Virus Production From Bench Scale To Industrial Scale: New Technologies That Intensify, Simplify Virus Manufacturing And Increase Overall Process Efficiency A. Onraedt , P. Lefebvre , M. Kremer (Pall Life Sciences) Contributing to the complexities of developing viral vaccines are the availability of purpose built process technologies to manufacture virus. Current upstream and downstream technologies either are scaled-out laboratory tools, including 2D growth surfaces for adherent cells, or are derived from other biotech processes, where the size of the molecule or its shear sensitivity are not as critical as in virus manufacturing. The streamlined viral manufacturing platform that will be discussed is based on technologies that are specifically developed for large-scale manufacture of virus for vaccine or gene therapy applications. The result is a process that simplifies virus manufacturing and reduces manufacturing footprint. It also increases the process efficiency and reduces cost-of-goods versus traditional approaches. For the adherent culture of the host cell line, the iCELLis ® fixed-bed bioreactor from Pall Life Sciences is designed to achieve high cell densities and good virus productivity by combining controlled growth conditions and a large growth surface area in a small reactor volume. This has been demonstrated on a wide range of viruses including MVA, Bovine Herpes, Paramyxovirus, Influenza, Adenovirus, rAAV and more in various production cell lines, including VERO, HEK293 and MDBK. Scale-up of the cell culture and virus production from benchtop iCELLis bioreactors to up to 500 m² has been realized. Specific virus productivity in the iCELLis bioreactors at benchtop and industrial scale was similar or better than that achieved in static culture or roller bottles. In combination with Pall’s virus downstream processing solution, the iCELLis bioreactor offers a versatile platform for industrial scale virus production. SUMMARY The single-use iCELLis bioreactor containes a compact fixed-bed with carriers made of medical grade polyester. This matrix provides a large growth surface area in a relatively small volume: the iCELLis design provides up to 500 m 2 in only 25 liters of fixed-bed. Commercially available iCELLis bioreactors range in size from 0.53 to 500 m 2 with two compaction densities of carriers (Table 1). Table 1 Commercially available iCELLis bioreactor size specifications iCELLis Nano Bioreactor iCELLis 500 Bioreactor Fixed-Bed (FB) (Process Development) (Large-Scale Production) Compaction Density FB Height (cm) FB Volume (L) FB Surface Area (m 2 ) FB Volume (L) FB Surface Area (m 2 ) 96 g/L 2 0.04 0.53 5 66 (C 1) 4 0.08 1.06 10 133 13 m 2 /L 10 0.2 2.67 25 333 144 g/L 2 0.04 0.8 5 100 (C 1.5) 4 0.08 1.6 10 200 19.5 m 2 /L 10 0.2 4.0 25 500 SUMMARY PALL VIRUS MANUFACTURING PLATFORM Virus analytics with BLI (Octet ® , Blitz ® ) Cell debris removal with V100P depth filter Final sterilizing filtration Further formulation & filling Purification with Mustang ® AEX and Cadence™ SU TFF Virus production in iCELLis 500 with BLI (Octet Virus analytics ) ® , Blitz ® with BLI (Octet Virus analytics with V100P depth filter Cell debris r with V100P depth filter emoval Cell debris r with V100P depth filter with V100P depth filter with V100P depth filter with V100P depth filter Final sterilizing filtration Final sterilizing filtration Final sterilizing filtration Further formulation & filling Final sterilizing filtration Further formulation & filling Final sterilizing filtration Further formulation & filling Final sterilizing filtration oduction in iCELLis 500 Virus pr oduction in iCELLis 500 oduction in iCELLis 500 AEX and Cadence™ SU TFF AEX and Cadence™ SU TFF Purification with Mustang AEX and Cadence™ SU TFF ® Purification with Mustang AEX and Cadence™ SU TFF Virus pr ICELLIS BIOREACTOR TRANSFER AND SCALE-UP OF A HEK293 CELL CULTURE PROCESS FOR PRODUCTION OF ADENOVIRUS RESULTS Table 2 Results of HEK293 cell culture four days after inoculation in small- and large-scale iCELLis bioreactors. Average Average Fixed Bed Fixed Bed Cell Density Total Bioreactor Surface Height Fixed Bed Reached Cells per Scale Area (m 2 ) (cm) Volume (L) (cells/cm 2 ) Bioreactor iCELLis nano 1.06 4 0.08 2.8 to 3.8 x 10 5 3.5 x 10 9 iCELLis 500 133 4 10 2.7 to 3.4 x 10 5 4.1 x 10 11 Analysis of glucose and lactate (Figure 3) at both scales in comparison to a 5-tray Cell Factory control indicated that cell metabolism was comparable between small and large scale iCELLis bioreactors and the standard 2-D process. Figure 3 Comparability of Glucose and Lactate Profiles of HEK293 culture in the iCELLis 133 m 2 bioreactor (Blue), iCELLis nano bioreactor 1.06 m 2 (Green) and 5-tray Cell Factory (Red). Figure 2 The iCELLis nano bioreactor system (from 0.53 to 4 m 2 ) Figure 1 The iCELLis 500 control system and bioreactor Evenly distributed medium circulation is powered by a built-in magnetic mixer, ensuring low shear stress. The cell culture medium flows through the fixed-bed from the bottom to the top and returns to the bottom of the bioreactor as a waterfall, resulting in a high mass transfer co-efficient (k L a). In the iCELLis bioreactors, the following parameters can be measured and controlled: pH, DO, biomass (using a biomass probe), temperature, gas flow rate, agitation, pressure and medium recirculation/perfusion rate. The unique waterfall oxygenation together with gentle agitation, biomass immobilization and process control provides a favored growth environment that can increase the cell specific productivity of the desired protein or virus. EXPERIMENTAL APPROACH Hardware iCELLis nano bioreactor (from 0.53 to 4 m 2 ) iCELLis 500 bioreactor (133 m 2 ) Metabolites were analyzed with a Bioprofile-100 bio-analyzer (Nova Biomedical, MA, USA) Biological Material HEK293 cells were grown in serum-supplemented medium Cell Culture Parameters An existing process using HEK293 cells for the production of adenovirus was first transferred from multi-tray systems to an iCELLis nano bioreactor (0.53 m 2 , 40 mL of fixed-bed) by keeping equivalent cell culture parameters: Temperature, pH and DO (% saturation with air) Multiplicity of infection (pfu/cell) Time of infection Cell seeding density (cells/cm 2 and cells/mL) Culture duration Additional experiments were performed with lower cell densities at inoculation in order to reduce the number of preculture steps at large scale. The following parameters were also optimized for cell growth and virus productivity: Compaction of carriers inside the fixed-bed (96 g/L or 144 g/L) Linear velocity of medium through the fixed-bed (cm/s) Fixed-bed height (2, 4 or 10 cm) 0 1 2 3 4 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5CS nano (1,06 m 2 ) large (133 m 2 ) Time (days) Glucose (g/L) 5 5 4 3 2 1 Glucose (g/L) 5CS nano (1,06 m large (133 m 5CS ) 2 nano (1,06 m ) 2 large (133 m 0.5 0 1 0 Glucose (g/L) 2 1.5 1 Time (days) 3.5 3 2.5 Time (days) 4.5 4 Table 3 Results for different viruses from various cell lines at different bioreactor scales. Surface Productivity Total Virus Scale Up of Control iCELLis SA. (Virus Titer per cm 2 ) Extrapolated to Process Required Cells Virus Tested (m 2 ) iCELLis Control Units 500 m 2 iCELLis to Match iCELLis 500 CEF MVA 0.07* 3.5E+06 1.0E+07 pfu/cm 2 1.7E+13 pfu 780 CF-10 MDBK Bovine 4 2.2E+07 1.3E+07 pfu/cm 2 1.1E+14 pfu 310 L MC (6g/L) Herpes 20* 1.7E+07 8.5E+13 pfu 240 L MC (6g/L) Virus 66 3.3E+07 1.6E+14 pfu 460 L MC (6g/L) A549 rAAV 0.53 5.0E+08 3.0E+08 vg/cm 2 2.6E+15 vg 1300 CF-10 Adenovirus 2.67 9.8E+09 9.6E+09 TCID50/cm 2 4.9E+16 TCID50 810 CF-10 Vero Influenza 4 3.8E+06 1.3E+06 TCID50/cm 2 1.9E+13 TCID50 540 L MC (6g/L) 20* 2.5E+06 1.3E+13 TCID50 360 L MC (6g/L) Paramyxovirus 2.67 6.4E+05 5.7E+04 TCID50/cm 2 3.2E+12 TCID50 8900 CF-10 2.9E+08 5.3E+07 Vp/cm 2 1.5E+15 vp 4300 CF-10 Undisclosed 40* Confidential 794 CF-10 Lytic Virus 133 (serum-free) 660* 1238 CF-10 (+serum) *Not a standard bioreactor size. Please refer to table 1 where all commercially available bioreactor sizes are specified VIRUS PRODUCTION RESULTS IN ICELLIS © 2015, Pall Corporation. Pall, , Blitz, Cadence, iCELLis, Mustang, and Octet are trademarks are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 3/15, GN15.6201 Contact: 800.717.7255 (USA and Canada) • 1.516.484.5400 (Outside USA and Canada) • www.pall.com/bioreactors • www.pall.com/vaccines • E-mail: [email protected] This summary of experiments demonstrates that the fixed-bed design of the iCELLis bioreactor enables high cell densities to be achieved and maintained in both small and large bioreactor volumes. Different processes have been easily scaled up by keeping cell culture conditions and process parameters identical to the standard 2-D cell culture process. The iCELLis bioreactor can be inoculated at a very low cell density, leading to a dramatic simplification of seed train operations and a significant reduction of development timelines. In conclusion, large biomass amplification and excellent virus productivities, combined with the advantages of a fully closed single-use system with low shear stress, make the iCELLis fixed-bed bioreactor a simple and straightforward solution for industrial production of viruses. In combination with Pall's virus downstream processing solution, it brings an industrial virus production platform with a reduced footprint that is fully automatable for better control and that brings overall process economics. Outer shell, top section with probes and ports Fixed-bed retaining ring Fixed-bed filled with 500 m 2 macrocarriers Fixed-bed support Pump housing Impeller Pump housing ring Outer shell, bottom section Double-jacket 0 5 10 15 20 25 30 0 0.5 1 1.5 2 2.5 Time (days) Lactate (mM) 3 3.5 4 4.5 5CS nano (1,06 m 2 ) large (133 m 2 ) 30 Lactate (mM) 30 25 20 15 10 5 5CS nano (1,06 m large (133 m 5CS ) 2 nano (1,06 m ) 2 large (133 m 0.5 0 5 0 Time (days) 2 1.5 1 3.5 3 Time (days) 2.5 4.5 4 CONCLUSION: VIRUS PRODUCTION INTENSIFICATION AND SIMPLIFICATION Sequence of automated, single-use technologies optimized for high virus yield and quality, process robustness and optimized process economics: