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Method for monitoring tetanus toxoid or diphtheria toxoid

A technology for tetanus toxoid and diphtheria toxoid, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems that there is no method for evaluating or monitoring formaldehyde detoxification process

Active Publication Date: 2020-10-30
SHIMADZU (CHINA) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no methods for evaluating or monitoring formaldehyde detoxification processes other than testing for toxicity and immunogenicity

Method used

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  • Method for monitoring tetanus toxoid or diphtheria toxoid
  • Method for monitoring tetanus toxoid or diphtheria toxoid
  • Method for monitoring tetanus toxoid or diphtheria toxoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The monitoring of embodiment 1 tetanus toxoid

[0088] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.

[0089] 2. Experimental reagents: standard tetanus toxoid, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, RapiGest TM , trypsin, tetanus toxoid samples.

[0090] 3. Detection conditions:

[0091] Chromatographic conditions:

[0092] Chromatographic column: stationary phase 1 (biocompatible C18 chromatographic column);

[0093] Mobile phase: A: acetic acid aqueous solution (the volume ratio of acetic acid to water is 1:1000); B: acetic acid-acetonitrile mixture (the volume ratio of acetic acid to acetonitrile is 1:1000);

[0094] Gradient: 0~8min, 5%B~40%B; 8~8.1min, 40%B~100%B; 8.1~10min, 100%B; 10~10.1min, 100%B~5%B; 10.1~ 15min, 5% B; column temperature: 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0095] Mass Spectrometry Cond...

Embodiment 2

[0121] The monitoring of embodiment 2 tetanus toxoid

[0122] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.

[0123] 2. Experimental reagents: standard tetanus toxoid, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, RapiGest TM , chymotrypsin, tetanus toxoid samples.

[0124] 3. Detection conditions:

[0125] Chromatographic conditions:

[0126] Chromatographic column: stationary phase 1 (biocompatible C18 chromatographic column);

[0127] Mobile phase: A: acetic acid aqueous solution (the volume ratio of acetic acid to water is 1:1000); B: acetic acid-acetonitrile mixture (the volume ratio of acetic acid to acetonitrile is 1:1000);

[0128] Gradient: 0~8min, 5%B~40%B; 8~8.1min, 40%B~100%B; 8.1~10min, 100%B; 10~10.1min, 100%B~5%B; 10.1~ 15min, 5% B; column temperature: 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0129] Mass Spectrometry...

Embodiment 3

[0156] Example 3 Monitoring of diphtheria toxoid

[0157] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.

[0158] 2. Experimental reagents: diphtheria toxoid standard, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, RapiGest TM , trypsin, diphtheria toxoid samples.

[0159] 3. Detection conditions:

[0160] Chromatographic conditions:

[0161] Chromatographic column: stationary phase 1 (biocompatible C18 chromatographic column);

[0162] Mobile phase: A: acetic acid aqueous solution (the volume ratio of acetic acid to water is 1:1000); B: acetic acid-acetonitrile mixture (the volume ratio of acetic acid to acetonitrile is 1:1000);

[0163] Gradient: 0~8min, 5%B~40%B; 8~8.1min, 40%B~100%B; 8.1~10min, 100%B; 10~10.1min, 100%B~5%B; 10.1~ 15min, 5% B; column temperature: 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0164] Mass Spectrometry Co...

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Abstract

The invention discloses a method for monitoring tetanus toxoid or diphtheria toxoid. The method comprises the following steps of: (1) carrying out enzymolysis of a standard substance; (2) carrying outsolid-phase extraction to enrich a target peptide fragment; (3) performing high performance liquid chromatography tandem mass spectrometry analysis; (4) drawing a standard characteristic peptide fragment table to obtain relative response intensity of each characteristic peptide fragment of a standard variety; (5) detecting a sample, and calculating to obtain the relative response intensity and arecovery rate of each characteristic peptide fragment in the sample; and (6) evaluating the detoxification effect according to the number of peptide fragments with good characteristic peptide fragmentrecovery rate. According to the monitoring method of the invention, the method for quantitatively monitoring the process stability of tetanus toxoid and diphtheria toxoid is implemented for the firsttime, the problem that a formaldehyde detoxification protein process lacks a monitoring method is solved, effective quantitative data is provided for production optimization of tetanus vaccine, tetanus-diphtheria bivalent vaccine, pertussis-diphtheria-tetanus triple vaccine, pentavaccine and proteoglycan protein conjugate vaccine and research and development of novel proteoglycan protein conjugate vaccine, and the vaccine quality improvement process and new product research and development are accelerated.

Description

technical field [0001] The invention relates to a monitoring method for tetanus toxoid (formaldehyde-detoxified tetanus protein) or diphtheria toxoid (formaldehyde-detoxified diphtheria protein), and belongs to the technical field of vaccine quality evaluation. Background technique [0002] Chemical detoxification is a common production process for vaccines. Through the interaction between formaldehyde or glutaraldehyde and proteins, the toxicity of proteins is reduced while retaining their immunogenicity. Chemically detoxified exotoxin proteins and outer membrane proteins can be directly used as the main components of vaccines, such as formaldehyde-detoxified diphtheria protein (diphtheria toxoid) and formaldehyde-detoxified tetanus protein (tetanus toxoid). The two are prepared in proportion with formaldehyde (glutaraldehyde) detoxified Bordetella pertussis protein to become DPT vaccine. Formaldehyde-detoxified proteins can also be used as raw materials for vaccine produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027G01N2030/062
Inventor 龙珍卫辰马霄李月琪谭亚军李茂光李长坤黄涛宏
Owner SHIMADZU (CHINA) CO LTD
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