Genetic Engineering & Biotechnology News

JUL 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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20 | JULY 2018 | Genetic Engineering & Biotechnology News | Maya Yovcheva, Kenneth Thompson, Ph.D., Sara Barnes, Katalyn Irvin, Melissa Cross, Natasha Lucki, Ph.D., Henry Chiou, Ph.D., and Jonathan F. Zmuda, Ph.D. The baculovirus expression vector system (BEVS) provides a versatile platform for the expression of individual recombinant pro- teins as well as multimeric protein complexes, virus-like particles, membrane proteins, and proteins that are toxic to mammalian cells. Re- cently, BEVS has demonstrated particular util- ity in the commercial-scale manufacture of vac- cines for human and veterinary applications. Among the advantages of BEVS are the ability to express proteins with enhanced post-translational modifications compared to bacteria, lower costs of reagents and fewer equipment needs compared to mammalian expression, and a demonstrated record of scalability for the commercial production of vaccines and cell therapy reagents. 1 BEVS platforms, however, continue to possess a number of shortcomings compared to plasmid-based mammalian transient sys- tems including: lot-to-lot variability of the expression media due to the presence of undefined components such as yeastolate, lower protein titers, and longer overall time to protein. These factors can significantly hamper product development at a time when manufacturers are under continual pressure to develop vaccines and other biologics more quickly and efficiently. Unlike many mammalian expression sys- tems that transitioned to chemically-defined media formulations more than a decade ago, insect expression systems have continued to rely on poorly defined, yeastolate-containing media formulations that can impart signifi- cant lot-to-lot variability on cell growth, bac- ulovirus production, and protein expression. Yeastolate-containing media are further limited in their capacity to sustain high-densi- ty cell growth and consequently support high- cell-density baculovirus infections, two criti- cal aspects required to increase biomass and improve protein titers on a per volume basis. Compared to mammalian transient pro- tein expression, the time from gene to protein for insect systems is significantly longer, typi- cally on the order of 3– 4 weeks, due primar- ily to the time required to generate a baculo- virus stock using adherent Sf9 cells followed by subsequent amplification of virus through P1, P2, and/or P3 stocks to obtain sufficient virus for protein expression runs. Eliminating Variability To address the shortcomings of tradi- tional insect expression systems, the Ex- piSf™ Expression System was developed to eliminate the variability associated with yeastolate-containing culture medium, to in- crease protein titers, and to reduce total time to protein—all to streamline research such as vaccine development from bench to clinic. Similar to the Expi293 and ExpiCHO mammalian expression systems, a systems- based approach was employed during devel- opment of the ExpiSf Expression System. As an initial step in this process, the first chemically defined (CD), yeastolate-free in- sect cell culture medium, ExpiSf CD Medi- um, was developed to support high-density cell growth, high-titer virus production, and enhanced protein expression. Multiple lots of ExpiSf CD Medium were formulated and compared for cell growth and protein expres- sion levels; consistent growth kinetics (Figure 1A) and protein titers (Figure 1B) were ob- tained across four different lots of ExpiSf CD Medium. Next, Gibco Sf9 cells were adapted into ExpiSf CD Medium through extensive long- term passaging to generate the high-density ExpiSf9 cell line. ExpiSf9 cell growth char- acteristics were compared to traditional Sf9 cells by culturing Sf9 cells in various yeast- olate-containing media for at least 10 pas- sages to ensure adaptation. Compared to Sf9 cells grown in yeasto- late-containing media, ExpiSf9 cells grown in ExpiSf CD Medium achieved higher peak vi- able cell densities in standard shake flask cul- tures (>20 × 10 6 cells/mL vs. 2–10 × 10 6 cells/ mL; Figure 1C), approximately double the peak density of the next highest density me- dia formulation. ExpiSf9 cells also possessed a broad log-phase growth range spanning from approximately 4–12 × 10 6 viable cells/ Employing the ExpiSf™ Chemically Defined Sf9 Insect Cell Expression System High-Titer Recombinant Protein Production Bioprocessing Tutorial Figure 1. Performance characteristics of the ExpiSf Expression System. (A) Four different lots of ExpiSf CD Medium demonstrated consistent growth of ExpiSf9 cells with peak viable cell densities (VCDs) of ~20 × 10 6 cells/mL. (B) Green fluorescent protein (GFP) titers were 4–5-fold higher in all lots of ExpiSf CD Medium tested compared to a traditional Sf9 workflow using yeastolate-containing (YC) medium. (C) ExpiSf9 cells in ExpiSf CD Medium exhibited superior cell growth compared to five yeastolate-containing insect cell media. (D) ExpiSf Enhancer, used in conjunction with ExpiSf CD Medium and ExpiSf9 cells, generated 3-fold higher GFP titers than a traditional Sf9 workflow; ExpiSf Enhancer nearly doubled protein titers compared to the ExpiSf System without enhancer addition. (E) The ExpiSf Expression System reduced the time required to go from bacmid DNA to protein expression by half by eliminating the need for virus amplification via direct generation of high-volume and high-titer P0 virus stocks. A D B E C Figure 2. Comparison of the ExpiSf Expression System to traditional Sf9 workflows using various yeastolate-containing media. (A) Expression levels of an Fc fusion protein, green fluorescent protein (GFP), and tumor necrosis factor-alpha (TNF-α) were on average >4, >3, or >5-fold higher in the ExpiSf expression system than those obtained using various yeastolate-containing media in a traditional Sf9 workflow. (B) Optimization of CB2 G-protein- coupled chemokine receptor harvest time and (C) post-infection viability kinetics in the ExpiSf Expression System. (D) Increased total CB2 expression levels obtained in the ExpiSf Expression System compared to traditional Sf9 workflow in Sf-900 II medium; increased expression of CB2 is due to both higher per cell expression as well as greater cell density in a given volume for the ExpiSf Expression System. A B C D Figure 3. Comparison of glycosylation patterns and biological activity for proteins expressed in the ExpiSf Expression System and by traditional Sf9 workflow. (A) Glycosylation patterns of secreted alkaline phosphatase (SEAP) were highly similar in the ExpiSf Expression System and in a traditional insect workflow using Sf900-II medium. (B) SDS-PAGE of SEAP purified from the ExpiSf Expression System and a traditional Sf9 workflow using Sf900-II medium. (C) The ExpiSf Expression System generated >4-fold higher TNF-α expression levels compared to a traditional Sf9 workflow. (D) TNF-α activity, as measured by luciferase-based NFκB reporter gene assay, showed equivalent biological response for protein generated in the ExpiSf Expression System and by traditional Sf9 workflow. B A C D

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