In vitro adventitious agents detection
Viruses can be present at low concentrations in cell lines and cell-derived products. Very sensitive assays involving detector cell lines are needed to detect viruses. To guarantee the absence of viral contamination in cell lines and their derivatives, Texcell has developed protocols (starting in 1987) that are fully compliant with regulatory guidelines. The number of erythrocyte species used is never reduced (human group O Rhesus Negative, chicken and guinea pig). Internal interference and inhibition controls are always included.
Lytic and/or haemadsorbing viruses are detected after inoculation of the sensitive indicator cell line with the test article. Indicator cells are examined regularly for signs of a cytopathic effect (CPE) over a period of 14 or 28 days (with a passage after a period of 14 days) and two haemadsorption assays are carried out with each of the three erythrocyte species.
For the evaluation of cell-line-derived biologicals used as medicinal products, the US FDA's note on "Points to Consider on the Characterisation of Cell Lines used to Produce Biologicals" recommends (as a minimum measure) that in addition to monolayer culture of the same species and tissue type as that used in production, a normal human diploid cell line (such as MRC-5) and a monkey kidney cell line (such as Vero) should be examined over a period of 14 days (3 detector cell lines are therefore used). For test materials that can support the growth of human cytomegalovirus, a 28-day culture period is recommended.
For serum or cell lines grown with FBS, Texcell provides you with the specific assay required in guidelines 9CFR 113.53 and CPMP/BWP/1793/02:
Protocol 0969: In vitro assay for the detection of bovine viruses (9CFR, 2 cells) |
Product to be tested: raw materials |
This assay includes two detector cell lines for screening adventitious viruses present into raw materials such as serum: Vero cell line (a simian cell line) and BT cell line (a bovine cell line). The test article is inoculated into both cell lines and the culture is monitored for CPE for three 7-day periods. At the end of the test, haemadsorption assays are carried out with erythrocytes (human group O Rhesus Negative, chicken and guinea pig) and the cells inoculated with the test article are fixed onto slides. The fixed cells are then examined for the presence of specific bovine viruses using fluorescent antibody staining techniques. 9 viruses are screened: bovine viral diarrhoea virus (BVDV); bovine adenovirus type 3 (BAV3); bovine parvovirus (BPV); bluetongue virus (BTV), BT-2 strain; bovine syncytial virus (BRSV), TN strain; reovirus type 3 (Reo3), Abney strain and rabies virus. The latter two are screened for using the Vero cell line. Bovine parainfluenza virus type 3 (PI3) and infectious bovine rhinotracheitis virus (IBRV) are screened for by haemagglutination and a CPE assay on BT cells, respectively |
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Following a specific request by a client, Texcell provides protocols including only the detection of lytic and/or haemadsorbing bovine viruses (such as lytic strains of bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3, Infectious bovine rhinotracheitis virus (IBRV), and Enterovirus species). In such a case, particular attention should be paid to bovine polyomavirus (BpyV) and non-cytopathic strains of BVDV. These viruses can then be detected using a specific Q-PCR protocol (See Q-PCR section )
