CN108387664A - Human serum albumin molecular size is distributed ultra performance liquid chromatography assay method - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of human serum albumin molecular sizes to be distributed ultra performance liquid chromatography assay method, the method includes with ultra performance liquid chromatography measure human serum albumin molecular size distribution the step of, ultra performance liquid chromatography analysis in using ACQUITY UPLC PROTEIN BEH SEC Column,1.7 μm of granularity, column length 150mm, column diameterChromatographic column, column temperature is room temperature, uses mobile phase for 0.2molL‑1, flow velocity 0.6mlmin‑1PBS buffer solution, using Waters UPLC TUV UV detector 280nm gathered datas, 10 μ l of sample feeding volume wash by force needle liquid product 200 μ l, weak to wash needle liquid product 600 μ l, quantitative loop option partial loop with needle overfill.The UPLC human serum albumin molecular size distribution determination methods that this research is established improve 10 times or more compared to former HPLC methods detection speed, and chromatography response improves 5 times or more；The Content of polymer that UPLC methods measure human serum albumin product is fast and convenient, reproducible；UPLC methods help to realize high-throughput human serum albumin poly body measurement, greatly improve human serum albumin checkability.
Description
Technical field
The present invention relates to human serum albumin determination techniques field more particularly to a kind of distribution of human serum albumin molecular size are super
High-performance liquid chromatogram determination method.
Background technology
After polymer mainly appears on Pasteur's virus inactivation technology in human serum albumin, with 60 DEG C of heatings, 10 hours diseases
The completion of malicious inactivation step, fraction albumin itself form polymer, also fraction and existing foreign protein after heating
Polymerization forms polymer, i.e., other than Pasteur's inactivation of virus influence factor, the purity of albumin can also influence Content of polymer.People
Blood albumin is mainly used for Hypoproteinemia caused by treating many reasons, wound of losing blood or the caused shock of burn etc..Country
Drug Administration's publication new edition approval and sign hair management method regulation in 2018, no matter domestic or import blood product is both needed to carry out
Approval and sign is sent out, i.e., drug inspection and abstract audit it is qualified obtain approval and sign issue licence it is bright after can carry out list marketing.Version in 2015《China
Pharmacopeia》Regulation will be to the poly of human serum albumin in three, approved import drugs registered standard or the national drug standards
Body content is detected, and content should meet approval and require.Nearest 2015~2017 human serum albumins sign and issue quantity difference
For 2415,2648,2879 batches, declares substantial amounts and rise year by year.
Foreign countries are required to control human serum albumin Content of polymer including European Pharmacopoeia, British Pharmacopoeia, Chinese Pharmacopoeia
System, to prevent Content of polymer from increasing the side reaction of initiation.Legal Inspection method such as version in 2015 at present《Chinese Pharmacopoeia》And
Foreign pharmacopeia includes《European Pharmacopoeia》、《British Pharmacopoeia》The HPLC methods that human serum albumins polymer assay method is traditional are waited,
Analytic process needs at least 60 minutes, and the time limit that this lets pass for the manufacture of cumulative year after year human serum albumin and approval and sign hair is examined is wanted
It asks generation conflict, Check-Out Time disadvantage of high cost to start to highlight, therefore especially needs to establish quick, accurate, reproducible
The new method of inspection.
Invention content
Based on the above the deficiencies in the prior art, the purpose of the present invention is to provide a kind of distributions of human serum albumin molecular size
Ultra performance liquid chromatography assay method establishes ultra performance liquid chromatography method according to molecular exclusion mechanism and measures human serum albumin point
Sub- size distribution, realize Content of polymer quickly, high throughput assay.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions：The present invention provides a kind of people's blood
Albumin molecule size distribution ultra performance liquid chromatography assay method, the method includes measuring people's blood with ultra performance liquid chromatography
The step of albumin molecule size distribution.
Wherein, ultra performance liquid chromatography analysis in using ACQUITY UPLC PROTEIN BEH SEC Column,1.7 μm of granularity, column length 150mm, column diameterThe chromatographic column of 4.6mm, column temperature are room temperature；Use mobile phase for
0.2mol·L-1, flow velocity 0.6mlmin-1PBS buffer solution；It is acquired using Waters UPLC TUV UV detector 280nm
Data；10 μ l of sample feeding volume, wash by force 200 μ l of needle liquid product, and weak needle liquid of washing accumulates 600 μ l, quantitative loop option partial
loop with needle overfill。
Further, this method further includes the preparation process of sample solution and reference material solution：
It is prepared by sample solution：According to the mark albumen concentration of human serum albumin sample, sample is diluted respectively with mobile phase
To 0.5mgmL-1、1.0mg·mL-1、2.0mg·mL-1、3.0mg·mL-1、4.0mg·mL-1、6.0mg·mL-1、8.0mg·
mL-1、10.0mg·mL-1、12.0mg·mL-1、16.0mg·mL-1、20.0mg·mL-1, totally 11 various concentrations；
It is prepared by reference material solution：It compares sample and filters standby inspection after mixing by similarly diluting according to mark albumen concentration.
Preferably, sample introduction concentration range is when ultra-performance liquid chromatography measures the distribution of human serum albumin molecular size
10mg·mL-1~16mgmL-1。
Preferably, it is 1.469min, dimer that ultra-performance liquid chromatography, which measures human serum albumin polymer retention time,
Retention time is 1.972min, and monomer peak retention time is 2.267min；The separating degree of dimer and monomer is 2.20, monomer peak
Tailing factor be 1.18.
Compared with prior art, the present invention has the advantages that：
1) UPLC measures the distribution of human serum albumin molecular size, in 10mgmL-1~16mgmL-1In concentration range (into
100 μ g of μ g~160 of sample amount) each component peak area percent keeps relative stability, and result error is small, reproducible.
2) to avoid the interference for occurring stray light under the conditions of 280nm from leading to result error, 11 sample introduction concentration
(0.5mg·mL-1~20mgmL-1) under, each component polymer, dimer, the peak height of monomer peak, peak area and sample introduction concentration
Between extraordinary linear relationship is presented, the linearly dependent coefficient square of each index is all higher than 0.9970.
3) in subsequent processes confirmation, to realize and the existing Chinese Pharmacopoeia HPLC method migrations of version in 2015 and mutual defaecation
Profit introduces human serum albumin National reference parallel determination, and as a result UPLC methods analysis human serum albumin National reference is more
Aggressiveness percentage composition result mean value is 5.62%, and relative standard deviation is 0.37% (n=10), the parallel determinations with HPLC
(5.58%) unanimously, and both meet the quality control clearance of the establishment of human serum albumin National reference, result reliability
It is high, reproducible.
4) HPLC, which confirms with UPLC method Conformance Assessments in experiment, determines 20 batches of people's blood from domestic and international 7 enterprises
As a result albumin sample shows that the relative coefficient that two methods measure polymer peak area percentage composition is 0.9960 (n=
20), there was no significant difference (P ＞ 0.05) for two methods.
Comprehensive all experiment analysis results, and preferably realize two method bridge joints and transfer, it establishes UPLC and measures people
The distribution of blood albumin molecular size uses a concentration of 12mgmL-1；The UPLC human serum albumin molecular sizes point that this research is established
Cloth (Content of polymer) assay method improves 10 times or more compared to former HPLC methods detection speed, and chromatography response improves 5
Times or more；UPLC rebuilding methods either measure human serum albumin National reference still with the method comparison of HPLC, all
Show very high accuracy, consistency；UPLC methods measure human serum albumin product Content of polymer it is fast and convenient, repeat
Property is good；UPLC methods help to realize high-throughput human serum albumin poly body measurement, greatly improve human serum albumin and examine effect
Rate.
Description of the drawings
It, below will be to required in embodiment or description of the prior art in order to illustrate more clearly of technical scheme of the present invention
The attached drawing used is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 is that UPLC methods measure human serum albumin molecular size distribution chromatogram；
Fig. 2 is that human serum albumin National reference HPLC and UPLC molecular sizes distribution chromatogram compares；
Fig. 3 is human serum albumin polymer and dimer peak area percent with sample introduction albumen concentration variation diagram；
Fig. 4 is human serum albumin monomer peak area percentage with sample introduction albumen concentration variation diagram；
Fig. 5 is the linear relationship chart of human serum albumin polymer and dimer peak height and sample introduction concentration；
Fig. 6 is the linear relationship chart of human serum albumin monomer peak height and sample introduction concentration；
Fig. 7 is the linear relationship chart of human serum albumin polymer and dimer peak area and sample introduction concentration；
Fig. 8 is the linear relationship chart of human serum albumin monomer peak area and sample introduction concentration.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art obtained under the premise of not making creative work it is all its
His embodiment, shall fall within the protection scope of the present invention.
Experimental method described in following embodiments is unless otherwise specified conventional method；The reagent and material, such as
Without specified otherwise, commercially obtain.Specific experimental method is as follows：
1. instrument and reagent
1.1 instrument
Waters ACQUITY UPLC systems, Waters TUV UV detector, 3 chromatographies of Waters Empower are soft
Part.
1.2 reagent
Sodium dihydrogen phosphate (NaH2PO4·2H2O), disodium hydrogen phosphate (Na2HPO4·12H2O), isopropanol (HPLC grades), super
Pure water etc..
2.UPLC chromatographic conditions
Chromatographic column：ACQUITY UPLC PROTEIN BEH SEC Column (Waters),1.7 μm of granularity, column
Long 150mm, column diameter4.6mm；Column temperature is room temperature；Mobile phase is 0.2molL-1PBS buffer solution；Flow velocity 0.6ml
min-1；Waters UPLC TUV UV detector 280nm gathered datas；10 μ l of sample feeding volume；Strong needle liquid of washing accumulates 200 μ
l；Weak needle liquid of washing accumulates 600 μ l；Quantitative loop option partial loop with needle overfill.
3. sample treatment
Mark albumen concentration (200mgmL according to human serum albumin sample-1), sample is diluted to respectively with mobile phase
0.5mg·mL-1、1.0mg·mL-1、2.0mg·mL-1、3.0mg·mL-1、4.0mg·mL-1、6.0mg·mL-1、8.0mg·
mL-1、10.0mg·mL-1、12.0mg·mL-1、16.0mg·mL-1、20.0mg·mL-1, totally 11 various concentrations.The white egg of people's blood
White National reference and method are compared filters standby inspection according to mark albumen concentration with 20 batches of samples by similarly diluting after mixing.
4.UPLC measures human serum albumin sample molecule size distribution
It is 1.469min that UPLC methods, which measure human serum albumin polymer retention time, and dimer retention time is
1.972min, monomer peak retention time are 2.267min；The separating degree of dimer and monomer is 2.20, the tailing factor of monomer peak
It is 1.18.UPLC measures human serum albumin molecular size and is distributed chromatogram referring to Fig. 1.
Confirm with repeatability 5. human serum albumin National reference is measured
Research introduces human serum albumin National reference of the early period through 11 laboratory cooperation calibration, further confirms
UPLC human serum albumin polymer percentage composition credible result.The National reference presses HPLC polymer peak area percent marks
Quality Control range should be 5.44% ± 0.58% (sample introduction concentration 12mgmL when showing-1When).
6.UPLC and HPLC measures human serum albumin National reference
Content of polymer (the concentration 12mgmL of human serum albumin National reference is measured by HPLC methods-1) result is
5.58%, by newly-built UPLC methods parallel determination, its polymer mean value is 5.62% (n=10,12mgmL-1), as a result consistency
Well, meet 5.44% ± 0.58% term of reference, HPLC is compared with UPLC chromatograms referring to Fig. 2.From Figure 2 it can be seen that UPLC
Method measures human serum albumin National reference polymer, dimer, monomer peak peak sequence and basic peak type and existing 2015
Version Chinese Pharmacopoeia HPLC method spectrum forms are consistent, and 60min of the UPLC run times compared to HPLC substantially foreshortens to 5min,
And chromatographic peak response improves 5 times or more, UPLC analysis results are more accurate, quick.
The confirmation of the best sample introduction concentration ranges of 7.UPLC
11 various concentration 0.5mgmL of human serum albumin-1~20.0mgmL-1The result shows that sample introduction concentration is different,
Content of polymer peak area percent result is different, and curve graph made of the two is drawn shows as first precipitous (0.5mgmL-1~
6.0mg·mL-1) gentle (8.0mgmL afterwards-1~20.0mgmL-1) form；It is painted with concentration by dimer peak area percent
Manufactured curve is similar with polymer situation, more than 6.0mgmL-1Switch to approach platform by rising when concentration, referring to Fig. 3.
Since the calculation of area normalization method is the percentage that purpose peak accounts for each component peak area summation, old friend's blood
Albumin content of monomer shows as increasing the curve form for occurring declining with sample introduction concentration, in 10.0mgmL-1When concentrations above
Decline slows down and gradually approaches platform, referring to Fig. 4.
8. human serum albumin molecular size is distributed each component peak height and 11 sample introduction concentration linear relationships confirm
To prevent presence of the sample in ultraviolet 280nm detections due to stray light, increases in sample size or albumen concentration increases
When occur component linear relationship deviation, study to 0.5mgmL-1~20.0mgmL-1Each group swarming under 11 various concentrations
The linear relationship of high result and sample introduction concentration is confirmed.The results show that for this parameter of peak height, 11 sample introduction concentration with
Good linear correlation is presented in polymer, dimer peak height, and two components correspond to the linearly dependent coefficient square of various concentration
Respectively 0.9994 and 0.9999, polymer, dimer peak height and the linear regression equation and linear relationship chart of sample introduction concentration are joined
See Fig. 5.
Human serum albumin monomer peak peak height and 11 sample introduction concentration are also presented good linear relationship, monomer peak peak height with into
The linearly dependent coefficient square of sample concentration is 0.9971, and linear regression equation and curve graph are referring to Fig. 6.
9. human serum albumin molecular size is distributed each component peak area and 11 sample introduction concentration linear relationships confirm
Research is to 0.5mgmL under UPLC chromatographic conditions-1~20.0mgmL-1Totally 11 difference faces sample introduction concentration Xia Feng
Linear relationship between product result and concentration is confirmed, the results showed that for this parameter of peak area, polymer, dimer
Peak area and sample introduction concentration present it is linearly related well, the linearly dependent coefficient square of the two be respectively 0.9994 and
0.9998, the linear regression equation and linear relationship chart of two components and sample introduction concentration are referring to Fig. 7.Human serum albumin monomer peak face
Good linear relationship, the linearly dependent coefficient of monomer peak peak area and different sample introduction concentration is also presented with 11 sample introduction concentration in product
Square it is 0.9998, the linear regression equation and linear relationship chart of monomer peak peak area and sample introduction concentration are referring to Fig. 8.
10. the reproducibility under different sample introduction concentration conditions
Research further investigates polymer peak area percent repeatability under 11 sample introduction concentration.As a result table
It is bright, polymer peak area percent low concentration be not so good as high concentration result favorable reproducibility, 10,12,16mgmL-1Three concentration polies
Body percentage composition RSD is respectively 0.00%, 0.10%, 0.10%, and concrete outcome is referring to table 1.
1 UPLC of table measures human serum albumin sample Content of polymer peak area percent under different albumen concentration
In summary Content of polymer percentage result, each component peak height and peak area based on different sample introduction concentration with
Factors, the UPLC methods such as the linear relationship of sample introduction concentration, the reproducibility of different sample introduction concentration measure human serum albumin molecular size point
The suggestion of sample introduction concentration range is 10mgmL when cloth-1~16mgmL-1(i.e. loading total amount is 100 μ g of μ g~160).
It should be appreciated that pharmacopoeia of each country measures the concentration model that molecular size distribution can all be suggested to HPLC under normal conditions
It encloses, such as European Pharmacopoeia suggests a concentration of 4~12mgmL of sample introduction to human serum albumin HPLC Content of polymer-1, and 2005
Version Chinese Pharmacopoeia three is then 4mgmL-1, subsequent comprehensive consideration separating degree, chromatography response, with other country's pharmacopeia intercommunity etc. because
Sample introduction concentration is promoted to 12mgmL-1 by element, versions in 2010 and version Chinese Pharmacopoeia in 2015.For realize create UPLC methods with
Existing pharmacopeia HPLC methods intercommunication convenience is studied in 10mgmL-1~16mgmL-112mgmL is selected in concentration range-1
Carry out subsequent authentication.
Confirm with repeatability 11. human serum albumin National reference is measured
Research introduces human serum albumin National reference of the early period through 11 laboratory cooperation calibration, further confirms
UPLC human serum albumin polymer percentage composition credible result.The National reference presses HPLC polymer peak area percent marks
Quality Control range should be 5.44% ± 0.58% (sample introduction concentration 12mgmL when showing-1When).
11.1UPLC and HPLC measures human serum albumin National reference
By HPLC methods measure human serum albumin National reference Content of polymer (concentration 12mgmL-1) result be
5.58%, by newly-built UPLC methods parallel determination, its polymer mean value is 5.62% (n=10,12mgmL-1), as a result consistency
Well, meet 5.44% ± 0.58% term of reference.
11.2UPLC measures human serum albumin National reference repeatability and confirms
10 (12mgmL are measured to human serum albumin National reference using UPLC methods-1Concentration), carry out method repetition
Property confirm.It is computed, polymer peak retention time mean value is 1.43min, and relative standard deviation RSD is 0.05% (n=10)；Peak
High mean value is 38115 μ AU, and relative standard deviation RSD is 0.61% (n=10)；Peak area mean value is 315719 μ AUSec, phase
It is 0.58% (n=10) to standard deviation RSD；Peak area percent content mean value is 5.62%, and relative standard deviation RSD is
0.40% (n=10).
The application of 12.UPLC methods and confirm again with the consistency of version Chinese Pharmacopoeia HPLC methods in 2015
The 12mgmL tentatively established according to the studies above result-1Sample introduction concentration conditions, research have selected more samples
The uniformity comparison of carry out method.Selection comes from domestic and international 7 producers totally 20 batch human serum albumins extensively, by mark albumen
Concentration dilution is to 12mgmL-1, sample is put down using version Chinese Pharmacopoeia HPLC methods in 2015 and newly-built UPLC methods
Row measures, and the polymer percentage composition comparison result that two methods measure is referring to table 2.
2 HPLC of table and 20 batches of human serum albumin polymer peak area percent content results consistency of UPLC parallel determinations
It compares
Statistical analysis shows the phase of both Content of polymer peak area percents that two methods of HPLC and UPLC measure
Relationship number is 0.9960 (n=20), and the double tail T check analyses of two methods pairing show two methods there was no significant difference (P ＞
0.05), probability P is 0.9607 (n=20).
Conclusion：UPLC human serum albumin molecular sizes distribution (Content of polymer) assay method is examined compared to former HPLC methods
Degree of testing the speed improves 10 times or more, and chromatography response improves 5 times or more；UPLC rebuilding methods either measure human serum albumin state
The method comparison of family's reference material or multiple batches of product and HPLC, all shows very high accuracy, consistency.This hair
It is more fast and convenient, repeated more preferable that UPLC methods in bright measure human serum albumin product Content of polymer method；It can realize
High-throughput human serum albumin Content of polymer analysis, is substantially improved checkability；Domestic and international human serum albumin ultra high efficiency is filled up
The blank of liquid chromatogram multimer analysis method.
The above is the preferred embodiment of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, it is noted that for those skilled in the art, without departing from the principle of the present invention, may be used also
To make several improvement and variation, these are improved and variation is also considered as protection scope of the present invention.
Claims (8)
1. a kind of human serum albumin molecular size is distributed ultra performance liquid chromatography assay method, which is characterized in that the method packet
Include the step of measuring the distribution of human serum albumin molecular size with ultra performance liquid chromatography.
2. human serum albumin molecular size according to claim 1 is distributed ultra performance liquid chromatography assay method, feature
Be, ultra performance liquid chromatography analysis in using ACQUITY UPLC PROTEIN BEH SEC Column,Granularity
1.7 μm, column length 150mm, column diameterChromatographic column, column temperature is room temperature.
3. human serum albumin molecular size according to claim 1 is distributed ultra performance liquid chromatography assay method, feature
It is, uses mobile phase for 0.2molL-1, flow velocity 0.6mlmin-1PBS buffer solution.
4. human serum albumin molecular size according to claim 1 is distributed ultra performance liquid chromatography assay method, feature
It is, uses Waters UPLC TUV UV detector 280nm gathered datas.
5. human serum albumin molecular size according to claim 1 is distributed ultra performance liquid chromatography assay method, feature
It is, 10 μ l of sample feeding volume, wash 200 μ l of needle liquid product by force, weak needle liquid of washing accumulates 600 μ l, quantitative loop option partial
loop with needle overfill。
6. according to any human serum albumin molecular size distribution ultra performance liquid chromatography measurement side in claim 1-5
Method, which is characterized in that this method further includes the preparation process of sample solution and reference material solution：
It is prepared by sample solution：According to the mark albumen concentration of human serum albumin sample, sample is diluted to respectively with mobile phase
0.5mg·mL-1、1.0mg·mL-1、2.0mg·mL-1、3.0mg·mL-1、4.0mg·mL-1、6.0mg·mL-1、8.0mg·
mL-1、10.0mg·mL-1、12.0mg·mL-1、16.0mg·mL-1、20.0mg·mL-1, totally 11 various concentrations；
It is prepared by reference material solution：It compares sample and filters standby inspection after mixing by similarly diluting according to mark albumen concentration.
7. human serum albumin molecular size according to claim 6 is distributed ultra performance liquid chromatography assay method, feature
It is, sample introduction concentration range is 10mgmL when ultra-performance liquid chromatography measures the distribution of human serum albumin molecular size-1~
16mg·mL-1。
8. human serum albumin molecular size according to claim 6 is distributed ultra performance liquid chromatography assay method, feature
It is, it is 1.469min that ultra-performance liquid chromatography, which measures human serum albumin polymer retention time, and dimer retention time is
1.972min, monomer peak retention time are 2.267min；The separating degree of dimer and monomer is 2.20, the tailing factor of monomer peak
It is 1.18.
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| CN110618202B (en) * | 2018-06-20 | 2022-07-08 | 成都康弘生物科技有限公司 | Method for detecting protein purity |
| CN113671081A (en) * | 2021-08-19 | 2021-11-19 | 天士力生物医药股份有限公司 | Method for detecting purity of recombinant human prourokinase |
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