CN107941983A - A kind of rhPTH albumen or the detection method of rhPTH protein formulations and the analysis method of purity - Google Patents
A kind of rhPTH albumen or the detection method of rhPTH protein formulations and the analysis method of purity Download PDFInfo
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Abstract
The present invention provides a kind of rhPTH albumen or the detection method of rhPTH protein formulations and the analysis method of purity, belong to medical test and Pharmaceutical Analysis field.The condition such as the detection method of rhPTH albumen or rhPTH protein formulations provided by the invention, proportioning, the concentration of sample, detection time, Detection wavelength and column temperature by optimizing mobile phase, further improves the specificity, test limit and durability of detection；Carrying out purity analysis using the detection method can also have more accurate testing result good and reliably detect data, have larger actual application value.
Description
Technical field
The present invention relates to medical test and Pharmaceutical Analysis field, in particular to a kind of rhPTH albumen or rhPTH eggs
The detection method of white preparation and the analysis method of purity.
Background technology
In the development process of medicine, the detection method of protein content and purity is the important of drug screening and quality control
Evaluating, this method must meet the requirement in terms of specificity, precision, accuracy method validation by method validation,
It so just can guarantee that the reliability of testing result, screening and the control of product quality for instructing medicine could be used for.
Human parathyroid hormone is one of important hormone for maintaining body blood calcium balance, and the effect of its basic physiological is to pass through increasing
Add osteoblast number, it is the active fragment of human endogenous parathyroid hormone to promote bone information and growth, rhPTH, daily note
Penetrate once can increase aggregation of the new bone in cancellous bone and cortical bone surface by stimulating osteoblast activity.
Since rhPTH products are in the development phase, currently for the detection method specificity of rhPTH contents and purity, inspection
Limit, durability and precision are surveyed all not enough, it is necessary to further improve.
The content of the invention
The first object of the present invention is to provide a kind of detection method of rhPTH albumen or rhPTH protein formulations, by this
Detection method, can preferably improve the specificity, test limit and precision of detection, there is provided accurately and reliably detect data.
The second object of the present invention is to provide a kind of analysis method of rhPTH albumen or rhPTH protein formulation purity, leads to
The analysis method is crossed, can preferably analyze the purity and content information of different samples, is preferably believed for offers such as quality controls
Breath ensures.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
A kind of detection method of rhPTH albumen or rhPTH protein formulations, comprises the following steps：
RhPTH albumen or rhPTH protein formulations are configured to solution to be measured, solution to be measured is injected into high performance liquid chromatography
Instrument, is detected in the following conditions：
Chromatographic condition：Chromatographic column is reverse-phase chromatographic column, and mobile phase includes mobile phase A and Mobile phase B, wherein, mobile phase A master
Will be by acetonitrile solution and metabisulfite solution according to 8-15:The volume ratio of 85-92 is mixed to prepare, and Mobile phase B is mainly by acetonitrile solution
With metabisulfite solution according to 45-55:45:55 volume ratios are mixed to prepare；
The pH value of mobile phase A and Mobile phase B is 1.9-2.5；
Elution requirement：The volume content of Mobile phase B is as follows in elution time and elution time：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%；
Detection wavelength is 210-220nm.
A kind of analysis method of rhPTH albumen or rhPTH protein formulation purity, configures the gradient of rhPTH protein standard substances
The solution of strength solution and rhPTH albumen or rhPTH protein formulations product to be checked, according to above-mentioned rhPTH albumen or rhPTH eggs
The detection method of white preparation carries out chromatography detection, and rhPTH albumen or rhPTH albumen systems is calculated according to the result that chromatography detects
The purity result of agent product to be checked.
Compared with prior art, beneficial effects of the present invention are：RhPTH albumen provided by the invention or rhPTH albumen systems
The condition such as the detection method of agent, proportioning, the concentration of sample, detection time, Detection wavelength and column temperature by optimizing mobile phase, into
One step improves specificity, test limit and the durability of detection；More essence can also be had by carrying out purity analysis using the detection method
True testing result is good and reliably detects data, has larger actual application value.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair
The restriction of scope, for those of ordinary skill in the art, without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the chromatogram for the existing detection method that experimental example 1 of the present invention provides；
Fig. 2 is the chromatogram that the original that experimental example 1 of the present invention provides grinds medicine；
Fig. 3 is the chromatogram for the elution requirement adjustment that experimental example 1 of the present invention provides.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
The conventional products that can be obtained by commercially available purchase.
Below to the detection method of a kind of rhPTH albumen or rhPTH protein formulations and point of purity of the embodiment of the present invention
Analysis method is specifically described.
A kind of detection method of rhPTH albumen or rhPTH protein formulations, comprises the following steps：
RhPTH albumen or rhPTH protein formulations are configured to solution to be measured, solution to be measured is injected into high performance liquid chromatography
Instrument, is detected in the following conditions：
Chromatographic condition：Chromatographic column is reverse-phase chromatographic column, and mobile phase includes mobile phase A and Mobile phase B, wherein, mobile phase A master
Will be by acetonitrile solution and metabisulfite solution according to 8-15:The volume ratio of 85-92 is mixed to prepare, and Mobile phase B is mainly by acetonitrile solution
With metabisulfite solution according to 45-55:45:55 volume ratios are mixed to prepare；
The pH value of mobile phase A and Mobile phase B is 1.9-2.5；
Elution requirement：The volume content of Mobile phase B is as follows in elution time and elution time：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%；
Detection wavelength is 210-220nm.
Further, in presently preferred embodiments of the present invention, the flow velocity of mobile phase A and Mobile phase B is 0.8mL/min-
1.3mL/min。
Further, in presently preferred embodiments of the present invention, reverse-phase chromatographic column is C18 columns or C8 columns.
Further, in presently preferred embodiments of the present invention, the sample-adding amount of solution to be measured is 45-60 μ L.
Further, in presently preferred embodiments of the present invention, the column temperature of chromatographic column is 38 DEG C -44 DEG C.
Further, in presently preferred embodiments of the present invention, the concentration of solution to be measured is 0.2mg/mL-0.3mg/mL.
Further, in presently preferred embodiments of the present invention, surveying solution has test mother liquor to be made, and test mother liquor is by rhPTH eggs
White or rhPTH albumen systems are made.
Further, in presently preferred embodiments of the present invention, the 2mg/mL-3mg/mL of mother liquor is tested.
A kind of analysis method of rhPTH albumen or rhPTH protein formulation purity, configures the gradient of rhPTH protein standard substances
The solution of strength solution and rhPTH albumen or rhPTH protein formulations product to be checked, according to above-mentioned rhPTH albumen or rhPTH eggs
The detection method of white preparation carries out chromatography detection, and rhPTH albumen or rhPTH albumen systems is calculated according to the result that chromatography detects
The purity result of agent product to be checked.
Further, in presently preferred embodiments of the present invention, the acquisition of the standard curve of rhPTH protein standard substances is further included.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides the detection method of a kind of rhPTH albumen or rhPTH protein formulations, specific detecting step are as follows：
RhPTH albumen to be checked or rhPTH protein formulations are configured to test mother liquor by 1.1, and the concentration for testing mother liquor is 2mg/
mL；
1.2 are diluted to the solution to be measured of rhPTH albumen or rhPTH protein formulations by mother liquor is tested；The concentration of solution to be measured
For 0.2mg/mL；
1.3 mobile phases include mobile phase A and Mobile phase B, and mobile phase A is mainly by acetonitrile solution and metabisulfite solution according to 8：
92 volume ratio is mixed to prepare, and the pH value of mobile phase A is 1.9, Mobile phase B mainly by acetonitrile solution and metabisulfite solution according to
45:55 volume ratios are mixed to prepare, and the pH value of Mobile phase B is 2.5；
45 μ L solution to be measured are added in chromatographic column in the detection of 210nm by 1.4 selection C18 columns as reverse-phase chromatographic column
It is detected under wavelength, 38 DEG C of column temperature；
The flow velocity of 1.5 mobile phases is 0.8mL/min.
Elution requirement is：The volume content of Mobile phase B is as follows in elution time and elution time：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%.
Embodiment 2
The present embodiment provides the detection method of a kind of rhPTH albumen or rhPTH protein formulations, specific detecting step are as follows：
RhPTH albumen to be checked or rhPTH protein formulations are configured to test mother liquor by 1.1, and the concentration for testing mother liquor is 3mg/
mL；
1.2 are diluted to the solution to be measured of rhPTH albumen or rhPTH protein formulations by mother liquor is tested；The concentration of solution to be measured
For 0.3mg/mL；
1.3 mobile phases include mobile phase A and Mobile phase B, mobile phase A mainly by acetonitrile solution and metabisulfite solution according to
15：85 volume ratio is mixed to prepare, and the pH value of mobile phase A is 2.5, and Mobile phase B is mainly pressed by acetonitrile solution and metabisulfite solution
According to 55:45 volume ratios are mixed to prepare, and the pH value of Mobile phase B is 1.9；
60 μ L solution to be measured are added in chromatographic column in the detection ripple of 220nm by 1.4 selection C8 columns as reverse-phase chromatographic column
It is detected under long, 44 DEG C of column temperatures；
The flow velocity of 1.5 mobile phases is 1.3mL/min.
Elution requirement is：The volume content of Mobile phase B is as follows in elution time and elution time：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%.
Embodiment 3
The present embodiment provides the detection method of a kind of rhPTH albumen or rhPTH protein formulations, specific detecting step are as follows：
RhPTH albumen to be checked or rhPTH protein formulations are configured to test mother liquor by 1.1, and the concentration for testing mother liquor is
2.5mg/mL；
1.2 are diluted to the solution to be measured of rhPTH albumen or rhPTH protein formulations by mother liquor is tested；The concentration of solution to be measured
For 0.25mg/mL；
1.3 mobile phases include mobile phase A and Mobile phase B, mobile phase A mainly by acetonitrile solution and metabisulfite solution according to
10：90 volume ratio is mixed to prepare, and the pH value of mobile phase A is 2.3, and Mobile phase B is mainly pressed by acetonitrile solution and metabisulfite solution
According to 50:50 volume ratios are mixed to prepare, and the pH value of Mobile phase B is 2.3；
50 μ L solution to be measured are added in chromatographic column in the detection of 210nm by 1.4 selection C18 columns as reverse-phase chromatographic column
It is detected under wavelength, 40 DEG C of column temperatures；
The flow velocity of 1.5 mobile phases is 1.0mL/min.
Elution requirement is：The volume content of Mobile phase B is as follows in elution time and elution time：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%..
Embodiment 4
The present embodiment provides a kind of rhPTH albumen or the analysis method of rhPTH protein formulation purity, comprise the following steps that：
The gradient concentration solution and rhPTH albumen or rhPTH protein formulations of 1.1 configuration rhPTH protein standard substances are to be checked
The solution of product；
1.2 detection methods provided according to embodiment 3 carry out chromatography detection；
The standard curve of rhPTH protein standard substances is calculated in 1.3 results detected according to chromatography；
1.4 are calculated rhPTH albumen or rhPTH protein formulations according to the standard curve technology of rhPTH protein standard substances
The purity of product to be checked.
Experimental example 1
Optimal Experimental of this experimental example to rhPTH detection methods.
(1) existing rhPTH purity of protein and related substance detecting method
Waters Symmetry 300C18 (3.9mm ID × 150mm, 5 μm,), Part.No.WAT106154
Mobile phase A：+ 0.1% trifluoroacetic acid of+5% acetonitrile of 95% water, Mobile phase B：+ 0.1% trifluoro of+95% acetonitrile of 5% water
Acetic acid
Analysis condition is：Mobile phase A and Mobile phase B gradient elution；Detecting instrument：HPLC, PDA detector；30 DEG C of column temperature,
Detection wavelength 220nm, records time 35min, 10 μ g of sample size (40 μ L), elution requirement is as shown in table 1.
1 existing chromatographic elution conditions of table
| 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 |
Corresponding chromatogram is as shown in Figure 1.
(2) rhPTH originals grind medicine quality standard moderate purity and related substance detecting method
Analyze chromatographic column：Agilent Zorbax 300SB-C18(4.6×150mm、5μm、),
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；
Analysis condition is：Mobile phase A and Mobile phase B gradient elution；Detecting instrument：HPLC, PDA detector；40 DEG C of column temperature,
Detection wavelength 214nm, records time 100min, 12.5 μ g of sample size (50 μ L), elution requirement is as shown in table 2.
Table 2rhPTH originals grind chromatographic condition
| 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 of chromatography detection is as shown in Figure 2.
(3) gradient optimization is carried out on the basis of original grinds medicine detection method：
Analyze chromatographic column：Agilent Zorbax 300SB-C18(4.6×150mm、5μm、),
Part.No.863973-902；
Mobile phase A：0.2M Na2SO4(pH2.3):Acetonitrile=90:10；Mobile phase B：0.2M Na2SO4(pH2.3):Second
Nitrile=50:50；
Analysis condition is：Mobile phase A and Mobile phase B gradient elution；Detecting instrument：HPLC, PDA detector；40 DEG C of column temperature,
Detection wavelength 214nm, records time 100min, 12.5 μ g of sample size (50 μ L)；Condition is respectively as shown in table 3-7.
Table 3 adjusts 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 adjusts 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 adjusts gradient
Table 6 adjusts 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 adjusts 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 of gradient adjustment is as shown in Figure 3.
From the separating degree (R of retention time, peak area, purity, main peak and impurity>1.5), tailing factor (0.8-1.5) and
The different chromatography testing results of dimensional comparison such as theoretical cam curve (being not less than 10000).The results are shown in Table 8.
The chromatographic results of 8 distinct methods of table compare
As can be seen from Table 8, the method for anti-phase C18 existing compared to this product, original grind medicine rhPTH in relation to the anti-phase of material
The flowing of C18 methods has been added 200mM Na2SO4Afterwards, peak type is effectively improved, tailing factor is close to 1.0, two chromatographic columns
It is closely sized to, Agilent chromatographic column particle diameter 3.5um, has improvement in separating degree.Therefore, by optimization, preferably chromatostrip is obtained
Part：Using Agilent chromatographic columns, by the adjustment of 5 gradients, the method total run time after optimization is 85min.Purity
Close with the result of existing anti-phase C18, for the separating degree before and after main peak all more than 1.5, theoretical cam curve is more than 10000.
Experimental example 2
This experimental example carries out rhPTH albumen and rhPTH albumen related preparations specificity, precision, test limit and durable
Property detection and analysis.The method of chromatographic condition, analysis method reference implementation example 3.Sample is as shown in table 9.
Table 9rhPTH albumen and rhPTH albumen related preparations
| Title | Lot number | Specification | Storage requirement |
| RhPTH working reference substances | 20141206 | 0.49mg/mL | - 20 DEG C, sealing |
| RhPTH parenteral solutions | 01-20170322-01 | 0.25mg/mL | Refrigeration, sealing |
| RhPTH albumen stostes | P20161201 | 1.43mg/mL | - 20 DEG C, sealing |
It is prepared by solution
Prescription buffer solution：8mmol/L acetate buffers (pH4.0), 45.4mg/mL mannitol, 3mg/mL metacresols mix
Solution is closed, 4 DEG C save backup；
250 μ g/mL working reference substance solution：Take 200 μ L rhPTH to compare product, add 192 μ L water to mix, exact dilution is extremely
250μg/mL。
Stoste test sample：The rhPTH albumen stostes for taking 100 μ L process chambers to prepare, add 472 μ L exact dilutions of water to 250 μ g/
ML, as stoste test sample.
System suitability sample：LmL working reference substances are taken, 1M HCl is added and is tuned into pH3.0, when 90 DEG C of water-baths 2 are small, use water
It is diluted to 0.25mg/mL and carries out system suitability confirmation as system suitability solution.Specificity experimental result is as shown in table 10.
10 specificity experimental result of table
As can be seen from Table 10, the inverse analysis method is preferable to the specificity for detecting rhPTH, and main peak tailing factor is averaged
Be worth for the auxiliary material in 1.30, rhPTH parenteral solutions it is noiseless to the purity testing of main ingredient.It is molten to system suitability with PDA detectors
Liquid carries out 3D scannings, and 1 angle of test sample main peak purity is respectively less than 1 threshold value of purity, shows that main peak purity is higher, is single component.
The testing result of test limit is as shown in table 11.
11 test limit experimental result of table
As can be seen from Table 11, gradient dilution is carried out to rhPTH main peaks, when working reference substance sample introduction concentration is 4 μ g/mL
Three pin signal-to-noise ratio average values be 8.6, more than 3:1, chromatographic peak is can't detect when working reference substance sample introduction concentration is 2 μ g/mL,
The concentration that can determine 4 μ g/mL test samples is the test limit of this method.
RhPTH albumen stostes are selected, carry out the experiment of 6 repetitions to carry out the inspection of Repeatability checking and Intermediate precision
Test, Repeatability checking result is as shown in table 12, and the inspection of Intermediate precision is as shown in table 13.
12 repeated experiment result of table
As can be seen from Table 12, the repetition experiment of continuous 6 times, the relative standard deviation RSD=0.009 of main peak purity are carried out
≤ 2%, therefore repeat sexual satisfaction verification and require.
13 Intermediate precision experimental result of table
As can be seen from Table 13, by 6 times it is continuous repeat to test, the relative standard deviation RSD=0.096 of main peak purity≤
2%, therefore Intermediate precision meets that verification requires.
System robustness is examined, by varying conditions such as mobile phase, pH value, column temperature and column temperatures, is to examine
The durability of system, inspection result are as shown in table 14.
14 system robustness experimental result of table
As can be seen from Table 14, when testing conditions change, main peak purity detecting result is from flow velocity, column temperature, mobile phase
The influence of pH value and salt ionic concentration ,≤2%, separating degree R is equal by RSD>1.5, tailing factor is respectively positioned between 0.8~1.5, is
Durability of uniting is preferable.
Experimental example 3
This experimental example main peak purity detecting result is from flow velocity, column temperature, the influence of flowing phase pH value and salt ionic concentration,
≤ 2%, separating degree R is equal by RSD>1.5, tailing factor is respectively positioned between 0.8~1.5, and system robustness is preferable.
In conclusion the detection method and purity analysis of rhPTH albumen provided in an embodiment of the present invention or protein formulation, lead to
The chromatography of efficient liquid phase is crossed, preferable experimental result can be provided, and detection and analysis method have preferable specificity,
Test limit is relatively low, can provide sensitive detection, preferably, Intermediate precision is also preferable, whole detection method for the repeatability of method
System robustness it is also higher；Illustrate the analysis of the detection method and purity of rhPTH albumen or protein formulation provided by the invention
Method, has higher practicality and higher application value.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
1. the detection method of a kind of rhPTH albumen or rhPTH protein formulations, it is characterised in that comprise the following steps：
RhPTH albumen or rhPTH protein formulations are configured to solution to be measured, the solution to be measured is injected into high performance liquid chromatography
Instrument, is detected in the following conditions：
Chromatographic condition：Chromatographic column is reverse-phase chromatographic column, and mobile phase includes mobile phase A and Mobile phase B, wherein, the mobile phase A master
Will be by acetonitrile solution and metabisulfite solution according to 8-15:The volume ratio of 85-92 is mixed to prepare, and the Mobile phase B is mainly by acetonitrile
Solution and metabisulfite solution are according to 45-55:45:55 volume ratios are mixed to prepare；
The pH value of the mobile phase A and the Mobile phase B is 1.9-2.5；
Elution requirement：The volume content of the Mobile phase B is as follows in elution time and the elution time：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%；
Detection wavelength is 210-220nm.
2. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 1, it is characterised in that the flowing
Phase A and the flow velocity of the Mobile phase B are 0.8mL/min-1.3mL/min.
3. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 2, it is characterised in that described anti-phase
Chromatographic column is C18 columns or C8 columns.
4. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 1, it is characterised in that described to be measured
The sample-adding amount of solution is 45-60 μ L.
5. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 1, it is characterised in that the chromatography
The column temperature of column is 38 DEG C -44 DEG C.
6. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 1, it is characterised in that described to be measured
The concentration of solution is 0.2mg/mL-0.3mg/mL.
7. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 6, it is characterised in that described to be measured
Solution is made by test mother liquor, and the test mother liquor is made by the rhPTH albumen or the rhPTH protein formulations.
8. the detection method of rhPTH albumen or rhPTH protein formulations according to claim 7, it is characterised in that the test
The 2mg/mL-3mg/mL of mother liquor.
A kind of 9. analysis method of rhPTH albumen or rhPTH protein formulation purity, it is characterised in that configuration rhPTH protein standards
The gradient concentration solution and rhPTH albumen of product or the solution of rhPTH protein formulations product to be checked, it is any according to claim 1-8
The detection method of rhPTH albumen or rhPTH protein formulations described in carries out chromatography detection, the result detected according to the chromatography
The purity result of the rhPTH albumen or rhPTH protein formulations product to be checked is calculated.
10. the analysis method of rhPTH albumen or rhPTH protein formulation purity according to claim 9, it is characterised in that also
The acquisition of standard curve including the rhPTH protein standard substances.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1900120A (en) * | 2006-03-31 | 2007-01-24 | 深圳市华生元基因工程发展有限公司 | Fusion protein containing haman parathyroxin 1-34 and its expression vector |
| MX2010001612A (en) * | 2007-08-09 | 2010-07-01 | Usv Ltd | Novel orthogonal process for purification of recombinant human parathyroid hormone (rhpth) (1-34). |
| CN105473610A (en) * | 2013-08-21 | 2016-04-06 | 卡迪拉保健有限公司 | Purification process for PTH |
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2018
- 2018-01-05 CN CN201810013592.9A patent/CN107941983B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1900120A (en) * | 2006-03-31 | 2007-01-24 | 深圳市华生元基因工程发展有限公司 | Fusion protein containing haman parathyroxin 1-34 and its expression vector |
| MX2010001612A (en) * | 2007-08-09 | 2010-07-01 | Usv Ltd | Novel orthogonal process for purification of recombinant human parathyroid hormone (rhpth) (1-34). |
| CN105473610A (en) * | 2013-08-21 | 2016-04-06 | 卡迪拉保健有限公司 | Purification process for PTH |
Non-Patent Citations (3)
| Title |
|---|
| CHARLES A.FROLIK 等: "Comparison of Recombinant Human PTH(1–34) (LY333334) with a C‐Terminally Substituted Analog of Human PTH‐Related Protein(1–34) (RS‐66271)：In Vitro Activity and In Vivo Pharmacological Effects in Rats", 《JOURNAL OF BONE AND MINERAL RESEARCH》 * |
| HALEH HAMEDIFAR 等: "A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli", 《 AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOG》 * |
| 陈秀 等: "HPLC法测定甲状旁腺激素（1-34）原料药的含量", 《中国药房》 * |
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