The production technology of Classical swine fever vaccine
Classical swine fever is a highly contagious viral disease. The morbidity and mortality can reach 100% in the acute form, which is caused by a virulent strain of virus, induces huge economic losses. The early-period vaccine for epidemic prevention was produced with spleen and lymph node tissues of rabbits (the lapinized vaccine). Later, due to animal welfare issues, the use of lapinized vaccine has been banned in Taiwan since 2022, and replaced by tissue cultured vaccine. In 2007, our institute obtained the production license of tissue culture live virus vaccine for classical swine fever (animal drug manufacturing license No. 08325), but this vaccine was not produced and sold at that period of time. In 2019, the establishment of large-scale production technology was completed, and since then, the vaccine started be produced and sold successfully. Furthermore, this tissue-culture vaccine was transferred to local private animal vaccine factory. In 2023, a vaccine pharmaceutical factory successfully obtained a production license for this live vaccine and has planned to export this vaccine for international markets.
The Recombinant Factor C Assay Technique for Bacterial Endotoxins in Animal Drugs
The main component of bacterial endotoxins is lipopolysaccharide (LPS), which makes up about 70% of the outer membrane of Gram-negative bacterial cell walls. During the bacterial growth stage, endotoxins are released in minute quantities, but when bacteria decompose or die, they are released in large amounts. Lipid A triggers immune responses in animals, producing cytokines, activating clotting reactions and complements, which cause symptoms such as fever, hypoglycemia, hypotension, or disseminated intravascular coagulation. The bacterial endotoxins test (BET) is currently included in the inspection standards for animal drugs to ensure the safety of injections. In addition to legally testing for bacterial endotoxins, domestic animal drug manufacturers should establish a risk assessment mechanism to consider factors such as raw materials, production equipment, environment, water quality, and packaging materials as potential sources of bacterial endotoxins, reduce the endotoxin limit in products. Currently, pharmacopoeias have three methods for bacterial endotoxin testing: gel-clot technique, turbidimetric technique, and chromogenic technique. These methods utilize the BET reagent made from the aqueous extract of horseshoe crab amoebocytes to form a gel characteristic when combined with bacterial endotoxins, allowing for the detection or quantification of bacterial endotoxins. However, due to the global decline in the number of horseshoe crabs and the international promotion of the 3R principle for animal testing, synthetic recombinant Factor C (rFC) or recombinant cascade reagent (rCR) are being used as alternatives to horseshoe crab source BET reagents. The 10.3 version of the European Pharmacopoeia updated in 2022 has officially listed rFC reagent as an official method. This study aims to establish the rFC assay technique for bacterial endotoxins in animal drugs. We followed the European Pharmacopoeia specifications to perform analytical method validation and tested 13 animal injectable products for bacterial endotoxins. In the preliminary test, the correlation coefficient (r) of the standard curve within the 0.005-5.0 EU/mL test range was all greater than 0.980, and the recovery rate of the interference factor test was between 50-200%, all in compliance with pharmacopoeia specifications. Therefore, the rFC assay can be used for testing in animal drugs. It is hoped that it can be promoted for use in domestic animal pharmaceutical factories in the future, reducing our reliance on horseshoe crab sourced reagents.