CN104458969B - The assay method of Triton X-100 residual quantity in the HBsAg stoste that recombinant Saccharomyces cerevisiae is expressed - Google Patents
The assay method of Triton X-100 residual quantity in the HBsAg stoste that recombinant Saccharomyces cerevisiae is expressed Download PDFInfo
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Abstract
Does the present invention disclose Triton in the HBsAg stoste that a kind of recombinant Saccharomyces cerevisiae expresses? the assay method of X-100 residual quantity, include reference substance solution preparation steps, product to be tested solution preparation step and Triton? X-100 determination step, described product to be tested solution preparation step comprises: the protein content in every group of product to be tested is detected, screen out protein content and be greater than the product to be tested that the product to be tested of a threshold value and protein content are less than described threshold value; Pass through solid-phase extraction column with 1 part of water respectively, wash for the first time assorted to every group of product to be tested; Protein content is greater than to the product to be tested of described threshold value, then with 1 part of water by solid-phase extraction column, again wash assorted to every group of product to be tested; The methanol solution that is 2%～70% by 1 part of volumetric concentration, by solid-phase extraction column, is washed assorted to every group of product to be tested for the third time. Can the present invention effectively remove protein macromolecule material in stoste and other interfering materials to Triton? the impact of the X-100 determination of residual amount, the rate of recovery is high, can measure exactly the Triton in stoste? X-100 residual quantity, its detectability can arrive 1 μ g/ml level.
Description
Technical field
The present invention relates to the preparation technique of recombination engineering hepatitis B vaccine, relate in particular to the assay method of TritonX-100 residual quantity in the HBsAg stoste that a kind of recombinant Saccharomyces cerevisiae expresses.
Background technology
China is the high Endemic Area of hepatitis B, every annual report morbidity people more than 50 ten thousand examples, and HB vaccination is effective, the most most economical method of prevention hepatitis B. Utilize recombinant Saccharomyces cerevisiae to express hepatitis B surface antigen (hepatitisBsurfaceantigen, HBsAg) large-scale industrial production hepatitis B vaccine, be a kind of culture medium, cost is low, productivity ratio is high hepatitis B vaccine production technology easily obtained, can meet the wilderness demand of China to hepatitis B vaccine.
Triton X-100 (TritonX-100) is a kind of nonionic surface active agent (or claiming detergent), and its energy solubilizing lipids, to increase the dissolubility of lipoprotein.
After the fermentation of recombinant Saccharomyces cerevisiae bacterium, through broken, purifying, obtain the HBsAg stoste for preparing hepatitis B vaccine, the protein content of HBsAg stoste is 20 μ g/ml～250 μ g/ml. In described purifying process, add TritonX-100 as albumen extract or eluant, eluent, and after antigen purification, needed to remove wherein residual TritonX-100. TritonX-100 residual quantity in HBsAg stoste is one of key index of stock solution quality control.
Regulation in existing " Chinese pharmacopoeia ", in recombinant hepatitis B vaccine production technology sample, the assay method of TritonX-100 residual quantity is that phenol reacts generation white opacity material with TritonX-100, adopts spectrophotometry. The method detectability can only reach 10 μ g/ml, and protein in sample has interference to TritonX-100 assay.
Adopting high performance liquid chromatography, go out the content of TritonX-100 according to the calculated by peak area of product to be tested, is the effective ways of TritonX-100 residual quantity in a kind of comparatively advanced detection of biological goods. This method is reproducible, accuracy is high, it is easy to save time, and has been applied to measuring the TritonX-100 residual quantity in blood plasma product, antitoxin/antiserum, the multiple biological products such as fine former.
Before adopting the content of TritonX-100 in some sample solutions that contain impurity of high effective liquid chromatography for measuring; in order to protect the accuracy of liquid-phase chromatographic column and guarantee measurement result; also need sample solution to carry out pretreatment, remove the interfering material in sample solution. In the prior art, to the goods such as blood plasma, antitoxin/antiserum, after the activation of employing solid-phase extraction column, sample solution is carried out loading, washes assorted, wash-out processing, can effectively remove the interfering material in sample solution.
But, inventor finds in research process of the present invention, the hepatitis B surface antigen that recombinant Saccharomyces cerevisiae bacterium is expressed, purified technique obtains the HBsAg stoste for preparing vaccine, its protein content is between 20 μ g/ml～250 μ g/ml, as macromolecular substances, the protein content of high concentration not only can produce severe jamming to the accuracy of high effective liquid chromatography for measuring TritonX-100 residual quantity, and, the solid phase extraction of prior art wash assorted method, can not effectively remove impurity disturbs, simultaneously in Syrups by HPLC step, the solvent of preparation reference substance is water, and product to be tested solution is methanol solution, system is inconsistent, affect accuracy.
Summary of the invention
Technical problem solved by the invention is, the assay method of TritonX-100 residual quantity in the HBgAs that a kind of recombinant Saccharomyces cerevisiae expresses is provided, and the method rate of recovery is high, detectability is low, can realize the Accurate Determining of TritonX-100 residual quantity in stoste.
In order to solve the problems of the technologies described above, the invention discloses following scheme:
The assay method of TritonX-100 residual quantity in the HBsAg stoste that a kind of recombinant Saccharomyces cerevisiae is expressed, include reference substance solution preparation steps, product to be tested solution preparation step and TritonX-100 determination step, in described product to be tested solution preparation step, successively by solid-phase extraction column activation, loading, wash assorted and elution step, product to be tested is carried out to SPE processing, before described loading step, also comprise:
Protein content detecting step, from HBsAg stoste to be detected, taking by volume every part as unit, get after some groups of products to be tested, protein content in every group of product to be tested is detected, screen out protein content and be greater than the product to be tested that the product to be tested of a threshold value and protein content are less than described threshold value, the span of wherein said threshold value is 70 μ g/ml-125 μ g/ml;
And described in wash assorted step and comprise:
Once wash assorted step, pass through solid-phase extraction column with 1 part of water respectively, wash for the first time assorted to every group of product to be tested;
Secondary is washed assorted step, protein content is greater than to the product to be tested of described threshold value, once washes after assorted step described, passes through solid-phase extraction column more respectively with 1 part of water, again washes assorted to every group of product to be tested;
Wash assorted step three times, wash assorted step/the second wash after assorted step described first, the methanol solution that is 2%～70% by 1 part of volumetric concentration respectively, by solid-phase extraction column, is washed assorted to every group of product to be tested for the third time.
Preferably, the value of described threshold value is 100 μ g/ml.
Preferably, described elution step comprises:
Carry out wash-out by solid-phase extraction column with 1 part of 90%～100% methyl alcohol, collect eluent, obtain product to be tested solution.
Preferably, described TritonX-100 determination step comprises:
Liquid chromatogram measuring step, carries out high-performance liquid chromatogram determination to reference substance solution described in every group and product to be tested solution respectively;
Establishing equation and solution procedure, in peak area by reference substance solution and reference substance solution, the concentration of TritonX-100 is carried out linear regression, Criterion curvilinear equation, by the peak area substitution calibration curve equation of product to be tested solution, calculates the residual quantity of TritonX-100 in product to be tested.
Preferably, in described liquid chromatogram measuring step, the chromatographic column of employing is WatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, taking methanol aqueous solution as mobile phase, adopt diode array detector monitoring, and the analytical wavelength range of TritonX-100 is at 220nm～240nm.
Preferably, in described methanol aqueous solution mobile phase, methyl alcohol is 70:30～85:15 with the ratio of water.
Preferably, methanol aqueous solution flows while being in a ratio of 80:20, and the flow rates of mobile phase is between 0.6ml/～1.0ml/ minute.
Preferably, described reference substance solution preparation steps comprises:
With 90% methanol solution preparation TritonX-100 reference substance solution, make the reference substance solution of the TritonX-100 of some groups of 1～20 μ g/ml.
Preferably, described activation step comprises:
Choose solid-phase extraction column, and pass through successively 1 part of methyl alcohol and 1 part of water in this solid-phase extraction column, carry out preactivated to it;
Described loading step comprises:
In solid-phase extraction column after respectively every group of product to be tested loading extremely being activated;
And in described product to be tested solution preparation step, liquid is no faster than 1ml/ minute by the flow velocity of solid-phase extraction column.
Preferably, in described activation step, the solid-phase extraction column of choosing is WatersHLB1cc solid-phase extraction column.
The invention has the beneficial effects as follows:
Embodiments of the invention are by being optimized SPE, before loading, detect the protein content in product to be tested, screen out the product to be tested of protein content higher than 100 μ g/ml left and right, and it is carried out to secondary and wash assorted, and utilize rp-hplc determination TritonX-100 content, thereby reached, the detectability of TritonX-100 is reduced to 1 μ g/ml, can measures exactly the effect of TritonX-100 residual quantity in stoste.
Detailed description of the invention
Describe an embodiment of the assay method of TritonX-100 residual quantity in the HBsAg stoste that recombinant Saccharomyces cerevisiae provided by the invention expresses below in detail; The mensuration that the present embodiment is realized TritonX-100 residual quantity in the HBsAg stoste that recombinant Saccharomyces cerevisiae expresses mainly comprises the following steps:
Include reference substance solution preparation steps, product to be tested solution preparation step and TritonX-100 determination step, in described product to be tested solution preparation step, successively by solid-phase extraction column activation, loading, wash assorted and elution step, product to be tested is carried out to SPE processing, before described loading step, also comprise:
Protein content detecting step, from HBsAg stoste to be detected, taking by volume every part as unit, get after some groups of products to be tested, protein content in every group of product to be tested is detected, screen out protein content and be greater than the product to be tested that the product to be tested of a threshold value and protein content are less than described threshold value, the span of wherein said threshold value is 70 μ g/ml-125 μ g/ml;
And described in wash assorted step and specifically comprise:
Once wash assorted step, pass through solid-phase extraction column with 1 part of water respectively, wash for the first time assorted to every group of product to be tested;
Secondary is washed assorted step, protein content is greater than to the product to be tested of described threshold value, once washes after assorted step described, passes through solid-phase extraction column more respectively with 1 part of water, again washes assorted to every group of product to be tested;
Wash assorted step three times, wash assorted step/the second wash after assorted step described first, the methanol solution that is 2%～70% by 1 part of volumetric concentration respectively, by solid-phase extraction column, is washed assorted to every group of product to be tested for the third time.
And described elution step specifically comprises:
Carry out wash-out by solid-phase extraction column with 1 part of 90%～100% methyl alcohol, collect eluent, obtain product to be tested solution.
In addition, described reference substance solution preparation steps can specifically comprise:
With 90% methanol solution preparation TritonX-100 reference substance solution, make the reference substance solution that some groups of concentration are the TritonX-100 of 1～20 μ g/ml.
In this step, prepare reference substance solution by the water that replaces prior art using methyl alcohol as solvent, thereby reach system unification, ensured the accuracy of measurement result.
Described activation step can specifically comprise:
Choose solid-phase extraction column, and pass through successively 1 part of methyl alcohol and 1 part of water in this solid-phase extraction column, carry out preactivated to it;
Described loading step can specifically comprise:
In solid-phase extraction column after respectively every group of product to be tested loading extremely being activated.
When specific implementation, in described product to be tested solution preparation step, liquid is no faster than 1ml/ minute by the flow velocity of solid-phase extraction column.
Described TritonX-100 determination step can specifically comprise:
Liquid chromatogram measuring step, carries out high-performance liquid chromatogram determination to reference substance solution described in every group and product to be tested solution respectively;
Establishing equation and solution procedure, in peak area by reference substance solution and reference substance solution, the concentration of TritonX-100 is carried out linear regression, Criterion curvilinear equation, by the peak area substitution calibration curve equation of product to be tested solution, calculates the residual quantity of TritonX-100 in product to be tested.
In described activation step, the solid-phase extraction column of choosing is WatersHLB1cc solid-phase extraction column.
In described liquid chromatogram measuring step, the chromatographic column of employing is WatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, taking methanol aqueous solution as mobile phase, adopt diode array detector monitoring, and the analytical wavelength range of TritonX-100 is 220nm～240nm.
And in described methanol aqueous solution mobile phase, methyl alcohol is 70:30～85:15 with the ratio of water.
Methanol aqueous solution flows while being in a ratio of 80:20, and the flow rates of mobile phase is between 0.6ml/～1.0ml/ minute.
In the present embodiment, each material part all by volume.
As a preferred embodiment of the present embodiment, in described protein content detecting step, the value of described threshold value can be chosen 100 μ g/ml, that is, screen out protein content and be greater than the product to be tested that the product to be tested of 100 μ g/ml and protein content are less than 100 μ g/ml; And wash in assorted step at described secondary, protein content is greater than to the product to be tested of 100 μ g/ml, once wash after assorted step described, pass through solid-phase extraction column with 1 part of water more respectively, every group of product to be tested again washed and mixed as good.
Technological means and the effect taked for reaching predetermined goal of the invention in order further to explain the present embodiment, in the HBsAg stoste that the present embodiment mensuration recombinant Saccharomyces cerevisiae is expressed, the concrete steps of TritonX-100 residual quantity, are described in detail as follows by instance data.
Example 1
Instrument:
WatersAlliance2695 high performance liquid chromatograph, WatersPDA2996 detector, WatersEmpower software, plum Teller-Tuo benefit XP205 electronic analytical balance.
Reagent and reagent:
TritonX-100 reference substance: purchased from Sigma company, lot number 100M0128V; Methyl alcohol: HPLC level, purchased from Merck company; Water: ultra-pure water.
Flow process:
In reference substance solution preparation steps, TritonX-100 reference substance is dissolved in 90% methanol solution, make series concentration and be respectively the reference substance solution of 1 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml.
In activation step, choose WatersHLB1cc solid-phase extraction column, first passes through with 1ml methyl alcohol, then passes through with 1ml water.
In protein content detecting step, detect that the protein content in product to be tested stoste A is 20 μ g/ml; Protein content in product to be tested stoste B is 250 μ g/ml;
Taking stoste A as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste A of 2 μ g/ml;
Taking stoste B as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste B of 2 μ g/ml.
In loading step, get the each 1ml of above-mentioned product to be tested, by preactivated good solid-phase extraction column.
Washing in assorted step, stoste A and PC-stoste A are cleaned after each 1 time with 1ml water, or stoste B and PC-stoste B are cleaned after each 2 times with 1ml water, the methanol solution 1ml that is 2% by volumetric concentration cleans 1 time above-mentioned product to be tested is each.
In elution step, use 1ml methanol-eluted fractions, collect eluent, eluent is product to be tested solution.
Wherein, the each step operation in SPE, needs to control flow rate of liquid, and flow velocity should be no faster than 1ml/ minute.
In TritonX-100 determination step, the product to be tested solution that the reference substance solution respectively described reference substance solution preparation steps being obtained and product to be tested solution preparation step obtain carries out after high effective liquid chromatography for measuring, in peak area by reference substance solution and reference substance solution, the concentration of TritonX-100 is carried out linear regression, Criterion curvilinear equation, and by the peak area substitution calibration curve equation of product to be tested solution, the TritonX-100 residual quantity calculating in product to be tested is respectively: stoste A and stoste B are < 1 μ g/ml, PC-stoste A is 1.84 μ g/ml, PC-stoste B is 1.90 μ g/ml. wherein, chromatographic column is WatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, methanol aqueous solution mobile phase is than 70:30, and flow velocity is 0.6ml/ minute, adopts diode array detector (PDA) monitoring, and the analytical wavelengths of TritonX-100 is 230nm.
Example 2
Instrument:
WatersAlliance2695 high performance liquid chromatograph, WatersPDA2996 detector, WatersEmpower software, plum Teller-Tuo benefit XP205 electronic analytical balance.
Reagent and reagent:
TritonX-100 reference substance, purchased from Sigma company, lot number 100M0128V; Methyl alcohol: HPLC level, purchased from Merck company; Water: ultra-pure water.
Flow process:
In reference substance solution preparation steps, TritonX-100 reference substance is dissolved in 90% methanol solution, make series concentration and be respectively the reference substance solution of 1 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml.
In activation step, choose WatersHLB1cc solid-phase extraction column, first passes through with 1ml methyl alcohol, then passes through with 1ml water.
In protein content detecting step, detect that the protein content in product to be tested stoste C is 20 μ g/ml; Protein content in product to be tested stoste D is 250 μ g/ml;
Taking stoste C as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste C of 2 μ g/ml;
Taking stoste D as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste D of 2 μ g/ml.
In loading step, get above-mentioned product to be tested 1ml, by preactivated good solid-phase extraction column.
Washing in assorted step, stoste C and PC-stoste C are cleaned after each 1 time with 1ml water, or stoste D and PC-stoste D are cleaned after each 2 times with 1ml water, the methanol solution 1ml that is 70% by volumetric concentration cleans 1 time above-mentioned product to be tested is each.
In elution step, with 90% methanol solution 1ml wash-out, collect eluent, eluent is product to be tested solution.
Wherein, the each step operation in SPE, needs to control flow rate of liquid, and flow velocity should be no faster than 1ml/ minute.
In TritonX-100 determination step, the product to be tested solution that the reference substance solution respectively described reference substance solution preparation steps being obtained and product to be tested solution preparation step obtain carries out after high effective liquid chromatography for measuring, in peak area by reference substance solution and reference substance solution, the concentration of TritonX-100 is carried out linear regression, Criterion curvilinear equation, by the peak area substitution calibration curve equation of product to be tested solution, the TritonX-100 residual quantity calculating in product to be tested is respectively: stoste C and stoste D are < 1 μ g/ml, PC-stoste C is 1.87 μ g/ml, PC-stoste D is 1.90 μ g/ml. wherein, chromatographic column is WatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, methanol aqueous solution flows and is in a ratio of 80:20, and flow velocity is 1.0ml/ minute, adopts diode array detector (PDA) monitoring, and the analytical wavelengths of TritonX-100 is 240nm.
Example 3
Instrument:
WatersAlliance2695 high performance liquid chromatograph, WatersPDA2996 detector, WatersEmpower software, plum Teller-Tuo benefit XP205 electronic analytical balance.
Reagent and reagent:
TritonX-100 reference substance: purchased from Sigma company, lot number 100M0128V; Methyl alcohol: HPLC level, purchased from Merck company; Water: ultra-pure water.
Flow process:
In reference substance solution preparation steps, TritonX-100 reference substance is dissolved in 90% methanol solution, make series concentration and be respectively the reference substance solution of 1 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml;
In activation step, choose WatersHLB1cc solid-phase extraction column, first passes through with 1ml methyl alcohol, then passes through with 1ml water.
In protein content detecting step, detect that the protein content in product to be tested stoste E is 20 μ g/ml; Protein content in product to be tested stoste F is 250 μ g/ml;
Taking stoste E as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste E of 10 μ g/ml;
Taking stoste F as dilution, being mixed with TritonX-100 content is the product to be tested PC-stoste F of 10 μ g/ml.
In loading step, get the each 1ml of above-mentioned product to be tested, by preactivated good solid-phase extraction column.
Washing in assorted step, stoste E and PC-stoste E are cleaned after each 1 time with 1ml water, or stoste F and PC-stoste F are cleaned after each 2 times with 1ml water, the methanol solution 1ml that is 5% by volumetric concentration cleans 1 time above-mentioned product to be tested is each.
In elution step, with methanol solution 1ml wash-out, collect eluent, eluent is product to be tested solution.
Wherein, the each step operation in SPE, needs to control flow rate of liquid, and flow velocity should be no faster than 1ml/ minute.
In TritonX-100 determination step, the product to be tested solution that the reference substance solution respectively described reference substance solution preparation steps being obtained and product to be tested solution preparation step obtain carries out after high effective liquid chromatography for measuring, in peak area by reference substance solution and reference substance solution, the concentration of TritonX-100 is carried out linear regression, Criterion curvilinear equation, by the peak area substitution calibration curve equation of product to be tested solution, the TritonX-100 residual quantity calculating in product to be tested is respectively: stoste E and stoste F are < 1 μ g/ml, PC-stoste E is 9.56 μ g/ml, PC-stoste F is 9.73 μ g/ml. wherein, chromatographic column is WatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, methanol aqueous solution flows and is in a ratio of 85:15, and flow velocity is 0.6ml/ minute, adopts diode array detector (PDA) monitoring, and the analytical wavelengths of TritonX-100 is 220nm.
The TritonX-100 residual quantity data of measuring by above-mentioned each example are known, and the present embodiment has reached that the rate of recovery is high, detectability is low, can realize the effect of the Accurate Determining of TritonX-100 residual quantity in stoste.
Above content is in conjunction with concrete preferred embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations. For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (9)
1. an assay method for TritonX-100 residual quantity in the HBsAg stoste that recombinant Saccharomyces cerevisiae is expressed,Include reference substance solution preparation steps, product to be tested solution preparation step and TritonX-100 determination step,In described product to be tested solution preparation step, successively by solid-phase extraction column activation, loading, wash assorted and wash-out stepSuddenly, product to be tested is carried out to SPE processing, it is characterized in that, before described loading step, also comprise:
Protein content detecting step, from HBsAg stoste to be detected, taking by volume every part as unit,Get after some groups of products to be tested, the protein content in every group of product to be tested is detected, screen out protein and containAmount is greater than the product to be tested that the product to be tested of a threshold value and protein content are less than described threshold value, wherein said threshold valueSpan be 70 μ g/ml-125 μ g/ml;
And described in wash assorted step and comprise:
Once wash assorted step, pass through solid-phase extraction column with 1 part of water respectively, every group of product to be tested carried out for the first timeWash assorted;
Secondary is washed assorted step, described protein content is greater than to the product to be tested of described threshold value, once washes describedAfter assorted step, pass through solid-phase extraction column with 1 part of water more respectively, again wash assorted to every group of product to be tested;
Wash assorted step three times, wash assorted step/the second described first and wash after assorted step, use respectively 1 part of volume denseDegree be 2%～70% methanol solution by solid-phase extraction column, wash for the third time assorted to every group of product to be tested;
Wherein, in described activation step, the solid-phase extraction column of choosing isHLB1cc is solidPhase extraction column.
2. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 1 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, the value of described threshold value is 100 μ g/ml.
3. TritonX-100 in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 1 or 2 is expressedThe assay method of residual quantity, is characterized in that, described elution step comprises:
Carry out wash-out by solid-phase extraction column with 1 part of 90%～100% methyl alcohol, collect eluent, treatedSurvey product solution.
4. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 3 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, described TritonX-100 determination step comprises:
Liquid chromatogram measuring step, carries out high-efficient liquid phase color to every group of reference substance solution and product to be tested solution respectivelySpectrum is measured;
Establishing equation and solution procedure, Triton in the peak area by reference substance solution and reference substance solutionThe concentration of X-100 is carried out linear regression, and Criterion curvilinear equation, by the peak area substitution mark of product to be tested solutionDirectrix curve equation, calculates the residual quantity of TritonX-100 in product to be tested.
5. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 4 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, in described liquid chromatogram measuring step, the chromatographic column of employing isWatersSymmetryShieldTMRP18,5 μ m3.9 × 150mm reverse-phase chromatographic columns, taking methanol aqueous solution asMobile phase, adopts diode array detector monitoring, and the analytical wavelength range of TritonX-100 exists220nm～240nm。
6. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 5 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, in described methanol aqueous solution mobile phase, and the proportioning of methyl alcohol and waterFor 70:30～85:15.
7. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 6 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, methanol aqueous solution flows while being in a ratio of 80:20, the flow velocity of mobile phaseScope is between 0.6ml～1.0ml/ minute.
8. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 1 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, described reference substance solution preparation steps comprises:
With 90% methanol solution preparation TritonX-100 reference substance solution, make some groups of 1～20 μ g/mlThe reference substance solution of TritonX-100.
9. in the HBsAg stoste that recombinant Saccharomyces cerevisiae as claimed in claim 1 is expressed, TritonX-100 is residualThe assay method of allowance, is characterized in that, described activation step comprises:
Choose solid-phase extraction column, and pass through successively 1 part of methyl alcohol and 1 part of water in this solid-phase extraction column, to itCarry out preactivated;
Described loading step comprises:
In solid-phase extraction column after respectively every group of product to be tested loading extremely being activated;
And in described product to be tested solution preparation step, liquid is no faster than 1ml/ by the flow velocity of solid-phase extraction columnMinute.
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013005820A (en) * | 2003-08-15 | 2013-01-10 | Univ Of South Florida | Material and method for capture of pathogen and removal of aurintricarboxylic acid from sample |
| CN102590427A (en) * | 2012-02-24 | 2012-07-18 | 玉溪九洲生物技术有限责任公司 | Determination method of TritonX-100 residual quantity in antitoxin/ antiserum |
| CN104215718A (en) * | 2014-09-22 | 2014-12-17 | 成都生物制品研究所有限责任公司 | High performance liquid chromatography detection method of Triton X-100 content |
Non-Patent Citations (3)
| Title |
|---|
| Determination of Triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography;G. Karlsson et al.;《Journal of Chromatography A》;20021231;163-168 * |
| Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities: yeastolates,triton X-100 and methotrexate;Shiping Fang et al.;《Journal of Chromatography B》;20111007;3612-3619 * |
| 血浆制品中Triton X-100残留量测定方法的建立;杨鹏云等;《中国生物制品学杂志》;20031231;第16卷(第4期);235-236 * |
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