Combining single-use bioreactor technology and TIPS method to make IC/BEVS- based productions more efficient

Insect cells (ICs) in conjunction with the baculovirus-expression vector system (BEVS) are a suitable production system for the development and manufacturing of recombinant protein products such as virus-like particle vaccines. In order to speed up vaccine development and production, and to react more rapidly to pandemic scenarios, new strategies for IC/BEVS-based production technologies need to be developed and transferred. Among these is the implementation of single-use (SU) bioreactors, which have been proven as cost-effective (48 % less capital costs), ready-to-use systems without time-intensive cleaning and sterilization processes when compared with reusable bioreactors [1]. A further increase in process efficiency can be achieved by using the titerless infected-cell preservation and scale-up (TIPS) method. Here, the infection is directly performed with baculovirus-infected insect cells (BIICs), which makes laborious, time-consuming virus determinations and virus amplifications obsolete, while providing product titers and product activities comparable to the standard method, the classical infection of non-infected ICs [2, 3]. In our investigations we demonstrated the advantageous realization of the TIPS approach in a scalable, orbitally shaken single-use bioreactor, Adolf Kuhner`s SB10-X, for the first time. Furthermore, we compared the results generated by producing the recombinant model protein secreted alkaline phosphatase (rSEAP) with those achieved in Sartorius Stedim`s stirred single-use bioreactor, the UniVessel SU 2L. All production processes ran for 6 days and were performed with Spodoptera frugiperda suspension cells (Sf-9) cultivated in Sf-900 III serum-free medium containing Pluronic F-68 and L-glutamine. Cell maintenance and seed inoculum production were carried out as described by Imseng et al. 2014 [4]. Samples were taken daily in order to determine cell growth (Cedex HiRes, Roche), substrate and metabolite concentrations (BioProfile 100plus, NOVA biomedical) as well as product formation [4]. When using the TIPS approach and working with a MOI of 0.01 pfu cell-1 and a CCI of 2 x 106 cells mL-1, we achieved comparable rSEAP activities in the orbitally shaken and stirred single-use bioreactor. Saving four days in comparison with the standard method, we produced 158 U mL-1 rSEAP in the OrbShake system operated with BIICs. These encouraging results represent the basis for scaling-up the TIPS approach to the orbitally shaken SB50-X in the future. References: [1] Eibl, R., Steiger, N., Wellnitz, S., Vicente, T., John, C., Eibl, D., Fast single-use VLP vaccine productions based on insect cells and the baculovirus expression vector system: influenza as case study. Disposable Bioreactors II, Springer, 99-125, 2014. [2] Wasilko, D. J., Lee, E. S., TIPS: titerless infected-cells preservation and scale-up. Bioprocess International. 5, 1-43, 2006. [3] Wasilko, D. J., et al., The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. Protein expression and purification, 65, 122-132, 2009. [4] Imseng, N., Steiger, N., Frasson, D., Sievers, M., et al., Single-use wave-mixed versus stirred bioreactors for insect-cell/BEVS-based protein expression at benchtop scale. Eng. Life Sci. 14, 264- 271, 201