Method for detecting molecular weight of group B streptococcus capsular polysaccharide

Abstract

The invention provides a method for detecting the molecular weight of group B streptococcus capsular polysaccharide, and belongs to the technical field of polysaccharide molecular weight detection. The method comprises the following steps: preparing a plurality of glucan standard substances with different molecular weights into a standard solution by using a sample solvent, detecting the standard solution by using high performance liquid chromatography, detecting the peak appearance condition of the standard solution by using a differential detector, recording the retention time, and drawing a standard curve by taking the retention time as a horizontal coordinate and a logarithm value of the molecular weight of the glucan as a vertical coordinate; replacing an original solvent of a to-be-detected sample with a sample solvent through ultrafiltration to obtain a to-be-detected sample solution, detecting the to-be-detected sample solution by using high performance liquid chromatography, detecting the peak appearance condition of the to-be-detected sample by using a differential detector, recording the retention time, and substituting the retention time of the to-be-detected sample into the standard curve to calculate the molecular weight of the to-be-detected sample. According to the detection method disclosed by the invention, R2 of a standard curve is equal to 0.996387, and the detection method has the characteristics of high accuracy, good repeatability, good stability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide molecular weight detection, and in particular relates to a method for detecting the molecular weight of group B streptococcal capsular polysaccharide. Background Art

[0002] Currently, there are a variety of vaccines for preventing group B streptococcal infection that are mainly composed of polysaccharides and combined with carrier proteins and are in Phase II clinical trials. Group B streptococcal capsular polysaccharide (CPS) is the main active ingredient of the vaccine. Studies have shown that the molecular weight of CPS directly affects the immunogenicity of the polysaccharide vaccine; and in combination vaccines, CPS as an intermediate product is also an important indicator of quality control.

[0003] Currently, Pfizer's hexavalent polysaccharide-CRM197 conjugate vaccine has completed phase II clinical trials. The vaccine has shown good tolerance, can produce sufficient maternal immune response and can effectively cross the placenta to reach the fetus, thereby protecting the fetus from GBS invasion. The phase II clinical results of GSK's trivalent polysaccharide-CRM197 conjugate vaccine showed that the specific antibodies in maternal and neonatal serum and the specific antibodies in maternal milk were significantly higher than those in the control group and lasted until 90 days after delivery. However, the "Chinese Pharmacopoeia" and "European Pharmacopoeia" have not yet included the manufacturing and identification process of Group B Streptococcus polysaccharide conjugate vaccines, and have not specified the molecular weight of polysaccharides. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for detecting the molecular weight of group B streptococcal capsular polysaccharide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting the molecular weight of group B streptococcal capsular polysaccharide, comprising the following steps:

[0007] 1) multiple dextran standards with different molecular weights are prepared into standard solutions with a sample solvent, the standard solutions are detected by high performance liquid chromatography, the peaks of the standard solutions are detected by a differential detector, the retention time is recorded, and a standard curve is drawn with the retention time as the abscissa and the logarithm of the molecular weight of the dextran as the ordinate;

[0008] 2) replacing the original solvent of the sample to be tested with the sample solvent by ultrafiltration to obtain a sample solution to be tested, detecting the sample solution by high performance liquid chromatography, detecting the peak of the sample to be tested by a differential detector, recording the retention time, and substituting the retention time of the sample to be tested into the standard curve of step 1) to calculate the molecular weight of the sample to be tested.

[0009] Preferably, the sample solvent consists of the following ingredients: 5-9 g / L disodium hydrogen phosphate, 4-8 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA.

[0010] Preferably, the mobile phase for high performance liquid chromatography detection consists of the following components: 18-24 g / L disodium hydrogen phosphate, 13-22 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA.

[0011] Preferably, the chromatographic column used for the high performance liquid chromatography detection is from Shodex Co., Ltd. of Japan, and an OHpak SB-G 6B guard column, an OHpak SB-805HQ liquid chromatography column, and an OHpak SB-804HQ liquid chromatography column are connected in series.

[0012] Preferably, the detection conditions of the HPLC are: detector temperature 35-45° C., flow rate 0.4-1 mL / min, sample injection volume 45-55 μL, and running time 70-80 min.

[0013] Preferably, the serotype of the group B streptococcus is type Ia, type Ib, type II, type III, type IV, type V, type VI, type VII, type VIII, or type IX.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The invention adopts high performance size-exclusion chromatography-differential refractometry (HPSEC-RI) to detect a polysaccharide molecular weight standard with properties similar to those of a sample to be tested, and uses this to draw a mass distribution curve of the molecular weight of the standard as a function of sample elution time or volume, records the corresponding elution index of the sample to be tested, and calculates its molecular weight through the mass distribution curve; thereby constructing a method for detecting the molecular weight of group B streptococcal capsular polysaccharide, which can detect CPS of various types of group B streptococci such as type Ⅰa, type Ⅰb, type Ⅲ, type Ⅴ, etc., and the R value of the standard curve of the detection method of the invention is 0.0447 W / m. 2 =0.996387. This detection method has the characteristics of high accuracy, good repeatability and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The standard curve diagram of the molecular weight standard in Examples 1, 2, 3, and 4;

[0017] Figure 2 This is the GPC diagram of type Ia CPS in samples from different batches in Example 1;

[0018] Figure 3 This is the GPC diagram of Type Ib CPS in samples from different batches in Example 2;

[0019] Figure 4 This is the GPC diagram of type III CPS in samples from different batches in Example 3;

[0020] Figure 5 This is the GPC diagram of type V CPS in samples from different batches in Example 4. DETAILED DESCRIPTION

[0021] The present invention provides a method for detecting the molecular weight of group B streptococcal capsular polysaccharide, comprising the following steps:

[0022] 1) multiple dextran standards with different molecular weights are prepared into standard solutions with a sample solvent, the standard solutions are detected by high performance liquid chromatography, the peaks of the standard solutions are detected by a differential detector, the retention time is recorded, and a standard curve is drawn with the retention time as the abscissa and the logarithm of the molecular weight of the dextran as the ordinate;

[0023] 2) replacing the original solvent of the sample to be tested with the sample solvent by ultrafiltration to obtain a sample solution to be tested, detecting the sample solution by high performance liquid chromatography, detecting the peak of the sample to be tested by a differential detector, recording the retention time, and substituting the retention time of the sample to be tested into the standard curve of step 1) to calculate the molecular weight of the sample to be tested.

[0024] In the present invention, multiple dextran standards of different molecular weights are prepared into standard solutions with sample solvents, and the standard solutions are detected by high performance liquid chromatography. A standard curve is drawn with retention time as the horizontal axis and the logarithm of the molecular weight of the dextran as the vertical axis. The dextran is preferably pullulan; the sample solvent is composed of the following components: 5-9 g / L disodium hydrogen phosphate, 4-8 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA; the amount of the disodium hydrogen phosphate is preferably 6-8 / L, the amount of the sodium dihydrogen phosphate is preferably 5-7 g / L, and the amount of EDTA is preferably 0.02-0.03 g / L; the sample solvent is preferably ultrafiltered using a PTFE filter membrane; the concentration of the standard solution is preferably 1-3 mg / mL, and more preferably 2 mg / mL; the mobile phase for high performance liquid chromatography detection is composed of the following components: 18-24 g / L disodium hydrogen phosphate, 13-22 g / L sodium dihydrogen phosphate, 0.01-0.04 g / L EDTA; the dosage of the disodium hydrogen phosphate is preferably 20-22 g / L, the dosage of the sodium dihydrogen phosphate is preferably 15-20 g / L, and the dosage of the EDTA is preferably 0.02-0.03 g / L; the mobile phase is preferably ultrafiltered with a PTFE filter membrane; the chromatographic column used for the high performance liquid chromatography detection is from Japan Shodex Co., Ltd., OHpak SB-G6B guard column, OHpak SB-805HQ liquid chromatography column, and OHpak SB-804HQ liquid chromatography column are connected in series; the detection conditions of the high performance liquid chromatography are: detector temperature 35-45°C, flow rate 0.4-1 mL / min, sample injection volume 45-55 μL, and running time 70-80 min; the temperature of the detector is preferably 38-42°C; the flow rate is preferably 0.5-0.8 mL / min; the sample injection volume is preferably 48-52 μL; the running time is preferably 72-78 min.

[0025] In the present invention, the original solvent of the sample to be tested is replaced with the sample solvent by ultrafiltration to obtain a sample solution to be tested, the sample solution to be tested is detected by high performance liquid chromatography, the peak of the sample to be tested is detected by a differential detector, the retention time is recorded, and the retention time of the sample to be tested is substituted into the standard curve of step 1) to calculate the molecular weight of the sample to be tested. The ultrafiltration membrane molecular weight cutoff is preferably 50 to 200 Da, and more preferably 80 to 150 Da; the sample solvent is composed of the following components: 5 to 9 g / L disodium hydrogen phosphate, 4 to 8 g / L sodium dihydrogen phosphate, 0.01 to 0.04 g / L EDTA; the dosage of the disodium hydrogen phosphate is preferably 6-8 g / L, the dosage of the sodium dihydrogen phosphate is preferably 5-7 g / L, and the dosage of EDTA is preferably 0.02-0.03 g / L; the sample solvent is preferably ultrafiltered using a PTFE filter membrane; after replacing the sample solvent, centrifugation, filtration, and ultrasound are performed; the centrifugation speed is preferably 10000-14000 rpm, and more preferably 11000-13000 rpm; the centrifugation time is preferably 5-15 min, and more preferably 8-12 min; the filtration preferably uses a 0.22 μm aqueous phase filter membrane; the ultrasonic conditions are preferably 30-50 kHz, 5-15 min, and more preferably 35-45 kHz, 8-12 min; the role of ultrasound is to remove bubbles in the solution. The mobile phase for the HPLC detection is composed of the following components: 18-24 g / L disodium hydrogen phosphate, 13-22 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA; the amount of the disodium hydrogen phosphate is preferably 20-22 g / L, the amount of the sodium dihydrogen phosphate is preferably 15-20 g / L, and the amount of EDTA is preferably 0.02-0.03 g / L; the mobile phase is preferably ultrafiltered with a PTFE filter membrane; the chromatographic columns used for the HPLC detection are OHpak SB-G6B guard column, OHpak SB-805HQ liquid chromatography column, and OHpak The SB-804HQ liquid chromatography column is connected in series; the detection conditions of the high performance liquid chromatography are: detector temperature 35-45°C, flow rate 0.4-1mL / min, sample injection volume 45-55μL, and running time 70-80min; the temperature of the detector is preferably 38-42°C; the flow rate is preferably 0.5-0.8mL / min; the sample injection volume is preferably 48-52μL; the running time is preferably 72-78min; the serotype of the group B streptococcus is type Ia, type Ib, type II, type III, type IV, type V, type VI, type VII, type VIII, and type IX.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Materials used in the present invention:

[0028] Group B Streptococcus capsular polysaccharide. Extracted and purified in our laboratory.

[0029] Group B Streptococcus type Ia, type Ib, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX.

[0030] Reagents and instruments: electronic balance (Sartoraus), high-performance liquid chromatograph (Waters).

[0031] Sodium dihydrogen phosphate, disodium hydrogen phosphate, and EDTA were purchased from DiKMA. Dextran standard was purchased from Shodex Co., Ltd., Japan (Cat. No. P-82).

[0032] Example 1

[0033] (1) Sample solvent: 1 L 50 mM phosphate buffer: weigh 7.1 g disodium hydrogen phosphate, 6.0 g sodium dihydrogen phosphate, and 0.0292 g EDTA, dilute to 1 L with deionized water, and ultrafilter using a PTFE filter membrane.

[0034] Mobile phase: 1L 150mM phosphate buffer solution: weigh 21.3g disodium hydrogen phosphate, 18.0g sodium dihydrogen phosphate, 0.0292g EDTA, dilute to 1L with deionized water, and ultrafilter using a PTFE filter membrane.

[0035] (2) Establishing a standard curve

[0036] Weigh 40 mg of P-20, P-50, P-100, P-200, P-400 and P-800 pullulan polysaccharide standards respectively into a 20 ml volumetric flask, dissolve with sample solvent and dilute to the mark, which is a 2 mg / ml standard solution. Take 350 μl of each and add it to the HPLC loading bottle for injection.

[0037] HPLC loading parameter settings: column temperature 40°C, mobile phase 150mM phosphate buffer solution flow rate 0.6ml / min, injection volume 50μl, running time 75min. The chromatographic columns used for the high performance liquid chromatography detection are from Japan Shodex Co., Ltd., OHpak SB-G 6B guard column, OHpak SB-805HQ liquid chromatography column, and OHpak SB-804HQ liquid chromatography column connected in series.

[0038] During the detection process, the differential display refractive signal is integrated according to the GPC reanalysis data processing method in the Shimadzu Labsolution software. The chromatogram is integrated and the logarithmic value of the pullulan calibrated weight average molecular weight (Da) in the standard solution is used as the ordinate and the retention time (min) is used as the abscissa to draw a cubic equation standard curve. The equation of the standard curve is: Log (molecular weight [n]) = 1.92 × 101-1.44 × 100 × T + 5.46 × 10-2 × T2-7.70 × 10-4 × T3, R2 = 0.996387, the standard curve is as follows Figure 1 shown.

[0039] (3) Preparation of Type Ia CPS Analysis Samples

[0040] The test sample was placed in an ultrafiltration tube and subjected to 100× ultrafiltration with the sample solvent. After the liquid was replaced, it was centrifuged at 12000 rpm for 10 min and filtered through a 0.22 μm aqueous phase filter membrane. The solution was ultrasonically vibrated (40 kHz, 10 min) to remove bubbles, and 350 μl was taken and added to the HPLC loading bottle.

[0041] (4) Detection of Group B Streptococcus Type Ia CPS

[0042] The HPLC detection method is the same as step (2). The peak retention time of the sample to be tested is detected and recorded. The standard curve and regression equation of step (2) are used to calculate the 1gMp of the sample, and further obtain its corresponding molecular weight (Mp).

[0043] Experimental results: Figure 2 The peak retention time of the sample to be tested was detected and recorded, and the molecular weight (MP) of the sample was calculated using the established standard curve and regression equation. The molecular weight of type Ⅰa capsular polysaccharide was concentrated around 150 kDa, which was consistent with the results reported in the literature.

[0044] Example 2

[0045] The difference from Example 1 is that the "type Ia CPS analysis sample" is replaced by the "type Ib CPS analysis sample".

[0046] Experimental results: Figure 3 The peak retention time of the sample to be tested was detected and recorded, and the molecular weight (MP) of the sample was calculated using the established standard curve and regression equation. The molecular weight of type Ib capsular polysaccharide was concentrated around 110 kDa, which was consistent with the results reported in the literature.

[0047] Example 3

[0048] The difference from Example 1 is that the "type Ia CPS analysis sample" is replaced by the "type III CPS analysis sample".

[0049] Experimental results: Figure 4 The peak retention time of the sample to be tested was detected and recorded, and the molecular weight (MP) of the sample was calculated using the established standard curve and regression equation. The molecular weight of type III capsular polysaccharide was concentrated around 80 kDa, which was consistent with the results reported in the literature.

[0050] Example 4

[0051] The difference from Example 1 is that the "type Ia CPS analysis sample" is replaced by the "type V CPS analysis sample".

[0052] Experimental results: Figure 5 The peak retention time of the sample to be tested was detected and recorded, and the molecular weight (MP) of the sample was calculated using the established standard curve and regression equation. The molecular weight of type V capsular polysaccharide was concentrated around 50 kDa, which was consistent with the results reported in the literature.

[0053] It can be seen from the above examples that the detection method of the present invention can detect CPS of various types of group B streptococci such as type Ⅰa, type Ⅰb, type Ⅲ, type Ⅴ, etc. The R value of the standard curve of the detection method of the present invention is 2 =0.996387. This detection method has the characteristics of high accuracy, good repeatability and good stability.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the molecular weight of group B streptococcal capsular polysaccharide, characterized in that: The following steps are involved: 1) multiple dextran standards of different molecular weights are prepared into standard solutions with a sample solvent, the standard solutions are detected by high performance liquid chromatography, the peaks of the standard solutions are detected by a differential detector, the retention time is recorded, and a standard curve is drawn with the retention time as the abscissa and the logarithm of the molecular weight of the dextran as the ordinate; 2) replacing the original solvent of the sample to be tested with the sample solvent by ultrafiltration to obtain a sample solution to be tested, detecting the sample solution by high performance liquid chromatography, detecting the peak of the sample to be tested by a differential detector, recording the retention time, and substituting the retention time of the sample to be tested into the standard curve of step 1) to calculate the molecular weight of the sample to be tested.

2. The detection method according to claim 1, characterized in that: The sample solvent consists of the following components: 5-9 g / L disodium hydrogen phosphate, 4-8 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA.

3. The detection method according to claim 1, characterized in that: The mobile phase for high performance liquid chromatography detection consists of the following components: 18-24 g / L disodium hydrogen phosphate, 13-22 g / L sodium dihydrogen phosphate, and 0.01-0.04 g / L EDTA.

4. The detection method according to claim 1, characterized in that: The chromatographic columns used for the high performance liquid chromatography detection are from Japan Shodex Co., Ltd., and are OHpak SB-G 6B guard column, OHpak SB-805HQ liquid chromatography column, and OHpak SB-804HQ liquid chromatography column connected in series.

5. The detection method according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography are: detector temperature 35-45° C., flow rate 0.4-1 mL / min, sample injection volume 45-55 μL, and running time 70-80 min.

6. The detection method according to claim 1, characterized in that: The serotypes of the group B streptococci are type Ia, type Ib, type II, type III, type IV, type V, type VI, type VII, type VIII, and type IX.

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