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Advances in the Drosophila Expression System W.A. de Jongh, CSO New Cells, New Vaccines VII 18 March, 2013 Overview • About ExpreS2ion Biotechnologies • Drosophila S2 and The ExpreS2 system • Two vaccine development case studies EXPRES2ION Biotechnologies • Founded in 2010 in Copenhagen, Denmark • A CRO spin-off from Affitech A/S • Proprietary technology platform for production of proteins based on Drosophila S2 insect cells • > 10 years experience with S2 cells from research to GMP production for human clinical trials Drosophila S2 insect cells • Derived from the late stages of Drosophila melanogaster (fruit fly) embryos (Schneider, 1972) • Doubling times of 18-24 hrs (both serum-containing and serum-free medium) • Cell size: 9-10 µm in diameter • Grow well between 5 and 350E6 cells/ml in suspension • Grow at 23ᵒC to 27ᵒC • Very robust The ExpreS2 System An integrated and proprietary protein expression platform for the biopharmaceutical industry Know-How Expression vectors Transfection reagent Expression Host Culture medium • • • • • From construct to protein in 6 weeks GLP protein production Vector and cell line development Upstream process development Scale-up and transfer to cGMP Advantages of the ExpreS2 System • High success rate for complex proteins and VLPs • High yields for secreted proteins • Very fast to protein and stable process • • • • • Very high cell densities without aggregation Straightforward scalability, from lab to cGMP Robust system Homogeneous product Regulatory acceptance & reduced risk of human viral contaminants Saves time in R&D Flexibility & Compliance in Production ExpreS2 Improves Yields Medium development Vector development 300 100 100 pExpreS2-1 90 RANKL Mg/L RANKL (mg/L) Mg/L RANKL pExpreS2-2 80 70 60 50 40 30 20 10 0 Hybrid_RNA+ Hybrid pAc5.1/V5 pMT/V5.His.A Control Med.1 Med.2 Med.3 Med.4 Med.5 Med.6 ExpreS2ion *Adaptation to individual media were performed Quick to Protein and Stable Process Transfection Stable polyclonal cell line - High stability Week 1 Week 3 Week 6 Purified protein candidates from bioreactor 1st screening of protein candidates High density frozen cell lines 3-4 Weeks What makes the ExpreS2 system different Compared to Baculovirus insect cell systems • Non-viral, Non-lytic • More homogeneous product • High reproducibility and consistency between batches • High yield for secreted proteins • Long-term stability of frozen cell stocks Compared to other S2 systems • • • • Increased yield Protocols for large-scale production Including Perfusion processes Support for process development and cGMP production More Homogeneous Product • Consistent Glycosylation BEVS produced Hemagglutinin A from H5N1 bird flu material in: Sf21 Sf9 Compared to: Stable S2 cell line 3xHA2 HA1 HA2 Effect of different MOI’s and harvest time points BEVS: Baculovirus Expression Vector System MOI: Multiplicity of infection Consecutive days of harvest from a bioreactor running perfusion rProteins Made in Insect Cells are Clinically Validated Baculovirus-based Products on the market gsk Dendreon Protein Sciences HPV vaccine –Cervarix® Prostate cancer – Provenge® Flublok Market Market FDA approved Drosophila S2 products in clinical development Pharmexa TxCells Merck Inc. Hawaii Biotech Pharmexa Copenhagen University HER-2+ breast cancer vaccine Crohn’s disease Tetravalent Dengue fever vaccine West Nile virus vaccine Bone metastatic cancer vaccine Placental malaria vaccine Phase 2 Phase 2 Phase 1 Phase 1 Late PC Late PC Role of ExpreS2 in Malaria Vaccine Development Copenhagen University Placental Malaria The Jenner Institute, Oxford University Blood stage malaria Vaccine development case study 1: Placental Malaria Vaccine Development Assc. Prof. Ali Salanti, Prof. Thor Theander Placental Malaria Vaccine VAR2CSA from Plasmodium falciparum was identified by CU as a possible vaccine target (2003) CU Malaria case study Antigen Selection and Expression VAR2CSA is a complex 350 KDa protein, with 7 domains. Using Biosensor technology, the core CSA binding site within the DBL2X and minimal binding region (ID1-DBL2Xb; 62 Kda) were identified (CU) CU Malaria case study Screening Antigen Variants with ExpreS2 Variant construction Shake-flask production (500ml) 1L-15L production CU Malaria case study • Screening capability: ~40 variants have been tested • Expression ability and level • Initial immune response • Immune response, cross-reactivity • Scalability Relative expression level of 34 VAR2CSA truncation variants Immunogenicity and Cross-Reactivity AB induction (rats) VAR2CSA fragments -ABs raised in rats can block binding Cross-strain reactivity VAR2CSA fragments induce cross-reactive ABs to parasite subtypes Cross reactivity of 2 variants shown . Y axe - Proportion of binding CU Malaria case study Application of Single-Use Technology • Flexiblity • Reduced cost of goods Current Status and Future Work Minimal VAR2CSA regions can be expressed as functional proteins in ExpreS2 The proteins induce functional antibodies in various animal species Variant selection is completed • Upstream & downstream development is ongoing • Scale-up and transfer to CMO for GMP production • Phase Ia and Ib within 3 years Vaccine development case study 2: Development of a Broadly-Neutralising Vaccine against Blood-Stage P. falciparum Malaria Dr. Simon Draper Next-Generation Anti-Merozoite Vaccines 1. Are there CONSERVED and HIGHLY SUSCEPTIBLE target antigens in the Mz? 2. Is it possible to develop a strain-transcending anti-Mz vaccine Rh5 Identified as Vaccine target MEROZOITE Erythrocyte Invasion by P. falciparum Merozoite RH5 RH5 is essential in culture – KOs failed RH5 has an essential interaction with BSG Basigin Red blood cell Parasitophorous Vacuole Formation and Invasion Inward Motion Driven by Actinomyosin Motor Highly conserved (Potentially highly cross-reactive) RBC Merozoite Attachment Protein in adjuvant vaccine100% protection in primate model Resealing of RBC and Parasitophorous Vacoule Membrane The Problem? • Rh5 is extremely hard to produce – Failure: • E.coli • Yeast • Baculovirus – Partial success in transient Hek293 • Low yields ~1mg/L • A fusion protein, not native Rh5 ExpreS2 system expresses Rh5 at high levels PfRh5 S2 supernatant 70 Rh5 55 TFF/buffer exchange/filtration S2 produced Rh5 binds Basigin Affinity Chromatography Kd = 0.9µM 1 Size Exclusion Chromatography & concentration Kathryn Hjerrild, Matt Higgins, Kate Wright 1 2 2 Initial Process Optimization Application of single use and perfusion technology Batch Fed Batch ATF-Perfusion The ATF (Alternating Tangential Flow) method concentrates cells and protein Production using Fed-batch and High-Density Perfusion Culture Fed-Batch ATF-Perfusion Process Dependent Rh5 Yields • Hek293 yield = 1mg/L • S2 yield ... Concentrated Perfusion/ Concentrated Fed-batch Take Home Messages • Successful expression of difficult proteins • Well suited to Vaccine manufacture (clinical track record) • High yields for secreted proteins (multiple processing options) • Fast (6 weeks) • Great for R&D (speed, ease of use, high success rate) Acknowledgments Assc. Prof Ali Salanti Prof. Thor Theander Assc. Prof. Morten Agertoug Nielsen Mafalda Dos Santos Marques Resende Besim Berisha Simon Draper Sandy Douglas Joe Illingworth Kathryn Hjerrild Charlotte Dyring Anette Strøbæk Stine Clemmensen Carsten Leisted Lars Poulsen Sancha Salgueiro Selected Customers and Collaboration Partners Questions? GLYCOSYLATION Insect (S2) CHO Human N-glycosylations found in RANKL based on MALDI-TOF analysis 0 N-glycosylation +1 N-glycosylation +2 N-Glycosylation Native Glycosylated RANKL1.12 - 5 different Mw’s N-deglycosylated RANKL1.12 34 C C+F C C C+F C C+F C+F The parasite expresses proteins on the surface of infected red blood cells, which allows it to bind different tissues in the body IRBC Infected erythrocyte VAR2CSA CSA Placental tissue