Perfusion filtration technology for the cultivation of VNK-21/13-13 cells for the manufacture of rabies vaccine

All-Russian Scientific Research and Technological Institute of Biological Industry, Biocombinat Street 17, Biocombinat City, 141142, Russia

^* Corresponding author: smolentsev82@mail.ru

Abstract

The possibility of practical use of perfusion cell culture technology VNK-21/13-13 was evaluated in the manufacture of rabies vaccine using bioreactors with a working volume of 5 liters and 100 liters. The purpose of this study is to develop a highly effective technology for perfusion continuous suspension cultivation of the transplanted BHK– 21/13-13 cell line, suitable for use as a cellular substrate in the manufacture of rabies vaccine. Using Cytiva filter elements made of PES membrane material with a retention rating of 0.65 microns, a cell density above 7× 106 cells/ml was achieved for the subsequent production of a viral suspension. The glycoprotein content measured by the ELISA method reached a maximum on the 4th day after infection, was 5 IU/ml. The maximum cell accumulation and glycoprotein content in the 5 L bioreactor was more than 2-3 times higher than for the serial production of rabies vaccine. When reproducing the rabies virus in a 100 l bioreactor after perfusion cultivation, the cell concentration was 16-20 million/ml with a viability of 95.4%. The highest content of the virus glycoprotein was also achieved in a bioreactor with a working volume of 100 liters 4 days after infection and amounted to 8 IU/ ml. Sequencing of the glycoprotein gene revealed the genetic stability of the main immunogenicity protein, which is an important indicator for controlling the stability of the virus during many passages in vaccine production using a perfusion system.