A bioprocess to produce reovirus: Serum-free media development and the relationship between intracellular nucleotide levels and virus production.

摘要

Mammalian cell culture has become a standard in the biotechnology industry for the production of therapeutics, including recombinant proteins, antibodies, and vaccines. New therapeutics are being discovered at a rapid rate, and some have unexpected applications. A recent study showed that reovirus, an enteric virus normally commonly found in the intestines of humans, can be used to treat cancer. It has shown promising results in the treatment of various forms of cancer and is now in phase I and II clinical trials for the treatment of brain, prostate, and breast cancer. These findings suggest the need to develop a cell culture process to produce the virus for human use. The requirements of such a process must include an acceptable cell line, a serum-free medium, and a scalable production process. We have successfully developed a culture process to produce reovirus that involves a regulated cell line, Vero; an animal-product free serum-free medium developed in our lab (M-VSFM); and microcarrier technology (Cytodex-1) to make it scalable. The microcarrier cultures produced high titers of both reovirus type 1-Lang and type 3-Dearing to > 10^9 PFU/mland yielded comparable titers to cells in serum-supplemented media (10% FBS-DMEM). It was shown to be superior to the standard mouse L cell process used to produce reovirus in batch cultures with 260% higher specific productivity. The amount of virus produced from these cultures was found to be highly dependent on the intracellular ATP, total adenylate, and the adenylate energy charge (AEC) at the time of infection. A threshold limit of ATP and adenylate exists where a drop below this limit resulted in a 95% reduction in production titer compared to the control. A Km for ATP and total adenylate was calculated to be 3.2 and 3.3 fmoles/cell, respectively, for virus production. The productivity of the virus is not dependent upon the growth state of the cell but rather the ATP and adenylate levels in the cell at the time of infection. The AEC shared a negative sigmoidal relationship with virus productivity where cultures with an AEC higher than 0.97 had significantly lower virus titers. It was found that reovirus consumes as much as 28% of the ATP and GTP within the cell to fuel replication of its progeny. This information can be useful for optimizing a large-scale process to produce reovirus and may be applicable to other virus production systems. (Abstract shortened by UMI.)