rhPTH protein preparation to be detected according to the standard curve technology of the rhPTH protein standard product.
Experimental example 1
This experimental example is an optimization experiment for the rhPTH detection method.
(1) Existing rhPTH protein purity and related substance detection method
Waters Symmetry 300C18(3.9mm ID×150mm，5μm，
)，Part.No.WAT106154
Mobile phase A: 95% water + 5% acetonitrile + 0.1% trifluoroacetic acid, mobile phase B: 5% water + 95% acetonitrile + 0.1% trifluoroacetic acid
The analysis conditions were: gradient elution of mobile phase A and mobile phase B; a detection instrument: HPLC, PDA detector; the column temperature was 30 ℃, the detection wavelength was 220nm, the recording time was 35min, the sample size was 10. mu.g (40. mu.L), and the elution conditions are shown in Table 1.
TABLE 1 Current chromatographic elution conditions
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 5 | 1.0 | 80 | 20 |
| 3 | 20 | 1.0 | 70 | 30 |
| 4 | 25 | 1.0 | 0 | 100 |
| 5 | 30 | 1.0 | 100 | 0 |
| 6 | 35 | 1.0 | 100 | 0 |
The corresponding chromatogram is shown in FIG. 1.
(2) Method for detecting purity and related substances in quality standard of original rhPTH medicine
Analyzing a chromatographic column: agilent Zorbax 300SB-C18 (4.6X 150mm, 5 μm,
)，Part.No.863973-902；
Mobile phase A: 0.2M Na2SO4(ph2.3) acetonitrile 90: 10; mobile phase B: 0.2M Na2SO4(ph2.3) acetonitrile 50: 50;
the analysis conditions were: gradient elution of mobile phase A and mobile phase B; a detection instrument: HPLC, PDA detector; the column temperature was 40 ℃, the detection wavelength was 214nm, the recording time was 100min, the sample size was 12.5. mu.g (50. mu.L), and the elution conditions are shown in Table 2.
TABLE 2 original chromatographic conditions for rhPTH
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 2 | 1.0 | 100 | 0 |
| 3 | 8 | 1.0 | 76 | 24 |
| 4 | 68 | 1.0 | 60 | 40 |
| 5 | 75 | 1.0 | 0 | 100 |
| 6 | 80 | 1.0 | 0 | 100 |
| 7 | 82 | 1.0 | 100 | 0 |
| 8 | 100 | 1.0 | 100 | 0 |
The chromatogram for chromatographic detection is shown in FIG. 2.
(3) Carrying out elution gradient optimization on the basis of the original detection method of the medicine:
analyzing a chromatographic column: agilent Zorbax 300SB-C18 (4.6X 150mm, 5 μm,
)，Part.No.863973-902；
Mobile phase A: 0.2M Na2SO4(ph2.3) acetonitrile 90: 10; mobile phase B: 0.2M Na2SO4(ph2.3) acetonitrile 50: 50;
the analysis conditions were: gradient elution of mobile phase A and mobile phase B; a detection instrument: HPLC, PDA detector; the column temperature is 40 ℃, the detection wavelength is 214nm, the recording time is 100min, and the sample injection amount is 12.5 mug (50 mug L); the conditions are shown in tables 3 to 7, respectively.
Table 3 adjustment of elution gradient 1
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 2 | 1.0 | 100 | 0 |
| 3 | 8 | 1.0 | 76 | 30 |
| 4 | 68 | 1.0 | 60 | 60 |
| 5 | 75 | 1.0 | 0 | 100 |
| 6 | 80 | 1.0 | 0 | 100 |
| 7 | 82 | 1.0 | 100 | 0 |
| 8 | 100 | 1.0 | 100 | 0 |
TABLE 4 adjustment of elution gradient 2
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 5 | 1.0 | 75 | 25 |
| 3 | 40 | 1.0 | 50 | 50 |
| 4 | 45 | 1.0 | 0 | 100 |
| 5 | 47 | 1.0 | 0 | 100 |
| 6 | 48 | 1.0 | 100 | 0 |
| 7 | 60 | 1.0 | 100 | 0 |
TABLE 5 adjustment of elution gradient
TABLE 6 adjustment of elution gradient
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 5 | 1.0 | 75 | 25 |
| 3 | 20 | 1.0 | 65 | 35 |
| 4 | 50 | 1.0 | 50 | 50 |
| 5 | 55 | 1.0 | 0 | 100 |
| 6 | 57 | 1.0 | 0 | 100 |
| 7 | 58 | 1.0 | 100 | 0 |
| 8 | 70 | 1.0 | 100 | 0 |
TABLE 7 adjustment of elution gradient
| Step | Time(min) | Flow(mL/min) | ％Solution A | ％Solution B |
| 1 | 0 | 1.0 | 100 | 0 |
| 2 | 5 | 1.0 | 75 | 25 |
| 3 | 15 | 1.0 | 65 | 35 |
| 4 | 65 | 1.0 | 50 | 50 |
| 5 | 70 | 1.0 | 0 | 100 |
| 6 | 72 | 1.0 | 0 | 100 |
| 7 | 73 | 1.0 | 100 | 0 |
| 8 | 85 | 1.0 | 100 | 0 |
The chromatogram for elution gradient adjustment is shown in FIG. 3.
Comparing different chromatographic analysis detection results from dimensions such as retention time, peak area, purity, separation degree (R is more than 1.5) of main peak and impurity, tailing factor (0.8-1.5), theoretical plate number (not less than 10000) and the like. The results are shown in Table 8.
TABLE 8 comparison of chromatographic results of different methods
As can be seen from Table 8, the flux of the reversed phase C18 method of the original rhPTH related substance was added with 200mM Na in comparison with the current reversed phase C18 method of the product2SO4And then, the peak pattern is effectively improved, the tailing factor is close to 1.0, the sizes of the two chromatographic columns are close, the grain diameter of the Agilent chromatographic column is 3.5um, and the separation degree is improved. Therefore, by optimization, better chromatographic conditions are obtained: the Agilent chromatographic column is adopted, and the total operation time of the optimized method is 85min after 5 times of elution gradient adjustment. The purity is similar to the result of the prior reversed phase C18, the separation degree before and after the main peak is more than 1.5, and the theoretical plate number is more than 10000.
Experimental example 2
The experimental example performs detection analysis on the specificity, precision, detection limit and durability of the rhPTH protein and the rhPTH protein-related preparation. Chromatographic conditions, analytical methods the method of example 3 was referenced. The samples are shown in Table 9.
TABLE 9rhPTH proteins and rhPTH protein-related formulations
| Name (R) | Batch number | Specification of | Storage conditions |
| rhPTH working reference substance | 20141206 | 0.49mg/mL | Sealing at-20 deg.C |
| rhPTH injection | 01-20170322-01 | 0.25mg/mL | Refrigerating and sealing |
| rhPTH protein stock solution | P20161201 | 1.43mg/mL | Sealing at-20 deg.C |
Preparation of solutions
Prescription buffer solution: 8mmol/L acetate buffer solution (pH4.0), 45.4mg/mL mannitol, 3mg/mL m-cresol mixed solution, and storing at 4 ℃ for later use;
250 μ g/mL working control solution: taking 200 mu L of rhPTH as a reference substance, adding 192 mu L of water, mixing uniformly, and accurately diluting to 250 mu g/mL.
Stock solution test sample: the rhPTH protein stock solution prepared in a process chamber with 100 mu L is accurately diluted to 250 mu g/mL by adding 472 mu L of water to be used as a stock solution test sample.
System applicability sample: lmL working control was added with 1M HCl to adjust pH to 3.0, and the mixture was subjected to a water bath at 90 ℃ for 2 hours, and diluted with water to 0.25mg/mL to prepare a system compatibility solution for system compatibility confirmation. The results of the specificity experiments are shown in table 10.
TABLE 10 results of the specificity experiments
As can be seen from Table 10, the reversed phase analysis method has good specificity for detecting rhPTH, the average value of the main peak tailing factor is 1.30, and the auxiliary materials in the rhPTH injection have no interference on the purity determination of the main drug. And 3D scanning the system applicability solution by using a PDA detector, wherein the main peak purity 1 angles of the sample are all smaller than the purity 1 threshold value, which indicates that the main peak purity is higher and is a single component. The results of detection of the detection limit are shown in Table 11.
TABLE 11 detection limit test results
As can be seen from Table 11, the mean value of the three-probe SNR of the rhPTH subjected to gradient dilution is 8.6 and is greater than 3:1 when the working reference sample concentration is 4 μ g/mL, and the chromatographic peak is not detected when the working reference sample concentration is 2 μ g/mL, so that the concentration of the 4 μ g/mL sample can be determined as the detection limit of the method.
rhPTH protein stock was selected and 6 replicates were run to perform the repeat tests and the intermediate precision tests, the results of which are shown in table 12 and the intermediate precision tests are shown in table 13.
TABLE 12 results of repeated experiments
As can be seen from table 12, the relative standard deviation RSD of the purity of the main peak is 0.009 ≦ 2% after 6 consecutive repetitions of the experiment, so the reproducibility satisfied the validation requirements.
TABLE 13 results of intermediate precision experiments
As can be seen from table 13, the relative standard deviation RSD of the purity of the main peak is 0.096 ≦ 2% through 6 consecutive repeated experiments, so the intermediate precision meets the validation requirement.
The durability of the system was examined by changing the conditions of mobile phase, pH, column temperature and the like, and the results are shown in table 14.
TABLE 14 System durability test results
As can be seen from Table 14, when the detection conditions are changed, the main peak purity detection result is not affected by the flow rate, the column temperature, the pH value of the mobile phase and the salt ion concentration, the RSD is less than or equal to 2%, the separation degree R is greater than 1.5, the tailing factors are all between 0.8 and 1.5, and the system durability is good.
Experimental example 3
The detection result of the main peak purity of the experimental example is not influenced by flow velocity, column temperature, mobile phase pH value and salt ion concentration, RSD is less than or equal to 2%, the separation degree R is greater than 1.5, tailing factors are all located between 0.8 and 1.5, and the system durability is good.
In summary, the detection method and purity analysis of the rhPTH protein or protein preparation provided by the embodiment of the present invention can provide a better experimental result through high performance liquid chromatography, and the detection and analysis method has better specificity, lower detection limit, can provide sensitive detection, and has better repeatability, better intermediate precision, and higher system durability of the whole detection method; the detection method of the rhPTH protein or the protein preparation and the analysis method of the purity are proved to have higher practicability and higher popularization and application values.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. A detection method of rhPTH protein or a rhPTH protein preparation is characterized by comprising the following steps:
preparing rhPTH protein or a rhPTH protein preparation into a solution to be detected, injecting the solution to be detected into a high performance liquid chromatograph, and detecting under the following conditions:
chromatographic conditions are as follows: the chromatographic column is a reversed phase chromatographic column, and the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 8-15:85-92, and the mobile phase B is prepared by mixing an acetonitrile solution and a sodium sulfate solution according to a volume ratio of 45-55: 45-55;
the pH value of the mobile phase A and the mobile phase B is 1.9-2.5;
elution conditions: elution time and volume content of the mobile phase B during the elution time were as follows: 0-5min, 0-25%; 5-15min, 25% -35%; 15-65min, 35% -50%; 65-70min, 50% -100%; 70-72min, 100%; 72-73min, 100% -0; 73-85min, 0%;
the detection wavelength is 210-220 nm;
the reverse phase chromatographic column is a C18 column or a C8 column.
2. The method of claim 1, wherein the flow rates of mobile phase a and mobile phase B are 0.8mL/min to 1.3 mL/min.
3. The method for detecting rhPTH protein or rhPTH protein preparation of claim 1, wherein the amount of the test solution to be added is 45 to 60. mu.L.
4. The method of detecting rhPTH protein or rhPTH protein formulation of claim 1, wherein the column temperature of said chromatographic column is 38 ℃ -44 ℃.
5. The method for detecting rhPTH protein or rhPTH protein preparation of claim 1, wherein the concentration of said test solution is from 0.2mg/mL to 0.3 mg/mL.
6. The method of claim 5, wherein the test solution is prepared from a test stock solution prepared from the rhPTH protein or the rhPTH protein preparation.
7. The method of claim 6, wherein the concentration of the test stock solution is from 2mg/mL to 3 mg/mL.
8. An analysis method for purity of rhPTH protein or rhPTH protein preparation is characterized in that a gradient concentration solution of a rhPTH protein standard product and a solution of a to-be-detected rhPTH protein or rhPTH protein preparation are prepared, chromatographic detection is carried out according to the detection method of the rhPTH protein or the rhPTH protein preparation of any one of claims 1-7, and a purity result of the to-be-detected rhPTH protein or the to-be-detected rhPTH protein preparation is calculated according to the result of the chromatographic detection.
9. The method of analyzing purity of rhPTH protein or rhPTH protein preparation of claim 8, further comprising obtaining a standard curve of said rhPTH protein standard.
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| CN1706947A (en) * | 2004-06-04 | 2005-12-14 | 南京大学生物制药工程研究中心 | Construction, expression and purification method of recombinant human parathyroid hormone in colibacillus |
| CN100484958C (en) * | 2006-03-31 | 2009-05-06 | 东莞太力生物工程有限公司 | Fusion protein containing haman parathyroxin 1-34 and its expression vector |
| WO2009019715A1 (en) * | 2007-08-09 | 2009-02-12 | Usv Limited | Novel orthogonal process for purification of recombinant human parathyroid hormone (rhpth) (1-34) |
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