CN110736796B - Method for detecting molecular weight of pneumococcal capsular polysaccharide - Google Patents

Method for detecting molecular weight of pneumococcal capsular polysaccharide Download PDF

Info

Publication number
CN110736796B
CN110736796B CN201911008923.0A CN201911008923A CN110736796B CN 110736796 B CN110736796 B CN 110736796B CN 201911008923 A CN201911008923 A CN 201911008923A CN 110736796 B CN110736796 B CN 110736796B
Authority
CN
China
Prior art keywords
mobile phase
sample
molecular weight
pnps
viscosity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911008923.0A
Other languages
Chinese (zh)
Other versions
CN110736796A (en
Inventor
马可
朱卫华
宗向坤
张
赵林飞
胡月凤
杜琳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anhui Zhifei Longcom Biopharmaceutical Co ltd
Chongqing Zhifei Biological Products Co Ltd
Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Original Assignee
Anhui Zhifei Longcom Biopharmaceutical Co ltd
Chongqing Zhifei Biological Products Co Ltd
Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anhui Zhifei Longcom Biopharmaceutical Co ltd, Chongqing Zhifei Biological Products Co Ltd, Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd filed Critical Anhui Zhifei Longcom Biopharmaceutical Co ltd
Priority to CN201911008923.0A priority Critical patent/CN110736796B/en
Publication of CN110736796A publication Critical patent/CN110736796A/en
Application granted granted Critical
Publication of CN110736796B publication Critical patent/CN110736796B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for detecting the molecular weight of streptococcus pneumoniae capsular polysaccharide (PnPs) by an HPSEC-RI method. The method comprises the following steps: preparing a mobile phase; accurately weighing PnPs, adding a mobile phase for dissolving, diluting with the mobile phase, detecting the viscosity of the sample to ensure that the viscosity value of the diluted sample is within a certain range, and filtering to obtain a sample solution; selecting a plurality of glucan standard substances with different molecular weights, diluting the glucan standard substances with a mobile phase respectively, injecting a sample, analyzing the sample by liquid chromatography, and establishing a molecular weight standard curve and a regression equation; detecting and recording the sample under the same test condition, and calculating the molecular weight of the sample by using the established standard curve and regression equation. The buffer system and the sample processing method have high accuracy and good repeatability.

Description

Method for detecting molecular weight of pneumococcal capsular polysaccharide
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting the molecular weight of pneumococcal capsular polysaccharide.
Background
Currently, the vaccines marketed to prevent streptococcus pneumoniae infection are the 23-valent pneumococcal polysaccharide vaccine and the 7-valent/13-valent pneumococcal polysaccharide conjugate vaccine. Pneumococcal capsular polysaccharide (PnPs) is a main effective component of the vaccine, and researches show that the molecular weight of the PnPs directly influences the immunogenicity effect of the polysaccharide vaccine; in combination vaccines, PnPs are also important indexes for quality control as intermediate products of the vaccines.
By 1 month in 2019, a 23-valent pneumococcal polysaccharide vaccine is on the market in China, and a plurality of vaccine manufacturers are researching and developing pneumococcal polysaccharide vaccines and pneumococcal polysaccharide conjugate vaccines, but the three parts (2015 edition) of Chinese pharmacopoeia does not include the manufacturing and detection rules of the 23-valent pneumococcal polysaccharide vaccine, and the detection rules of the vaccine recorded 7.0 edition of European pharmacopoeia prescribes that the distribution coefficient (K & ltk & gt) of various PnPs samples in a chromatographic column is measured by using gel exclusion chromatography (SEC)D) And K is specified for each monovalent polysaccharideDThe method has the defects of time consumption, labor consumption, poor repeatability and the like. In the aspect of molecular weight determination, as the high performance liquid chromatography technology is mature, a polysaccharide molecular weight standard substance with similar properties to a sample to be measured is detected by a high performance gel exclusion chromatography-differential refraction method (HPSEC-RI), a mass distribution curve of the molecular weight of the standard substance along with the change of the elution time or the volume of the sample is drawn according to the polysaccharide molecular weight standard substance, the corresponding elution index of the sample to be measured is recorded, and the molecular weight of the sample to be measured is measured and calculated through the mass distribution curve.
A large number of experiments prove that when the HPSEC-RI method is used for detecting PnPs, the lower the dilution concentration of some serotype PnPs is, the larger the detection molecular weight result is, and the detection result does not change along with the concentration after the PnPs are diluted to a certain concentration. And the concentration and the viscosity of the PnPs have a positive correlation, and the correlation between the viscosity and the molecular weight of all the PnPs is counted to obtain a molecular weight result which is detected when the viscosity value of the solution is between 1.0 and 1.3cp and tends to be uniform. The traditional HPSEC-RI method does not consider the influence of viscosity factors when detecting PnPs, has larger errors, optimizes a mobile phase, and increases viscosity control in the sample dilution process, thereby improving the accuracy of the HPSEC-RI method in detecting the PnPs.
Disclosure of Invention
The invention aims to provide a method for detecting the molecular weight of pneumococcal capsular polysaccharide.
The molecular weight of the treated sample is detected by a method for detecting streptococcus pneumoniae capsular polysaccharide (PnPs) by a HPSEC-RI method, so that the molecular weight is more accurate.
The detection method comprises the following steps:
(1) preparing 0.1mol/L phosphate buffer solution as a mobile phase;
(2) accurately weighing PnPs, adding the PnPs into a mobile phase, dissolving, diluting with the mobile phase, and filtering to obtain a sample solution;
(3) selecting a plurality of glucan standard substances with different molecular weights, diluting the glucan standard substances to 1mg/ml by using a mobile phase, injecting a sample, analyzing by using liquid chromatography, and establishing a molecular weight standard curve and a regression equation;
(4) detecting and recording the sample under the same test condition, and calculating the molecular weight of the sample by using the established standard curve and regression equation.
Wherein the mobile phase is phosphate buffer solution, and the formula comprises 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride, and is prepared by adding deionized water to 1000 g.
Wherein, the chromatographic column used for the liquid chromatographic analysis is a silica gel chromatographic column, the chromatographic column is an analytical column of TSK G5000PWXL (7.8mm ID multiplied by 30cm) manufactured by TOSOH corporation of Japan, and the detection conditions of HPSEC-RI are as follows: the monitor temperature is 30 deg.C, the flow rate is 0.5ml/min, the molecular weight standard is prepared into 1mg/ml solution with mobile phase, 100 μ l of each solution is injected.
Wherein, the different viscosities of different serological type PnPs are diluted to 1mg/ml-0.02mg/ml by a mobile phase, the viscosity value of the solution is between 1.0 cp and 1.3cp, and the molecular weight is detected by high performance gel exclusion chromatography-differential refraction method (HPSEC-RI).
Among these, the method is applicable to all streptococcus pneumoniae PnPs.
Wherein, the streptococcus pneumoniae serotypes are PnPs with relatively small viscosity such as types 1, 2, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 12F, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and the viscosity is measured and should be between 1.0 and 1.3 cp.
Wherein, the streptococcus pneumoniae serotype 3 PnPs are diluted to 0.05 mg/ml-0.2 mg/ml, and the viscosity is detected to be between 1.0-1.3 cp.
Preferably, the detection method of the present invention comprises the following steps:
(1) preparing 0.1mol/L phosphate buffer solution as a mobile phase;
(2) accurately weighing PnPs, adding a mobile phase for dissolving for 12-24 hours, diluting with the mobile phase, wherein the concentration of polysaccharide is 0.2mg/ml, the concentration of type 3 polysaccharide is 0.05mg/ml, shaking up, filtering, and detecting the viscosity to be 1.0-1.3CP, namely a sample solution;
(3) selecting a plurality of glucan standard substances with different molecular weights, diluting the glucan standard substances to 1mg/ml by using a mobile phase, injecting a sample, analyzing by using liquid chromatography, and establishing a molecular weight standard curve and a regression equation; the detection conditions of the liquid chromatogram are as follows: the test temperature is 30 ℃, the mobile phase of the system is 0.1mol/L phosphate buffer solution, and the flow rate is 0.5 ml/min;
(4) detecting and recording the sample under the same test condition, and calculating the molecular weight of the sample by using the established standard curve and regression equation.
Further preferably, the detection method of the present invention comprises the steps of:
(1) formulating mobile phase
0.1mol/L phosphate buffer solution, taking the preparation of 1000ml total solution as an example, the formula is that 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride are weighed, and deionized water is added to reach 1000 g.
(2) Establishing standard curve and regression equation
Preparing the molecular weight standard substance into a solution with the concentration of 0.2mg/ml by using a mobile phase, and respectively preparing the sample to be detected into corresponding concentrations by using the mobile phase, and sampling 100 mu l of the solution.
The HPSEC-RI detection conditions are as follows: the test temperature is 30 ℃, the mobile phase of the system is 0.1mol/L phosphate buffer solution, and the flow rate is 0.5 ml/min.
The refractive index detection signal during the sample detection was collected on-line and the peak retention time of each sample on the RI detector was recorded using the Empower 3 software. A series of standard products with known molecular weights are detected through HPSEC-RI, peak tip retention time is used as an abscissa, and a logarithmic value (lgMp) of each standard product Mp is used as an ordinate to establish a molecular weight distribution standard curve and a regression equation.
(3) Streptococcus pneumoniae capsular polysaccharide dilution
Dissolving PnPs12-24 h to be detected by using the diluent, uniformly mixing, and then diluting the polysaccharide solution by using a mobile phase until the polysaccharide concentration is 0.2mg/ml, and diluting the type 3 by using the mobile phase until the polysaccharide concentration is 0.05 mg/ml. The viscosity is measured by a viscometer, and the viscosity at 25 ℃ is 1.0-1.3 CP.
(4) Molecular weight detection
The HPSEC-RI detection conditions are as follows: the test temperature is 30 ℃, the mobile phase of the system is 0.1mol/L phosphate buffer solution, and the flow rate is 0.5 ml/min. And detecting and recording the peak retention time of the sample to be detected under the same test condition with the standard substance, calculating the lgMp of the sample by using the established standard curve and regression equation, and further obtaining the corresponding molecular weight.
The detection method of the invention has the beneficial effects that: the conventional HPSEC-RI method does not consider the influence of viscosity factors when detecting PnPs, and has larger errors.
Compared with the existing method, the detection method provided by the invention has the characteristics of high accuracy, good repeatability, good stability and the like.
Drawings
FIG. 1 is a graph of the molecular weight standards of example 1.
Figure 2 is a GPC diagram of PnPs serotype 8 diluted to different concentrations in example 1.
FIG. 3 is a GPC chart of serotype 3 PnPs diluted to different concentrations in Experimental example 1.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.
Materials referred to in the following examples:
streptococcus pneumoniae capsular polysaccharides
Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 12F, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, for a total of 24 serotypes of PnPs.
Reagent and instrument
Viscometer, electronic balance, high-phase liquid chromatograph, diluent and mobile phase, and dextran standard.
Example 1:
1) instrument and reagent
Model JA2003 electronic balance was purchased from Shunhui scientific instruments, Inc., Shanghai; DV2T type viscometer available from BROOK FIELD Inc., spindle model number CPA-40Z; e2695 high performance liquid system pump, Waters 2414 differential refractometer, available from Waters; high performance liquid chromatography columns were TSK PWXL Guard column and TSK G5000PWXL (7.8 mmID. times.30 cm) analytical column from TOSOH corporation of Japan.
Reagent: sodium dihydrogen phosphate dodecahydrate, disodium hydrogen phosphate dihydrate, sodium chloride, dextran Standards were purchased from Waters Corporation and American Polymer Standards Corporation.
0.1mol/L phosphate buffer solution is prepared by weighing 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride, and adding deionized water to 1000 g.
2) Preparation of type 8 serum PnPs analysis sample
Accurately weighing 50mg of 8-type serum PnPs, adding 10ml of mobile phase to dissolve for 18 hours, and diluting a sample to 2mg/ml, 0.4mg/ml and 0.08mg/ml by using the mobile phase, wherein the specific concentration is shown in Table 1; shaking, filtering, and detecting viscosity to be 1.0-1.3CP to obtain sample solution;
3) detection of serum type PnPs of Streptococcus pneumoniae type 8
And detecting and recording the peak retention time of the sample to be detected, calculating the lgMp of the sample by using the established standard curve and a regression equation, and further obtaining the corresponding molecular weight (Mp) of the sample. The viscosity of the 2mg/ml PnPs sample was 1.50CP, and beyond the viscosity detection range, the molecular weight (Mp) results were seen to be smaller and were discarded. The viscosity of the PnPs samples at 0.4mg/ml and 0.08mg/ml is between 1.0 and 1.3CP, and the average molecular weight (Mp) of both results is 1488KD, as shown in Table 1. The GPC chart of PnPs type 8 is shown in FIG. 3.
TABLE 1 results of measuring viscosity and Mp value of serotype 19 type A PnPs
Figure BDA0002243592740000051
Example 2:
1) instrument and reagent
Model JA2003 electronic balance was purchased from Shunhui scientific instruments, Inc., Shanghai; DV2T type viscometer available from BROOK FIELD Inc., spindle model number CPA-40Z; e2695 high performance liquid system pump, Waters 2414 differential refractometer, available from Waters; high performance liquid chromatography columns were TSK PWXL Guard column and TSK G5000PWXL (7.8 mmID. times.30 cm) analytical column from TOSOH corporation of Japan.
Reagent: sodium dihydrogen phosphate dodecahydrate, disodium hydrogen phosphate dihydrate, sodium chloride, dextran Standards were purchased from Waters Corporation and American Polymer Standards Corporation.
0.1mol/L phosphate buffer solution is prepared by weighing 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride, and adding deionized water to 1000 g.
2) Preparation of 33F type serum PnPs analysis sample
Accurately weighing 50mg of 33F type serum PnPs, adding 10ml of mobile phase to dissolve for 16 hours, diluting the sample to 0.2mg/ml by using the mobile phase, and detecting the viscosity to be 1.0-1.3CP to obtain a test solution;
3) detection of Streptococcus pneumoniae type 33F serous PnPs
And detecting and recording the peak retention time of the sample to be detected, calculating the lgMp of the sample by using the established standard curve and a regression equation, and further obtaining the corresponding molecular weight (Mp) of the sample. The measurements were carried out 2 times in succession, and the two results were averaged to give a molecular weight (Mp) of 2766kD, as shown in Table 2.
TABLE 2 results of viscosity and Mp values of PnPs of serotype 33F
Figure BDA0002243592740000061
Test example 1: comparative experiment
Comparing the detection method with the existing method, the experimental data proves the superiority of the invention: the sample loading concentration of the conventional HPSEC-RI method is fixed when the molecular weight of PnPs is detected, the concentrations used in different laboratory detections are not completely the same, but the viscosity of different serotype PnPs is greatly different, the same serotype PnPs and different batches are also different, and the viscosity has a great influence on the detection molecular weight of the HPSEC-RI method in the experiment.
This test example simulates the prior HPSEC-RI assay with different loading concentrations of type 3 serum PnPs, and compares it with the results of the invention with increased viscosity control. In the range of 0.25-2.0mg/ml, the viscosity value is 1.45-5.54CP, the difference of the HPSEC-RI method in the detection of the molecular weight Mp value of PnPs is large, and the RSD value is 23.01%; in the range of 0.03125-0.125mg/ml, the viscosity value is in the control range (1.0-1.3CP), the HPSEC-RI method has no obvious difference in the result of detecting the molecular weight Mp value of PnPs, and the RSD value is 1.19%. See table 5. According to the invention, viscosity control is added in the sample dilution process, so that the accuracy of the HPSEC-RI method in detecting PnPs is improved.
1) Instrument and reagent
Model JA2003 electronic balance, available from Shunhu constant science instruments, Inc., Shanghai; DV2T type viscometer available from BROOK FIELD Inc., spindle model number CPA-40Z; e2695 high performance liquid system pump, Waters 2414 differential refractometer, available from Waters; high performance liquid chromatography columns were TSK PWXL Guard column and TSK G5000PWXL (7.8 mmID. times.30 cm) analytical column from TOSOH corporation of Japan.
Reagent: sodium dihydrogen phosphate dodecahydrate, disodium hydrogen phosphate dihydrate, sodium chloride, dextran Standards were purchased from Waters Corporation and American Polymer Standards Corporation.
0.1mol/L phosphate buffer solution is prepared by weighing 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride, and supplementing deionized water to 1000 g.
2) Preparation of type 3 serum PnPs analysis sample
Accurately weighing 50mg of 3-type serum PnPs, adding 10ml of mobile phase to dissolve for 18 hours, diluting a sample from 5mg/ml to 0.03125mg/ml by using the mobile phase, wherein the specific concentration is shown in Table 3; then, the sample is diluted to 0.05mg/ml by using the mobile phase, and 10 parts of the sample are respectively marked as type 3-sample 1 to type 3-sample 10, which is shown in a table 4; shaking, filtering, and detecting viscosity to be 1.0-1.3CP to obtain test solution;
3) detection of streptococcus pneumoniae serotype 3 PnPs
And detecting and recording the peak retention time of the sample to be detected, calculating the lgMp of the sample by using the established standard curve and the regression equation, and further obtaining the corresponding molecular weight. The difference between different concentrations of the sample molecular weight (Mp) is large, the Mp is not obviously different when the concentration is reduced to below 0.125mg/ml, and the corresponding viscosity is 1.26CP, see Table 3.
4) Precision survey
The same batch of type 3 was examined for 10 different samples and the results showed: the method detects serotype 3 PnPs with the concentration of 0.05mg/ml, the viscosity of 1.20-1.23, the difference of the molecular weight Mp results is small, the RSD value is 1.29%, and the precision is good, which is shown in Table 4.
TABLE 3 results of viscosity and Mp values for serotype 3 PnPs
Figure BDA0002243592740000071
TABLE 4 results of measuring viscosity and Mp values of 0.05mg/ml serotype 3 PnPs
Figure BDA0002243592740000072
Figure BDA0002243592740000081
Test example 2: screening experiments
The core point of the invention is that the invention is mainly improved in that the viscosity of the detected sample is controlled in a certain range by dilution, so that the detection result is more accurate. The most preferable detection method is obtained by screening through detecting Mp values of different viscosities of 24 serotype PnPs, the viscosity of a detection sample is controlled to be 1.0-1.3CP, and the method is high in result accuracy, good in repeatability and good in stability.
Taking type 1 PnPs, type 3 PnPs and type 19A PnPs as examples, the viscosity of polysaccharide samples of three serotypes is reduced along with the reduction of concentration, the Mp value is increased along with the reduction of viscosity when the viscosity is more than 1.3, and the viscosity is not obviously changed between 1.0 and 1.3 CP. Calculating the precision, the concentration interval of 1 type PnPs viscosity between 1.0 and 1.3 is 0.03125 to 0.5mg/ml, and the RSD value is 1.84 percent; the viscosity of the type 3 PnPs ranges from 1.0 to 1.3, the concentration range is 0.03125 to 0.125mg/ml, and the RSD value is 1.19 percent; the viscosity of the 19A type PnPs ranged from 1.0 to 1.3 at a concentration ranging from 0.03125 to 0.5mg/ml, and the RSD value was 4.85%, as shown in Table 5. Therefore, it is the most preferable method to control the viscosity of the test sample to 1.0 to 1.3.
TABLE 51 type PnPs, 3 type PnPs, and 19A type PnPs viscosity and Mp value results
Figure BDA0002243592740000082
Figure BDA0002243592740000091

Claims (5)

1. A method for detecting the molecular weight of pneumococcal capsular polysaccharide comprises the following steps: preparing a mobile phase; accurately weighing PnPs, adding the PnPs into a mobile phase, dissolving, diluting with the mobile phase, and filtering to obtain a sample solution; selecting a plurality of glucan standard substances with different molecular weights, diluting the glucan standard substances to 1mg/ml by using a mobile phase, injecting a sample, analyzing by using liquid chromatography, and establishing a molecular weight standard curve and a regression equation; detecting and recording a sample to be tested under the same test condition, and calculating the molecular weight of the sample by using the established standard curve and regression equation;
wherein the mobile phase is 0.1mol/L phosphate buffer solution;
wherein, the chromatographic column used for the liquid chromatographic analysis is a silica gel chromatographic column, the chromatographic column is a TSK G5000PWXL 7.8mm ID multiplied by 30cm analytical column of Japan TOSOH company, and the detection conditions of HPSEC-RI are as follows: the temperature of the monitor is 30 ℃, the flow rate is 0.5ml/min, the molecular weight standard substance is respectively prepared into solutions with the concentration of 1mg/ml by using the mobile phase, and 100 mul of the solutions are respectively injected;
wherein, aiming at different viscosities of different serotype PnPs, the solution is diluted to 0.02 mg/ml-1 mg/ml by a mobile phase, the viscosity value of the solution is between 1.0 cp and 1.3cp, and then the molecular weight is detected by high performance gel exclusion chromatography-differential refraction method (HPSEC-RI).
2. The method of claim 1, wherein: this method is applicable to all streptococcus pneumoniae PnPs.
3. The method of claim 2, wherein: wherein, the streptococcus pneumoniae serotype 3 PnPs are diluted to 0.05mg/ml to 0.2mg/ml, and the viscosity is detected to be between 1.0 and 1.3 cp.
4. The method of claim 1, comprising the steps of:
(1) preparing 0.1mol/L phosphate buffer solution as a mobile phase;
(2) accurately weighing PnPs, adding a mobile phase to dissolve for 12-24 hours, diluting with the mobile phase, wherein the concentration of polysaccharide is 0.2mg/ml, diluting streptococcus pneumoniae serotype 3 PnPs in the PnPs to 0.05mg/ml, shaking up, filtering, and detecting that the viscosity is 1.0-1.3CP, namely a test solution;
(3) selecting a plurality of glucan standard substances with different molecular weights, diluting the glucan standard substances to 1mg/ml by using a mobile phase, injecting a sample, analyzing by using liquid chromatography, and establishing a molecular weight standard curve and a regression equation; the detection conditions of the liquid chromatogram are as follows: the test temperature is 30 ℃, the mobile phase of the system is 0.1mol/L phosphate buffer solution, and the flow rate is 0.5 ml/min;
(4) detecting and recording the sample under the same test condition, and calculating the molecular weight of the sample by using the established standard curve and regression equation.
5. The method of claim 1, comprising the steps of:
(1) formulating a mobile phase
The preparation method of 0.1mol/L phosphate buffer solution comprises weighing 0.4g of sodium dihydrogen phosphate dodecahydrate, 6.0g of disodium hydrogen phosphate dihydrate and 9.0g of sodium chloride, and adding deionized water to 1000 g;
(2) establishing standard curve and regression equation
Preparing the molecular weight standard substance into a solution with the concentration of 0.2mg/ml by using a mobile phase matching method, preparing a sample to be detected into corresponding concentrations by using the mobile phase matching method respectively, and sampling 100 mu l of the solution;
the HPSEC-RI detection conditions are as follows: the test temperature is 30 ℃, the mobile phase of the system is 0.1mol/L phosphate buffer solution, and the flow rate is 0.5 ml/min;
collecting a refractive index detection signal in the sample detection process on line, and recording the peak retention time of each sample on an RI detector by using Empower 3 software; detecting a series of standard products with known molecular weight by HPSEC-RI, and establishing a molecular weight distribution standard curve and a regression equation by taking peak retention time as an abscissa and logarithmic values lgMp of all the standard products as an ordinate;
(3) streptococcus pneumoniae capsular polysaccharide dilution
Dissolving PnPs12-24 hours to be detected by using a diluent, uniformly mixing, and then diluting a polysaccharide solution by using a mobile phase until the concentration of polysaccharide is 0.2mg/ml, wherein the concentration of streptococcus pneumoniae serotype 3 PnPs in the PnPs is diluted to 0.05mg/ml, and the viscosity is detected by using a viscometer and is 1.0-1.3CP at the temperature of 25 ℃;
(4) molecular weight detection
The HPSEC-RI detection conditions are as follows: the test temperature is 30 ℃, the flow rate of a system mobile phase is 0.1mol/L phosphate buffer solution, the flow rate is 0.5ml/min, the peak retention time of a sample to be tested is detected and recorded under the same test condition with a standard substance, the lgMp of the sample is calculated by applying an established standard curve and a regression equation, and the corresponding molecular weight is further obtained.
CN201911008923.0A 2019-10-23 2019-10-23 Method for detecting molecular weight of pneumococcal capsular polysaccharide Active CN110736796B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911008923.0A CN110736796B (en) 2019-10-23 2019-10-23 Method for detecting molecular weight of pneumococcal capsular polysaccharide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911008923.0A CN110736796B (en) 2019-10-23 2019-10-23 Method for detecting molecular weight of pneumococcal capsular polysaccharide

Publications (2)

Publication Number Publication Date
CN110736796A CN110736796A (en) 2020-01-31
CN110736796B true CN110736796B (en) 2022-07-01

Family

ID=69270875

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911008923.0A Active CN110736796B (en) 2019-10-23 2019-10-23 Method for detecting molecular weight of pneumococcal capsular polysaccharide

Country Status (1)

Country Link
CN (1) CN110736796B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113030304B (en) * 2021-02-25 2022-07-19 中国食品药品检定研究院 Method for detecting polysaccharide by HPSEC-RI method and correlating with Sepharose CL-4B method
CN112986457B (en) * 2021-02-25 2022-07-12 中国食品药品检定研究院 Method for detecting polysaccharide by HPSEC-MALS method and correlating polysaccharide with Sepharose CL-4B method

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109336989A (en) * 2018-10-22 2019-02-15 北京智飞绿竹生物制药有限公司 Method for preparing pneumococcal capsular polysaccharide through viscosity control

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109336989A (en) * 2018-10-22 2019-02-15 北京智飞绿竹生物制药有限公司 Method for preparing pneumococcal capsular polysaccharide through viscosity control

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Molecular size analysis of capsular polysaccharid preparations from Streptococcus pneumoniae;Bohumil Bednar et al.;《Carbohydrafe Research》;19931231;第115-130页 *
肺炎链球菌荚膜多糖分子大小及分子量分析方法的比较;张轶 等;《中国生物制品学杂志》;20150930;第947-955,960页 *

Also Published As

Publication number Publication date
CN110736796A (en) 2020-01-31

Similar Documents

Publication Publication Date Title
CN110736796B (en) Method for detecting molecular weight of pneumococcal capsular polysaccharide
CN105067727A (en) Method for measuring content of polyethylene glycol in polycarboxylate superplasticizer macromonomer
US20240044851A1 (en) Method for detecting content of active ingredients of compound sophorae flavescentis radix injection and fingerprint spectrum thereof
CN113341028A (en) Method for measuring sodium hyaluronate molecular weight and molecular weight distribution by size exclusion chromatography
CN106226304A (en) A kind of method utilizing acid-base indicator method to measure oligochitosan deacetylation
CN107831127A (en) A kind of method of sugared content in detection feed
CN105911081B (en) A kind of method for differentiating mannatide
CN107421975A (en) The detection method of glycerol content in a kind of injection
CN106168610A (en) Fluorimetric HPLC measures the method for concentration of Clozapine in blood plasma
US6411383B1 (en) Method for measuring the 2nd virial coefficient
CN103808686A (en) Method for measuring weight ratio of SBS modifier in SBS modified asphalt
CN110274972A (en) A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution
CN104181124A (en) Method for testing hydroxyl value of BGAP
CN107084929B (en) The quantitative detecting method of pneumococal polysaccharide
CN113092640B (en) Method for detecting benzyl alcohol and benzaldehyde in heparin sodium injection
CN104155388A (en) Method for determining content of acetic acid in triacetin
CN114646708A (en) Method for measuring content of sodium hyaluronate
CN110824047B (en) Method for judging fluff beating proportion of folium artemisiae argyi
CN104111293A (en) Measuring method of content of sugar in glycosidic surfactant product
CN102243215A (en) Detection method for water-soluble glucomannan
CN104280494B (en) Methyl vinyl phenyl raw-silastic continuously MPVQ molecular weight determinations
CN101059483B (en) Sugar-ferric hydroxide composite molecular weight and molecular weight distribution determination method
CN105842352A (en) Simple method for determination of content of sodium caprylate in human albumin through GC method
CN111307990A (en) Method for simultaneously determining content of cystine and methionine in artificial tiger bone powder
CN111579660A (en) Detection method for measuring average molecular weight and distribution coefficient of polyethylene glycol 4000 and polyethylene glycol powder

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant