Samy Majoul

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells.

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines

IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated.

DEVELOPMENT OF A NOVEL PROCESS TO PRODUCE A VETERINARY RABIES VACCINE IN BHK-21 CELLS GROWN ON MICROCARRIERS IN A 20 LITER BIOREACTOR

We studied BHK-21 cells growth in a 2 liter bioreactor and investigated the effects of microcarriers concentration, nature of growth medium, culture mode and serum concentration. We have reached a cell density of 4×106 cells/ml under the following growth conditions: Medium: MEM supplemented with Hank’s salts, non essential amino acids and 5% FCS, Culture mode: perfusion, Microcarriers concentration: 4 g/l of Cytodex 3

Assay of Serum and Animal Protein Free Media for the Production of a Veterinary Rabies Vaccine by BHK-21 Cells

Rabies encephalo-myelitis is often fatal. Sixty thousand death and 10 millions post-exposure treatment are reported yearly among human. This viral infection remains a serious public health concern particularly in developing countries where dog, especially stray dog, is the main reservoir. Thus, mass vaccination of dogs seems to be the best way to prevent the contamination of man in these countries.