Preparation method of diphtheria toxoid vaccine

Abstract

The invention discloses a preparation method of a diphtheria toxoid vaccine. The diphtheria toxoid vaccine is prepared by taking a diphtheria bacillus strain as a raw material and then carrying out the steps of diphtheria toxoid culture, bacteria-liquid separation, ultrafiltration and concentration, two-time salting out, ultrafiltration and desalination and the like. According to the preparation method, the thalli of the culture solution is removed by using a hollow fiber filter membrane and then refining is carried, so that the pore blockage caused by the accumulation of the thalli and other impurity fragments in the subsequent filter process is prevented; through an improved salting out method, the toxin proteins and other allergens in the culture solution are effectively removed; and a tangential flow ultrafiltration method is used for both the concentration of the culture solution and the desalting method after the salting out, so that the antigen damage caused by the shearing to the toxin proteins is decreased and the precipitation of the proteins is avoided. According to the preparation method, the preparation time of the diphtheria toxoid vaccine is shortened and the production efficiency is improved.

Description

technical field [0001] The invention relates to a preparation method of a diphtheria toxoid vaccine. Background technique [0002] The diphtheria toxoid vaccine is made from diphtheria bacillus. The toxin secreted by the diphtheria bacillus is a single polypeptide chain without sugar groups and prosthetic groups. The protein is composed of 560 amino acids and has a molecular weight of 62kDa. It is a zymogen , Only under the action of protease, it can be cut into the gap toxin, it has biological activity. The isoelectric point is about pH4.1. At pH6, it will lose its activity and solidify quickly when heated to 55°C. Diphtheria toxoid includes two fragments, A and B, among which the B fragment is a receptor-binding and membrane-penetrating fragment, and the A fragment is a toxic fragment, which finally causes cell death by inhibiting the enzyme activity in protein synthesis. It can also be toxic to distal tissues and organs, especially the heart and the peripheral and centr...

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Problems solved by technology

[0003] The diphtheria toxoid vaccine obtained by the existing preparation method has poor selectivity, low quality, low purity (400-600Lf / mg TN), dark color, and is still a mixture of multiple components when checked by polyacrylamide electrophoresis

In particular, the separation of bacteria liquid, the concentration of diphtheria toxin solution, the purification of diphtheria toxin, and desalination are relatively simple and extensive. Some processes do not meet the requirements of GMP and are eliminated, and some processes will be restricted in the use of large-scale production conditions.

For example, in the bacterial liquid separation process, the volume to be processed in general large-scale production must reach more than 500L. If centrifugation is used, it is not suitable, the processing time will be very long, and a lot of labor is consumed; if canvas filtration is used for sterilization, the filtration is not complete. And the canvas material does not meet the defects of GMP requirements; the disadvantage of plate and frame filtration is that the filter plate is easy to block, and the filtration time may be too long; tangential flow filtration is not easy to block, and the filtration speed is ideal, but the shear force on bacteria and proteins larger

The concentration of diphtheria toxoid solution can be precipitated by a precipitant, filtered with silk cloth to obtain the precipitate, and then resuspended, but the use of silk cloth in production does not meet the GMP requirements; tangential flow filtration also has the effect of shearing the protein Problems with higher forces and slower filtration rates

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