Method for determining binding rate of proteoglycan protein binding site in proteoglycan protein binding vaccine
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A polysaccharide-protein-conjugated vaccine technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problem of no testing method
Active Publication Date: 2020-10-30
SHIMADZU (CHINA) CO LTD +1
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Although the detoxification of the carrier protein and the binding rate of the polysaccharide-protein binding si
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Embodiment 1
[0083] Example 1 Determination of Polysaccharide Protein Binding Site Binding Rate in Polysaccharide Protein Binding Vaccine Taking Tetanus Toxoid as Carrier
[0084] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.
[0089] Mobile phase: A: acetic acid aqueous solution (the volume ratio of acetic acid to water ...
Embodiment 2
[0134] Example 2 Determination of the binding rate of the polysaccharide protein binding site in the polysaccharide protein conjugate vaccine with CRM197 protein as the carrier
[0135] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.
[0136] 2. Experimental reagents: CRM197 protein standard (100 μg / L), TCEP, iodoacetamide (IAA), ammonium bicarbonate, urea, trypsin, meningitis vaccine A, W, C, Y with CRM197 protein as the carrier , Hib vaccine, 13-valent pneumonia vaccine 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, 19A and 23F.
[0140] 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 1:100...
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Abstract
The invention discloses a method for determining a binding rate of a proteoglycan protein binding site in a proteoglycan protein conjugate vaccine. The method comprises the following steps of: (1) carrying out enzymolysis on a standard substance; (2) carrying out solid-phase extraction to enrich a target peptide fragment; (3) performing high performance liquid chromatography tandem mass spectrometry analysis; (4) drawing a standard curve to obtain a standard working curve equation; (5) detecting a sample, and calculating to obtain the concentration of each characteristic peptide fragment in the sample; and (6) calculating a proteoglycan binding rate of each characteristic peptide fragment and a protein residue rate of each site. According to the method, the multi-site quantitative monitoring of the proteoglycan protein binding rate of the proteoglycan protein conjugate vaccine is achieved, the tetanus protein relates to 20 monitoring sites, and the CRM197 relates to 16 monitoring sites; the method not only can detect the residual protein content, but further can evaluate the binding rate of the proteoglycan protein binding site of the proteoglycan protein conjugate vaccine, can beused for researching polysaccharide or proteoglycan protein conjugate vaccines with the same protein residual rate but different activity and toxicity, and can provide a direct monitoring method for improving a proteoglycan protein binding process.
Description
technical field [0001] The invention relates to a method for determining the binding rate of polysaccharide-protein binding sites in polysaccharide-protein binding vaccines, belonging to the technical field of vaccine quality evaluation. Background technique [0002] Polysaccharide-protein conjugate vaccines are a type of vaccine formed by combining polysaccharides with proteins through chemical bonds. Proteins used in polysaccharide-protein conjugate vaccines are called protein carriers. Commonly used protein carriers include CRM197 protein, diphtheria toxoid (ie formaldehyde-detoxified diphtheria protein) and tetanus toxoid (ie formaldehyde-detoxified tetanus protein). Common polysaccharide-protein conjugate vaccines include quadrivalent meningitis vaccine (A, C, W, Y), Hib vaccine and 13-valent pneumonia polysaccharide vaccine (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C , 19F, 19A and 23F). Compared with polysaccharide vaccines, polysaccharide-protein conjugate vaccines have ...
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