Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.