Application of Tricine|CAS:5704-04-1| in Rabies Vaccine

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）The semi-finished products are prepared into finished products after sub packaging and freeze-drying.

The rabies vaccine product in the patent has no additives such as bovine serum, animal derived trypsin, gelatin and dextran, and has low DNA and protein residues in Vero cells and high safety. Moreover, the potency of vaccine products can still be maintained above 6.6IU/dose after 28 days at 37 ℃, with high thermal stability. In addition, each tank of 30L bioreactor can produce 160000 agents, with high process production efficiency.

Reference

CN111840535B The preparation of rabies vaccine using serum-free Vero cells and rabies virus