Pilot Lot Production of a Chromatographically Purified Vero Cell Rabies Vaccine.

PALACIOS C.A.; CHIAPPINI S.; DE NICHILO A.; NUGUER E.; LARGHI O.P.; PAROLA A.D.

Fort Collins, Colorado.

Congreso; XXVI Reunión Internacional de Rabia en Las Américas (RITA).; 2015

Rabia en Las Américas (RITA).

Rabies vaccine production in Latin America is stillbased on nerve tissue substrate (NTV), it is well known that these vaccines arenot safe inducing adverse reactions, and are less immunogenic than cell culturederived 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 rabiesvaccine (CPRV), describing generation and characterization of viraland cell banks, viral production in spinner flasks and a purification process involvingdifferent chromatographic steps. The aim of this study is to scale up bothupstream and downstream steps of a CPRV production. In this research, Verocells were grown in a five-liter stirred tank bioreactor (BIOSTAT Aplus,Sartorius Stedim Biotech) using Cytodex 1 microcarriers. The cell culture was startedby seeding 0.20-0.25 x 106 cells/ml in M199 cell culture medium, and infected at a MOIof 0.1 ID50/mlonce 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 postinfection. Cell culture was monitored for cell density, glucose andlactate. Viral production was followed by glycoprotein content and viral titer determination.Cells were also observed by immunofluorescence to check viral infection. Thedescribed process yielded a volumetric productivity of 2,500 glycoprotein IU/L,potentially reaching 11,000 vaccine doses without considering loss duringdownstream. Additionally, partially purified and b-propiolactoneinactivated virus showed protective levels in NIH assay, demonstrating that theobtained antigen induces a specific immune response. Finally, the purificationprocess involved clarification using depthfilters, concentration by ultrafiltration/diafiltration, and twochromatographic steps (cation exchangefollowed by gel filtration). The purified andinactivated viral particles complied with European Pharmacopoeia requirements.