Scale-down of an orbital shaken bioreactor: High cell density cultivation in perfusion mode and virus production

Application of single-use bioreactors has been commonly shown for several cell culture-based production systems including commercial vaccine production. Compared to stainless steel bioreactors, competitive cell growth characteristics as well as virus yields can be reached [1]. In addition to conventional stirred tank reactors (STR), wave bioreactors or orbital shaken bioreactors (OSBs) are available that rely on alternative mixing regimes. For small-scale screening of clones and media, cell maintenance and process optimization, OSBs are the most widely used system. Besides their simple design and ease of handling, OSBs allow for robust processes due to reduced mechanical stress caused by stirring and aeration [2]. Furthermore, scale-up (£ 2500 L) is simplified as larger OSBs rely on the same basic principles for mixing and aeration (e.g. bubble-free surface gassing). Particularly for high cell density (HCD) processes, high oxygen transfer rates, short mixing times, and low shear stress are beneficial. Until now, the step from spin tubes or shake flasks into larger OSBs was rather large, as only the OSB SB10-X (Kühner AG, Switzerland) with a minimum working volume (wv) of 4-5 L was available. In this study, a novel scale-down 3 L vessel module (wv = 1-3 L) for the OSB SB10-X was evaluated for cultivation of suspension BHK-21 cells (CEVA, Germany) in perfusion mode to HCD. Cultivation was carried out in serum-free medium in a 3 L and 10 L single-use standard bag with 3 L and 5 L initial wv and 100 and 70 rpm shaking frequency with a shaking diameter of 50 mm, respectively. For perfusion, an alternating tangential flow system (ATF2, Repligen) with a cut-off of 0.4 µm (SB10-X) and 0.5 µm (SB3-X), respectively, was used. Following an initial batch phase of 2-3 days, perfusion was initiated. After a complete media exchange, cells in the 3 L vessel module were infected with a fusogenic oncolytic virus (rVSV-NDV, recombinant vesicular stomatitis virus-Newcastle disease virus) at a cell concentration of 44.5x106 cells/mL at a multiplicity of infection (MOI) of 10-4. The obtained data were compared to a cultivation of BHK-21 cells in the standard SB10-X module (infection at a cell concentration of 12.5x106 cells/mL with yellow fever virus WHO 17D-213/77 with an MOI of 10-3) and to a cultivation in a 1 L STR. The novel 3 L vessel module allowed for a successful and direct scale-down utilizing the SB10-X backbone without the need for further optimization. For both the SB10-X and the 3 L vessel module, the ATF system was successfully coupled and cell concentrations of 32.7x106 cells/mL and 45.9x106 cells/mL were reached with high viabilities above 98%, respectively. A faster doubling time (tD=22 h) was observed in the 3 L vessel module compared to the SB10-X system (tD=27 h). For rVSV-NDV production, similar infectious virus titers were reached compared to perfusion cultivations of BHK-21 cells in a 1 L STR. Volumetric media consumption was significantly reduced in the 3 L vessel module, facilitating the implementation of OSB systems in non-industrial research environments. All in all, we demonstrated the adaptability and scalability of the single-use OSB system for the production of various viruses in HCD perfusion mode. References [1] Gallo-Ramirez, L. E., A. Nikolay, Y. Genzel, and U. Reichl. 2015. Bioreactor concepts for cell culture-based viral vaccine production. Expert Rev Vaccines 14 (9):1181-95. doi: 10.1586/14760584.2015.1067144 [2] Klöckner W, Diederichs S, Büchs J. Orbitally shaken single-use bioreactors. Adv Biochem Eng Biotechnol. 2014;138:45-60. doi: 10.1007/10_2013_188. PMID: 23604207

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging