PILOT LOT PRODUCTION OF A CHROMATOGRAPHICALLY PURIFIED VERO CELL RABIES VACCINE

CARLOS PALACIOS; SANTIAGO CHIAPPINI; ANALÍA DE NICHILO; EZEQUIEL NUGUER; OSCAR LARGHI; ALEJANDRO PAROLA

Fort Collins, Colorado

Congreso; 26TH ANNUAL RABIES IN THE AMERICAS CONFERENCE; 2015

RITA

Rabies vaccine production in Latin America is still based on nerve tissue substrate (NTV), it is well known that these vaccines are not safe inducing adverse reactions, and are less immunogenic than cell culture derived vaccines. More than three decades ago, WHO has recommended NTVs discontinuation. In this regard, in a previous report, we showed the development of a chromatographically purified Vero cell rabies vaccine (CPRV), describing generation and characterization of viral and cell banks, viral production in spinner flasks and a purification process involving different chromatographic steps. The aim of this study is to scale up both upstream and downstream steps of a CPRV production. In this research, Vero cells were grown in a five-liter stirred tank bioreactor (BIOSTAT Aplus, Sartorius Stedim Biotech) using Cytodex 1 microcarriers. The cell culture was started by seeding 0.20-0.25 x 106 cells/ml in M199 cell culture medium, and infected at a MOI of 0.1 ID50/ml once cell density reached levels between 2,5 and 3,5 x 106 cells/ml. Harvests were taken every 48 h, during at least 14 days post infection. Cell culture was monitored for cell density, glucose and lactate. Viral production was followed by glycoprotein content and viral titer determination. Cells were also observed by immunofluorescence to check viral infection. The described process yielded a volumetric productivity of 2,500 glycoprotein IU/L, potentially reaching 11,000 vaccine doses without considering loss during downstream. Additionally, partially purified and β-propiolactone inactivated virus showed protective levels in NIH assay, demonstrating that the obtained antigen induces a specific immune response. Finally, the purification process involved clarification using depth filters, concentration by ultrafiltration/diafiltration, and two chromatographic steps (cation exchange followed by gel filtration). The purified and inactivated viral particles complied with European Pharmacopoeia requirements.