Tricine (CAS:5704-04-1) is an excellent amphoteric biological good's buffer. The main purpose of Tricine is to effectively inhibit Gram-positive bacteria, Gram- negative bacteria, yeasts and molds, and it is also a pH stabilizer for many vaccine culture media. The application of Tricine in rabies vaccine is introduced in CN111840535B patent, in which Tricine is a component of serum-free culture medium, mainly used as PH value stabilizer. This week, YACOO editor introduced the specific preparation process of rabies vaccine. The specific process is as follows:

1. Preparation of stock solution

（1）Cell culture: Vero cells were perfused in serum-free medium in the bioreactor to 0.6~0.7×10⁷cells/ml, start virus inoculation, wherein the Vero cell serum free culture medium contains 2～4mmol/L glutathione, 0.5～2.0g/L fructose and 0.5～2.0g/L Tricine.

（2）Virus inoculation: inoculate rabies virus rPV-2061 into cells at 0.0025-0.01 MOI, and culture for more than 6 hours. Start to use serum free virus maintenance solution for perfusion culture. The formula of Vero cell serum free maintenance solution contains 2～4mmol/L glutathione, 0.5～2.0g/L fructose, 0.5～2.0g/L Tricine and 2～3g/L sodium bicarbonate.

（3）Virus collection: after virus inoculation, the virus will be harvested from the 3-17 days, and the virus harvest liquid will be obtained

（4）Inactivation: use β- Lactone inactivates the virus.

（5）Enzymatic digestion: The host DNA of Vero cells is digested with nuclease

（6）Purification: Purify the virus by molecular sieve chromatography. After purification, add 3% (m/v) human serum albumin and 5% (m/v) maltose as stabilizers to prepare the original solution

2. Finished product preparation

（1）Add an appropriate amount of freeze-drying protective agent as the stabilizer to obtain semi-finished products

（2 ） Die Halbfabrikate werden nach Unterverpackung und Gefriertrocknung zu Endprodukten verarbeitet.

Das Tollwutimpfstoffprodukt im Patent enthält keine Zusätze wie Rinderserum, tierisches Trypsin, Gelatine und Dextran und weist geringe DNA- und Proteinrückstände in Vero-Zellen und eine hohe Sicherheit auf. Darüber hinaus kann die Wirksamkeit von Impfstoffprodukten bei hoher thermischer Stabilität nach 28 Tagen bei 37 °C immer noch über 6,6 IE/Dosis gehalten werden . Darüber hinaus kann jeder Tank eines 30-Liter-Bioreaktors 160.000 Wirkstoffe mit hoher Prozessproduktionseffizienz produzieren.

Referenz

CN111840535B Die Herstellung eines Tollwutimpfstoffs unter Verwendung von serumfreien Vero-Zellen und dem Tollwutvirus rPV-2061.