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BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 36 of 151 Figure 1(abstract P18) BioPAT®Trace equipped with the single use Tube set. Introduction: Insect cells have been widely used for the production of recombinant proteins using recombinant baculovirus for gene delivery [1]. To simplify protein production in insect cells, we have previously described a method, based on transient gene expression (TGE) with cultures of suspension-adapted Sf9 cells using polyethylenimine (PEI) for DNA delivery [2]. Expression of GFP has been realized at high efficiency and a tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc) was produced at a level of 40 mg/L. However, the efficiency of the insect cells TGE system has not been studied and further optimization may improve protein titers. Here, we studied the efficiency of PEI for plasmid delivery in Sf9 cells. Methods: Cell culture: Sf-9 cells were maintained in suspension in TubeSpin® bioreactor 600 at 28°C [3]. Sf-9 cells Transfection: Sf9 cells were transfected as described before [2] using 25 kDa polyethylenimine PEI (Polysciences, Warrington, PA) and an expression vector for GFP or TNFR-Fc. GFP-specific fluorescence was measured 48 h post-transfection using the GUAVA EasyCyteTM flow cytometer (Millipore, Billerica MA, USA). TNFR-Fc was measured by sandwich ELISA [4]. Estimation of plasmid copy number: Total DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen AG, Hombrechtikon, Switzerland) according to the manufacturer’s protocol. PCR was executed using the Absolute qPCR SYBR Green ROX reaction mix (Axon Lab AG, Baden-Dättwil, Switzerland) with total cellular DNA as template. The PCR was performed using LightCycler® 480 real-time PCR system (Roche Applied Science, Basel, Switzerland). The plasmid copy number was estimated from the standard curve according to the threshold cycle (Ct) of each sample [4]. Cell cycle analysis: Cells at different times post-transfection were centrifuged and washed with PBS before fixation in 70% ethanol. Fixed cells were washed with PBS and then stained with Guava Cell Cycle Reagent and analyzed by the GUAVA EasyCyteTM flow cytometer. Cells treated with nocodazole(50ng/mL,16h)andmimosine(1mM,24h)wereusedas references for determining the positions of the G1 and G2/M phases [5]. Results: Plasmid delivery efficiency in Sf9 cells: To measure the time course of plasmid DNA delivery, cells were transfected with a GFP expression vector. At different times post-transfection, a complete medium exchange was performed. The percentage of GFP-positive cells was determined for all cultures including a control for which a medium exchange was not performed. All cultures exhibited similar levels of GFPpositive cells meaning that DNA uptake into cells occurred within 10 min of DNA addition (Figure 1A). To measure the amount of DNA uptake, Sf9 cells were transfected in two different ways with a TNFR-Fc expression vector and the amount of intracellular plasmid was measured by quantitative PCR. On the day of transfection more than 80% of the plasmid DNA was present within cells with the control transfection while 40% of the DNA was present within cells following a high-density transfection (Figure 1B). It has been reported that improved plasmid delivery can result in an increase in specific and volumetric productivity for HEK 293 cells transfected at high-density [6]. However, in our high-density protocol, plasmid delivery was diminished in comparison to the control (Figure 1B). Plasmid delivery was not improved, but cell growth was inhibited in an optimized TGE process: Improvement in TGE yields from Chinese hamster ovary cells was achieved by reducing the cell growth rate [5,7]. When the cell growth curve of the optimal TGE process with Sf9 cells was compared with that of the control protocol, we observed a significant decrease of viable cell number, within 24 h post-transfection (Figure 1C). This suggested a deregulation in the cell cycle in the initial phase of transfection. The cell cycle distribution was analyzed and an increase of the percentage of cells in the G2/M phase was observed for the high-density protocol early after transfection (Figure 1D). However, the growth inhibition was attenuated by 24 h post-transfection (Figure 1D). Nevertheless, the temporary cell growth inhibition contributed to yield improvement in our optimal protocol. Conclusion: A previously described method for the transient transfection of Sf9 cells was improved. The increase in recombinant protein yield was not due to an increased plasmid delivery after transfection. However, high-density transfection resulted in a significant percentage of cells being blocked in the G2/M phase of the cell cycle for the first 24 h posttransfection. References 1. Kost TA, Condreay JP, Jarvis DL: Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 2005, 23:567-575. 2. Shen X, Michel PO, Xie Q, Baldi L, Wurm FM: Transient transfection of insect Sf-9 cells in TubeSpin® bioreactor 50 tubes. BMC Proc 2011, Suppl 8: P37. 3. Xie Q, Michel PO, Baldi L, Hacker DL, Zhang X, Wurm FM: TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension. Biotechnol Lett 2011, 33:897-902. 4. Matasci M, Baldi L, Hacker DL, Wurm FM: The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability. Biotechnol Bioeng 2011, 108:2141-2150. 5. Wulhfard S, Tissot S, Bouchet S, Cevey J, De Jesus M, Hacker DL, Wurm FM: Mild hypothermia improves transient gene expression yields several fold in Chinese hamster ovary cells. Biotechnol prog 2008, 24:458-465. 6. Backliwal G, Hildinger M, Hasija V, Wurm FM: High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 2008, 99:721-727. 7. Gorman CM, Howard BH, Reeves R: Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic acids res 1983, 11:7631-7648. P20 Development of a Drosophila S2 insect-cell based placental malaria vaccine production process Wian A de Jongh 1 , Mafalda dos SM Resende 2 , Carsten Leisted 1 , Anette Strøbæk 1 , Besim Berisha 2 , Morten A Nielsen 2 , Ali Salanti 2 , Kathryn Hjerrild 3 , Simon Draper 3 , Charlotte Dyring 1* 1 ExpreS 2 ion Biotechnologies, Horsholm, Denmark, 2970; 2 Centre for Medical Parasitology, Copenhagen University, Copenhagen, Denmark, 1356; 3 The Jenner Institute, University of Oxford, Oxford, UK, OX3 7DQ BMC Proceedings 2013, 7(Suppl 6):P20 Background: Malaria during pregnancy is the cause of 1500 neonatal deaths a day. Moreover, 40% of all low weight births are caused by pregnancy associated malaria. Researchers at Copenhagen University have identified the VAR2CSA protein as a potential protective recombinant placental malaria vaccine. ExpreS 2 ion Biotechnologies is responsible establishment of cell lines expressing VAR2CSA variants and for developing the protein production process based on VAR2CSA. The ExpreS 2 System is a one-for-all protein expression system based on Drosophila S2 cells that is excellent in all phases of Drug Discovery, R&D and manufacturing due to high-level transient transfections, easy establishment of stable polyclonal pools that provides continuous high protein expression levels without selection pressure, and simple cloning procedure. It is a novel, non-viral, insect-cell based expression technology applied to the development of a critically needed vaccine. The VAR2CSA protein, which the vaccine is based on, is hard to express and comparison studies between
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 37 of 151 Figure 1(abstract P19) Study of the Sf9 TGE process. (A) Sf9 cells were transfected with EGFP-coding plasmid DNA and PEI at a starting cell density of 4×10 6 cells per ml. Media of the transfected culture were exchanged at 10, 30, 60, 90, 120, 180 minutes post-transfection. EGFP-positive cells were measured on day 2. (B) Average intracellular plasmid copy number on day of transfection and day 3 post-transfection of cultures transfected using control protocol and high-density TGE protocol were analyzed by quantitative PCR. (C) Cell growth of Sf9 cells transfected using the two different protocols were compared. Cell cycle distribution during the first 24 hours post-transfection of those two TGE culture were analyzed (D). C: control transfection at 4×10 6 cells/mL; H: high-density Sf9 transfection; h: hours. insect, bacteria and yeast have shown that an insect cell system is the only one leading to a clinically useful immune response. Process optimization is also critically important, as the cost of manufacture must be as low as possible to allow the vaccine to be used in the countries where it is most needed. Aim: The choice and cost of a manufacturing platform is one of the most important strategic decisions in recombinant subunit vaccine development. Furthermore, the geographic distribution of malaria and the philanthropic funding sources involved requires production to be as cost-effective as possible. Single-use provides manufacturing flexibility and economic advantages, both highly desirable in this type of process. We therefore aim to develop cost-effective Drosophila S2basedPlacentalandBlood-stage malaria vaccine production processes combining the ExpreS 2 constitutive insect cell expression system with single-use bioreactor technology. Materials and methods: Thirty-four truncation variants of the VAR2CSA placental malaria vaccine antigen and full-length PfRh5 were cloned into pExpreS 2 vectors and transfected into Drosophila S2 insect cells. Stable cell lines were established in three weeks in T-flask culture, which were then inoculated at 8E6 cells/ml in shake flasks, or batch or fed-batch production in DasGip Bioreactors and harvested after 3 and 7 days respectively. The cultures were harvested by centrifugation and filtration, where after the proteins were purified using Ni ++ affinity columns and gel filtration. Bioreactor optimisation were performed in 1L DasGip mini-bioreactors, 2L Braun glass bioreactor, and the single-use CellReady3L bioreactor. Alternating Tangential Flow (ATF) technology from Refine was also employed for perfusion production tests. The bioreactor conditions were 25°C, pH6.5, Dissolved Oxygen 20%, 110 rpm stirrer speed using a Marine impeller. The perfusion rate was set to 0.5 to 3 Reactor Volumes (RV) per
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