Genetic Engineering & Biotechnology News

JUL 2018

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Page 22 of 33 | Genetic Engineering & Biotechnology News | JULY 2018 | 21 mL, with a stable doubling time of ~24 hours. Taking advantage of the higher densi- ties that ExpiSf9 cells achieve in ExpiSf CD Medium, an expression enhancer (ExpiSf Enhancer) was developed to work in con- junction with the ExpiSf CD Medium to allow for consistent, high-density infection of ExpiSf9 cells at 5 × 10 6 cells/mL, leading to multifold improvements in protein titers compared to traditional Sf9 workflows, in which cells are infected at 1–2 × 10 6 cells/mL in yeastolate-containing media (Figure 1D). Since optimal baculovirus infection of Sf9 cells is thought to occur when cells are in log- phase growth, the extended log-phase growth range of the ExpiSf9 cells, in conjunction with the ExpiSf Enhancer, allows for ExpiSf9 cells to be infected at significantly higher densities than traditional insect workflows, thereby in- creasing protein titers on a per volume basis. Lastly, to reduce overall time to protein, the ExpiFectamine™ Sf Transfection Re- agent was developed to allow for gentle, non- toxic transfection of high-density ExpiSf9 suspension cultures with large bacmid DNA. Incorporating suspension-based transfec- tion for baculovirus generation enabled easy preparation of 100 mL (or greater) of high ti- ter (1–5 × 10 9 infectious virus particles/mL), high-quality P0 virus, completely eliminating the need for additional virus amplification for typical liter and subliter workflows. Additionally, suspension-based bacmid transfection shortens overall time to protein by up to 50% (Figure 1E) by eliminating the need for virus amplification while at the same time removing the risk of deleterious virus passaging effects (whereby gene incorpora- tion/protein expression levels decrease during generation of P1 + virus stocks), ensuring that the highest quality baculovirus is used for protein expression runs. Comparison of ExpiSf to Traditional Sf9-Based Workflows The expression levels of three different proteins—green fluorescence protein, human Fc fusion protein, and tumor necrosis factor- alpha (TNF-a)—were compared between the ExpiSf system and a traditional Sf9 workflow in which cells are infected at 1–2 × 10 6 cells/ mL in yeastolate-containing media. Compared to five different yeastolate- containing media tested using traditional Sf9 workflows, the high-density ExpiSf Expres- sion System generated 3–5-fold improve- ments in expression levels across the three proteins tested (Figure 2A). G-protein-coupled receptors are com- monly expressed in insect cells, in part be- cause of the potential for toxicity in mam- malian cells, as well as the desire for reduced glycosylation for structural biology studies. Cannabinoid receptor 2 (CB2) was ex- pressed in the ExpiSf system and in a tradi- tional Sf9 workflow. Optimal cell harvest time was determined to be 48 hours post infection in the ExpiSf corresponding to a cell viability of 70% at the time of harvest; longer infection times led to decreased viability without improving CB2 expression per cell (Figures 2B & 2C). CB2 expression (as measured by total num- ber of CB2 molecules per cell by quantitative flow cytometry) was 10-fold higher in the ExpiSf Expression System compared to the traditional Sf9 workflow (Figure 2D). This improvement was due to both per cell increases in expression levels as well as the significantly higher density of ExpiSf9 cells in a given volume compared to the tra- ditional Sf9 workflow. Glycosylation Patterns and Protein Functionality Although high protein yields are desir- able, resultant proteins are less valuable if they are aggregated, misfolded, degraded, or improperly glycosylated. The quality of the proteins expressed in the ExpiSf system was compared to the quality of the same proteins expressed using a traditional Sf9 workflow with yeastolate-containing medium. Using secreted alkaline phosphatase (SEAP) as a model protein, glycosylation patterns generated in the ExpiSf were shown to be highly comparable to those of a traditional Sf9 insect workflow, with the predominate glycoforms being Man3, Man3F, and Man6 in both instances (Fig- ure 3A). SDS-PAGE showed a single band with a molecular weight of ~57 kD for both workflows (Figure 3B). The biological activity of TNF- a ex- pressed in the ExpiSf system and by tradi- tional Sf9 workflow was assessed using an NF-κB luciferase reporter gene assay. HIS- tagged TNF-a was expressed and purified by Ni-NTA, and its concentration was de- termined by A280. TNF-a was expressed at >4-fold higher levels in ExpiSf compared to the traditional Sf9 workflow (Figure 3C); reporter gene assay results demonstrated equivalent biological activity (relative lumi- nescence units; RLUs) for the proteins, re- spectively (Figure 3D). In summary, the ExpiSf Expression Sys- tem represents a significant advance in insect cell protein expression in terms of media consistency, protein yield, and time. It en- ables researchers to streamline protein ex- pression and vaccine development to shorten time lines from bench to clinic. Reference 1. Felberbaum RS. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnol. J. 2015. 10: 702–714. Learn how to accelerate your pilot-scale media manufacturing at *Non-GMP pilot production. Additional time for shipping. © 2017 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company. MC8392 FOR SCALABLE, ONE-STOP CELL CULTURE MEDIA PRODUCTION, TURN TO BD. BD continually advances solutions to support process development and manufacturing for scientists. BD ™ Rapid Media Solutions delivers a 10-business-day * turnaround on developmental medium production. Each custom formulation is evaluated by our team of cell culture media development experts to ensure manufacturing suitability at both pilot- and full-scale production. For consistency, we develop every formulation as a hydratable-to-liquid powder in our full-service rapid media pilot facility, which replicates the equipment and processes of our large-scale media manufacturing plant. The result? A fast and reliable one-stop solution for every stage of media development from initial testing through clinical trials. Discover the difference of a faster turnaround time and full-service solution. Discover the difference of BD. Bioprocessing Maya Yovcheva is an R&D scientist and R&D lead for the ExpiSf Expression system at Thermo Fisher Scientific. Her colleagues are R&D scientists Kenneth Thompson, Ph.D., Sara Barnes, Katalyn Irvin, and Melissa Cross. Natasha Lucki, Ph.D., is a product manager, Henry Chiou, Ph.D., is an associate director, product management, and Jonathan F. Zmuda, Ph.D. (Jon.Zmuda, is a director of R&D. The ExpiSf™ Expression System is for Research Use Only. Not for use in diagnos- tic procedures. For additional information about the ExpiSf system, please go to:

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