Preparation Of Rabies Vaccine By Perfusion Culture Of Vero Cells On Microcarriers

Rabies is a natural infectious viral disease of mammals most often transmitted through the bite of a rabid animal, with 100% of mortality. The only effective way to prevent the spread of the disease is the inoculation of rabies vaccine. Vero cell line is recommended by WHO for the production of human-used rabies vaccine. Rabies vaccine is routinely generated by culturing rabies virus in roller-bottles. Due to its limited surface area and culture condition, roller-bottle protocol produce low-density of cells, labor intensive, ease of contamination, therefore, it is hard to produce the vaccine products with good quality. Since the emerging of the micro-carrier bioreactor technology, many institutions throughout the world have done an extensive researches on the application of this technology in vaccine production, including batch culture, fed-batch culture, and semi-continuous feeding culture. However, the combination of micro-carrier bioreactor and Perfusion culture has not yet been investigated. Perfusion culture is a dynamic culture system, which constantly feeds in the fresh medium and pumps out waste medium without disturb the cells in the bioreactor, thus providing the continuous nutritious environment during the cell culture. By controling the perfusion speed supply enough nutrients, and to remove the toxic metabolites, the culture environment maintains a stable condition with low inhibition activity, therefore, it not only increase the cell density, but also improve the refinement procedure. We have further studied the application of perfusion culture in mix round Bioreactor, and have obtained a significant improvement.To compare the effect of different pre-treatment of micro-carrier on the cell attachment to the wall, we pre-incubate micro-carrier with either FBS-containing medium or distilled water for 24 hours. Our results showed that pretreatment of micro-carrier could increase its adsorption ability of cells, but did not have effect on the overall cell attachment. The cell attachment with pre-incubation of FBS medium was significantly higher than that with pre-incubation of distilled water within 120minutes, and then reduced to the equal level after 4 hours. We also observed the significant effect of stirring speed on the cell attachment, the optimal speed is at around 50rpm that resulted 80% of cell attachment within 4 hours, and when stirring speed was at 150rpm ,it caused the cell in un-attachment.In the bioreactor experiments, the initial concentration of 1 * 104/mL >. lxl 05/ml, and lxlO6/mL of cells were seeded, 90% of cells attached to bio-carrier after 6 hours of cell culture. It takes 12, 7, and 3 days to reach cell density peak (6-7x106/mL) for l*104/mL. lx105/mL, and lxlO6/mL groups respectively. The cells in lowest concentration group maintained long stable growth state, while the cells in the highest concentration group underwent fast senescence, resulting in a significant cell deprivation from micro-carrier.To study the effect of multiplicities of infection on output of virus, we inoculate O.OIMOI or 0.001MOI of rabies virus on day 7, when cell density reached 6xlO6/mL. Our results demonstrated that the virus titer reached to the peak between day 3 and day 7, and then gradually dropped. There is no significant difference between O.OIMOI and 0.001MOI groups. The titer in O.OIMOI group is overall slightly higher than in 0.001MOI group, and the former group reached the peak than the latter.We have studied the states of glucose consumption and the outcome of lactic acid and ammonia during virus culture in the bioreactor. Our results illustrated that the consumption of glucose and the production of lactic acid and ammonia maintained at very level at the early stage, but greatly increased in accordance with the fast cell replication. After inoculation of virus, the consumption of glucose and the production of lactic acid decline and then maintenance low level, but the production of ammonia is not affected. The input amount of perfusion medium is determined by the quantity of residue glucose in the bioreactor during the virus culture. The perfusion speed is positively proportional to the increase of cell number and the consumption of glucose, and later gradually decreased to low speed due to the slow-down of metabolic speed and the senescence of cells. The optimal condition is that perfusion volume is 0-3 times of working volume per day, and total volume is 8times of working volume for the whole procedure. After inoculation of virus, by changing the perfiision speed and recovery speed, we could collect a total of 8 times working volume of conditional medium during the whole procedure.Our study showed that the initial cell seeding of 105/mL in 5L bioreactor could result in 6X106/mL after 6-7 days culture, with infection index of 0.01MOI for 12-day continuous recovery of virus conditional medium, the final virus titer is above 6.01ogLD5o/mL. In the two batches of vaccine from trial experiments, after collection of virus conditional medium for 11-12 days, the total collection of conditional medium was subjected to the procedure including concentration, inactivation, and centrifugation, followed by the purification by column, the titers of the resultant products were 5.63IU/mL and 4.89IU/mL respectively. The residue of fetal bovine serum was less than 50ng/mL, and residue Vero DNA was less than 1 Ong/mL.The rabies vaccine production procedure developed in this research combined the perfiision culture of high-density cells and the continuous perfusion during the whole production period. This strategy not only can stabilize the culture condition during cell proliferation, but also provides sufficient nutrients to cells after the inoculation of rabies virus, at the same time, the virus titer can be regulated to favor the virus amplification.In summary, the combination of bioreactor and perfusion culture is suitable for the Vero cell-based rabies vaccine production. Through continuous perfusion of fresh medium, the PH, nutrients supplies, lactic acid, and ammonia can be monitored, re-feed, or excluded, providing the cells a better environment for proliferation. In addition, this approach reduced the number of professional staffs and GMP production space, comparing those in roller-bottle production line, therefore, significantly reducing the production cost. In conclusion, this lab-scale method of preparing rabies vaccine by perfusion culture on microcarries in bioreactor is viable.