CN106872633B - A kind of rp-hplc analysis method of recombinant human lysozyme - Google Patents
A kind of rp-hplc analysis method of recombinant human lysozyme Download PDFInfo
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Abstract
The invention discloses a kind of recombinant human lysozyme rp-hplc analysis methods, this method using 20~50nm of aperture, 2~10 μm of grain size octadecylsilane chemically bonded silica as chromatographic column filler, not only this high molecular weight protein of recombinant human lysozyme can have been allowed to have certain retention time on column, but also the albumen will not be allowed to lead to not elute with filler in conjunction with too strong；By adding sodium chloride in mobile phase, to ensure the dissolubility of recombinant human lysozyme during rp-hplc analysis, it can be with mobile phase complete miscibility, ensure that the pedal number of chromatographic peak, symmetry, repeatability, quantitative analysis accuracy, can be used for the analysis of recombinant human lysozyme and quantitative detection.
Description
Technical field
The present invention relates to a kind of rp-hplc analysis methods of recombinant human lysozyme.
Background technology
Defined mobile phase is pumped into the chromatographic column equipped with filler by high performance liquid chromatography system using high pressure pump, right
Test sample carries out the chromatographic process of separation determination.
The test sample of injection, is brought by mobile phase in column, when the mixture flow carried in mobile phase is through stationary phase, with
Stationary phase interacts.Due to difference of each component in property and structure in mixture, generated between stationary phase
The size of active force, strong and weak different, with the movement of mobile phase, mixture is alternate by repeated multiple times partition equilibrium two,
So that each component is fixed the time difference mutually retained and realizes each group in mixture to be taken out of in a certain order by mobile phase
Point separation and sequentially enter detector, detection is recorded by integrator or data processing system and processing chromatographic signal.
Reversed-phased high performace liquid chromatographic：The polarity of mobile phase is more than the polarity of stationary phase, i.e., is mutually solid with non-polar linkage
Determine phase (often using 18 silane C18, octane C8, methyl C1, phenyl etc. as stationary phase)；Methanol, second is added in water or buffer solution
The organic solvent miscible with water such as nitrile, isopropanol, acetone, tetrahydrofuran is mobile phase to the efficient of separation detection related substances
Liquid chromatography.
It is widely used in target substance in biological medicine and protein detection analysis field, reversed-phased high performace liquid chromatographic
Analysis and quantitative detection.There is this protein of recombinant human lysozyme its special physicochemical property, inventor to find to use reverse phase
High-efficient liquid phase chromatogram technique analysis has the following problems when detecting：(1) recombinant human lysozyme molecular weight is 14000Da, molecular weight phase
To larger, often there is albumen with the analysis of common reverse phase C18 columns and lead to not elute with filler in conjunction with too strong, it can not be normal
It analyzes and damages chromatographic column；(2) recombinant human lysozyme is salting-in protein, molten in the sodium-chloride water solution of 100mM or more
Solution property is good, will present suspended state less than recombinant human lysozyme solution in the sodium-chloride water solution of 100mM；Common reverse phase is high
The sodium chloride of high concentration is not used in the mobile phase of effect liquid phase chromatogram, this will cause recombinant human lysozyme in analytic process to be in not
It is completely dissolved state, can be damaged if its dose-effect relationship and the albumen of endless fully dissolved can not be accurately reflected having chromatographic peak
Bad chromatographic system.
Invention content
The object of the present invention is to provide the reverse phases of a kind of repeatability and the high recombinant human lysozyme of accuracy of quantitative analysis
HPLC analytical method.
The technical solution that above-mentioned technical problem uses is solved to be made of following step：
1, recombinant human lysozyme standard items are dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, are prepared
At recombinant human lysozyme standard solution, standard solution is analyzed using reversed-phase high performance liquid chromatography, chromatography item
Part：Chromatographic column is C18 reverse-phase chromatographic columns, and filler is the octadecylsilane bonded silica of 20~50nm of aperture, 2~10 μm of grain size
Glue, mobile phase A are the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, Mobile phase B
It is acetonitrile and ultrapure water volume ratio is 6:Sodium chloride and volume in 4 mixed liquor and mixed liquor containing mass concentration for 0.9%
A concentration of 0.1% trifluoroacetic acid carries out gradient elution, eluent gradient A:B is by (90~0):(10~100) (50~0) are arrived:
(50~100), Detection wavelength 280nm, flow velocity are 1mL/ minutes；Chromatography main peak area is drawn with recombinant human lysozyme concentration to become
The standard curve of change.
2, recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, is used
Membrane filtration is collected filtrate, is analyzed filtrate using reversed-phase high performance liquid chromatography, chromatographiccondition is identical as step 1.
3, the chromatography main peak area obtained according to step 2 can be obtained in conjunction with the linear equation for the standard curve that step 1 determines
To the concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
In above-mentioned steps 1, the eluent gradient preferably 0 to 30 minutes A:B is by 55:45 to 5:95.
In above-mentioned steps 1, the preferred aperture 30nm of the filler, 5 μm of grain size octadecylsilane chemically bonded silica.
Beneficial effects of the present invention are as follows：
1, the present invention uses the octadecylsilane chemically bonded silica of 20~50nm of aperture, 2~10 μm of grain size as chromatographic column
Filler, can not only allow this high molecular weight protein of recombinant human lysozyme to have certain retention time on column, but also will not allow the egg
Lead to not elute with filler in conjunction with too strong in vain.
2, the present invention in mobile phase by adding sodium chloride, to ensure during rp-hplc analysis
The dissolubility of recombinant human lysozyme with mobile phase complete miscibility, can ensure that the pedal number of chromatographic peak, symmetry, again
Renaturation, quantitative analysis accuracy.
3, sodium chloride is first completely dissolved in ultra-pure water by the present invention when preparing Mobile phase B, then miscible with acetonitrile, to keep away
It is insoluble in acetonitrile to exempt from sodium chloride, used proportioning ensure that Mobile phase B good dissolubility and during global analysis
A, good compatibility between B mobile phases.
4, the repeatability of the method for the present invention is good, and accuracy is high, and detection limit is low, repeatability, system suitability, filler hole
Diameter grain size selects, and the formula of mobile phase meets《Pharmacopoeia of People's Republic of China》The three efficient liquid of annex III B of version in 2015
Phase chromatography requirement can be used for the analysis of recombinant human lysozyme and quantitative detection.
Description of the drawings
Fig. 1 is the relation curve of recombinant human lysozyme concentration and chromatography main peak area in linearity and range experiment.
Fig. 2 is the relation curve of recombinant human lysozyme concentration and chromatography main peak area in accuracy test.
Specific implementation mode
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
1, accurate weighing recombinant human lysozyme standard items 2896.0mg, wherein 150.0mg containing recombinant human lysozyme, is added matter
It measures a concentration of 0.9% sodium chloride ultra-pure water to be settled in 50.0mL volumetric flasks, it is a concentration of to be configured to recombinant human lysozyme
The standard solution of 3.0mg/mL；Then 5.0mL volumetric flasks are used, the sodium chloride ultra-pure water solution precision that mass concentration is 0.9%
8 sample concentrations are diluted, respectively：0.30mg/mL、0.60mg/mL、0.90mg/mL、1.20mg/mL、1.50mg/mL、
1.80mg/mL, 2.10mg/mL, 2.40mg/mL divide the standard solution after dilution using reversed-phase high performance liquid chromatography
Analysis, chromatographiccondition：Chromatographic column is C18 reverse-phase chromatographic columns, and filler is the octadecylsilane key of aperture 30nm, 5 μm of grain size
Silica gel is closed, mobile phase A is the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, stream
Dynamic phase B is acetonitrile and ultrapure water volume ratio is 6:In 4 mixed liquor and mixed liquor containing mass concentration be 0.9% sodium chloride and
The trifluoroacetic acid that volumetric concentration is 0.1%, carries out gradient elution, and eluent gradient selects 0 to 30 minutes A:B is by 55:45 to 5:
95, Detection wavelength 280nm, flow velocity are 1mL/ minutes, and applied sample amount is 20 μ L；It calculates the number of plates successively according to testing result, drag
The tail factor, chromatographic peak main peak area, the results are shown in Table 1.
1 recombinant human lysozyme linearity and range chromatography detection statistics result of table
The standard curve that chromatography main peak area changes with recombinant human lysozyme concentration, knot are drawn according to the result of calculation of table 1
Fruit sees Fig. 1.The linear equation of standard curve is：Y=2435x+29044, x represents recombinant human lysozyme concentration in formula, and y represents color
Compose main peak area, R2=0.99964, it is linear good, and pedal number, symmetry meet《Pharmacopoeia of People's Republic of China》2015
The requirement of year three annex III B high performance liquid chromatographies of version.
Accurate weighing recombinant human lysozyme standard items 96.53mg, wherein 5.0mg containing recombinant human lysozyme, it is dense to be added quality
Degree is settled to for 0.9% sodium chloride ultra-pure water solution in 5.0mL volumetric flasks, is configured to a concentration of 1.0mg/ of recombinant human lysozyme
The standard solution of mL；It is diluted to concentration with 5.0mL volumetric flasks, the sodium chloride ultra-pure water solution precision that mass concentration is 0.9%
Respectively：1.0×10-3mg/mL、1.0×10-4mg/mL、1.0×10-5mg/mL、1.0×10-6Mg/mL, using reversed phase high efficiency
Liquid chromatogram analyzes the standard solution after dilution, and chromatographiccondition is same as described above, and testing result is shown in Table 2.
2 recombinant human lysozyme of table detection limit and quantitative limit chromatography detection statistics result
As can be seen from Table 2, recombinant human lysozyme a concentration of 1.0 × 10-5When mg/mL, main peak retention time is 14.86min, high
Degree is 0.43mV, and baseline noise height is about 0.04mV, and signal-to-noise ratio is about 11:1, recombinant human lysozyme a concentration of 1.0 × 10- 6When mg/mL, main peak retention time is 14.86min, is highly 0.26mV, baseline noise height is about 0.04mV, and signal-to-noise ratio is about
It is 6:1, illustrate under the chromatographic condition, recombinant human lysozyme applied sample amount is linear good, recombined human in 6.0~48.0 μ g ranges
Quantifying for lysozyme is limited to 1.0 × 10-5μ L, that is, the 0.2ng of mg/mL × 20, detection are limited to 1.0 × 10-6The μ of mg/mL × 20 L are
0.02ng。
2, recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, is used
Membrane filtration is collected filtrate, is analyzed filtrate using reversed-phase high performance liquid chromatography, chromatographiccondition is identical as step 1.
3, the chromatography main peak area obtained according to step 2 can be obtained in conjunction with the linear equation for the standard curve that step 1 determines
To the concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
In order to prove that the repeatability and accuracy of analysis method of the present invention, inventor have carried out a large amount of laboratory research examination
It tests, specific test situation is as follows：
1, repetitive test
Precision prepares the standard solution of a concentration of 1.5mg/mL of recombinant human lysozyme, continuous sample introduction 8 times, each applied sample amount
20 μ L are analyzed according to the chromatographic condition of 1 step 1 of embodiment, according to corresponding main peak area numerical value, calculate standard deviation (SD
Value STDEV), arithmetic mean of instantaneous value (Xbar AVERAGE), relative standard deviation (RSD)=standard deviation (SD)/arithmetic mean of instantaneous value
(Xbar) × 100%, the relative standard deviation of peak area measurement value should be not more than 1.0%.
3 recombinant human lysozyme repetitive test result of table
4 recombinant human lysozyme repetitive test result statistical analysis of table
By the result of table 4 as it can be seen that continuous 8 sample introductions, the relative standard deviation of main peak area is 0.52715%, is less than
1.0%, illustrate that the repeatability of analysis method of the present invention is good.
2, accuracy test
Accurate weighing 2559.7mg test samples (the recombinant human lysozyme sample (20151125 batches) demarcated through content, wherein
The mass content of recombinant human lysozyme is 5.86%), wherein 150.0mg containing recombinant human lysozyme, the chlorine that mass concentration is 0.9%
Change sodium ultra-pure water solution to be settled in 50.0mL volumetric flasks, the standard items for being configured to a concentration of 3.0mg/mL of recombinant human lysozyme are molten
Liquid；5 sample concentrations are diluted with 5.0mL volumetric flasks, the sodium chloride ultra-pure water solution that mass concentration is 0.9%, respectively：
0.90mg/mL, 1.20mg/mL, 1.50mg/mL, 1.80mg/mL, 2.10mg/mL, according to the chromatographic condition of 1 step 1 of embodiment
It is analyzed, the results are shown in Table 5.
5 recombinant human lysozyme standard curve chromatography detection statistics result of table
According to the testing result of table 5, the standard curve that chromatography main peak area changes with recombinant human lysozyme concentration is drawn, knot
Fruit sees Fig. 2.The linear equation of standard curve is：Y=2306.9x+222118.3, x represents recombinant human lysozyme concentration, y in formula
Represent chromatography main peak area, R2=0.99924, it is linear good.
The above-mentioned test sample 170.65mg of accurate weighing, wherein 10.0mg containing recombinant human lysozyme, mass concentration, which is added, is
0.9% sodium chloride ultra-pure water solution is settled in 10.0mL volumetric flasks, is configured to a concentration of 1.0mg/mL of recombinant human lysozyme
Test solution is distributed into 1mL/ branch (every 1.0mg containing recombinant human lysozyme, A value) with pipette, distinguishes again in every sample
17.15mg test samples (1.0mg containing recombinant human lysozyme, B value) are added in accurate weighing, totally 7 samples, by 1 step 1 of embodiment
Chromatographic condition is analyzed.According to the chromatography main peak area and standard curve y=2306.9x+222118.3 measured, actual measurement is acquired
Concentration obtains measured value C, and recovery of standard addition is calculated according to following formula：
Rate of recovery %=(C-A)/B × 100%
A is recombinant human lysozyme component contained by test sample in formula；B is that reference substance amount is added；C is measured value.It the results are shown in Table
6。
6 recombinant human lysozyme chromatographic detection Accuracy Verification statistical result of table
By table 6 as it can be seen that the accuracy of the method for the present invention is 95.8%~100.0%, it is accurate to meet Chinese Pharmacopoeia 2015 editions
The requirement 95%~102% is spent, the detection of recombinant human lysozyme content is can be used for.
Claims (2)
1. a kind of rp-hplc analysis method of recombinant human lysozyme, it is characterised in that it is made of following step：
（1）Recombinant human lysozyme standard items are dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, are configured to weight
Group human lysozyme standard solution, analyzes standard solution using reversed-phase high performance liquid chromatography, chromatographiccondition：Color
Spectrum column is C18 reverse-phase chromatographic columns, and filler is the octadecylsilane chemically bonded silica of 20~50 nm of aperture, 2~10 μm of grain size, stream
Dynamic phase A is the ultra-pure water solution that sodium chloride mass concentration is 0.9%, trifluoroacetic acid volumetric concentration is 0.1%, and Mobile phase B is acetonitrile
It is 6 with ultrapure water volume ratio:In 4 mixed liquor and mixed liquor containing mass concentration be 0.9% sodium chloride and volumetric concentration be
0.1% trifluoroacetic acid, carries out gradient elution, and eluent gradient selects 0 to 30 minutes A:B is by 55:45 to 5:95, Detection wavelength
For 280 nm, flow velocity is 1 mL/ minutes；Draw the standard curve that chromatography main peak area changes with recombinant human lysozyme concentration；
（2）Recombinant human lysozyme sample to be measured is dissolved in the sodium chloride ultra-pure water solution that mass concentration is 0.9%, uses filter membrane
Filtering is collected filtrate, is analyzed filtrate using reversed-phase high performance liquid chromatography, chromatographiccondition and step（1）It is identical；
（3）According to step（2）Obtained chromatography main peak area, in conjunction with step（1）The linear equation of determining standard curve
Obtain the concentration of recombinant human lysozyme in recombinant human lysozyme sample to be measured.
2. the rp-hplc analysis method of recombinant human lysozyme according to claim 1, it is characterised in that：
Step（1）In, the filler is the octadecylsilane chemically bonded silica of 30 nm of aperture, 5 μm of grain size.
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| CN111751468A (en) * | 2020-07-03 | 2020-10-09 | 上海艾魁英生物科技有限公司 | Method for purifying lysozyme dimer and preparing standard substance thereof |
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