Techno-economic analysis of semicontinuous production of - PDF document

4/4/2018 1 Techno-economic analysis of semicontinuous production of recombinant butyrylcholinesterase in transgenic rice cell suspension cultures Jasmine M. Corbin 1 , Matthew J. McNulty 1 , Karen A. McDonald 1,2 , and Somen Nandi 1,2 . 1. Department of Chemical Engineering, University of California, Davis, CA, USA 2. Global HealthShare Initiative, University of California, Davis, CA, USA ACS 255 th National Meeting: Disruptive Bioprocessing & Process Integration March 21, 2018 Plant Cell Suspension Cultures 2  Problem: Need for reliable, safe, and scalable methods for production of biologics  Approach: Plant cell suspension cultures Limitations: Advantages: • Immune response to • Inexpensive plant glycans • Intrinsically biosafe • Low growth rate and • Complex products protein expression (post-translational • Not well characterized modifications) or developed 1

4/4/2018 Butyrylcholinesterase from Transgenic Rice Cell Suspension Cultures 3 Butyrylcholinesterase (BChE):  Bioscavenger for nerve agents  Human BChE from plasma = $20k per 400 mg dose  340 kDa tetramer Top view  14 disulfide bonds  36 N-glycosylation sites Rice cell suspension culture:  Dedifferentiated cells from Oryza sativa  Codon optimized human BChE gene Side view  Inducible promoter activated by sugar starvation Pan YM, et al . 2009. J Phys Chem. Semicontinuous Bioreactor Operation 4 1. Expression level: ~60 µg BChE/g FW (4 mg/L culture) in 10L bioreactor 2. Continuous 5/5 day cycling for >6 months with no decrease in expression level Corbin JM, et al. 2016. Frontiers in Plant Science. 7:1-9. 2

4/4/2018 Laboratory Scale Downstream Processing 5 Homogenization Clarification Microfiltration SDS-PAGE >95% purity of active BChE Concentration & Diafiltration (TFF) Affinity Chromatography AEX Chromatography (Hupresin) (DEAE) Western blot Corbin JM, et al. 2018. Biotechnology and Bioengineering:1-10. Design Parameters and Assumptions: Upstream 6 Production Target: 25 kg pure BChE year Current Projected (Laboratory Scale) (Manufacturing Scale) Initial culture density: 7 10 g FW/L Final culture density: 70 100 g FW/L Doubling time: 4 3 days Time, Initial to final density: 13 10 days Growth duration: 5 4 days Expression duration: 5 3 days Total cycle time: 10 7 days 60 200 mg BChE/kg FW Expression level: 4 20 mg BChE/L culture 3

4/4/2018 Design Parameters and Assumptions: Downstream 7 Overall Downstream BChE Recovery: 60% Current Projected (Lab Scale, Experimental*) (Manufacturing Scale) Step Step Process Step Process Recovery Recovery Recovery Recovery Crude Extract 100% 100% 100% 100% Clarification 100% 100% 96% 96% UF/DF (TFF) 95% 95% 97% 93% IEX (DEAE) 75% 71% 80% 73% Affinity (Hupresin) 60% 43% 85% 62% UF/DF (TFF) 95% 41% 95% 59% *Corbin JM, et al. 2018. Biotechnology and Bioengineering:1-10. Upstream: Seed Train 8 Each seed train step: • Inoculate at 10 g/L • Grows to 100 g/L over 10 days (τ D = 3 days) • Used to inoculate next step at 10 g/L 4

4/4/2018 Upstream: Production Reactor 9 4x 20kL production reactors • Grow 4 days (20kL) • Medium exchange (20kL) • Express 3 days (20kL) • Harvest (10kL) • Medium exchange (20kL) • Repeat for 6 months before shutdown, CIP, SIP Downstream: Clarification and Filtration 10 • Extract at 1:3 ratio (biomass to buffer) • Concentrate 10x • Diafilter 4x 5

4/4/2018 Downstream: Chromatography and Formulation 11 • All chromatography bind and elute • DEAE recovery = 80% • Hupresin recovery = 85% Final product: • 59% Recovery • 95% Purity Economics: CAPEX, OPEX, COGS 12 Annual Operating Cost Parameter Unit Section Base Case Breakdown Upstream 141.6 Consumables CAPEX $ million Downstream 24.4 8% Total 166.0 Laboratory/QC/QA 6% Upstream 2.6 $ OPEX million/ Downstream 32.0 Labor-Dependent year Total 34.6 17% Upstream 115 Raw Materials $/g COGS Downstream 1,432 69% BChE Total 1,547 $620 per 400 mg dose 6

4/4/2018 Conclusions and Future Work 13  Developed base case model for  Refine costs, process times, manufacturing scale production process parameters of recombinant BChE in  Minimize equipment down transgenic rice cell suspension time culture  Alternate cases Cost per 400  Reactor scheduling BChE Source mg Dose  Clarification equipment >30-fold Human plasma $20,000  Sensitivity analysis reduction in cost Rice cell culture $620  Expression level  Developed tool for future process  Affinity resin binding capacity, design and analysis recovery Acknowledgements References 14 Corbin JM , et al . 2016. Semicontinuous Bioreactor  Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures. Frontiers in Plant Science. 7 :1–9. Corbin JM , et al . 2018. Purification,  characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures. Biotechnology and Bioengineering :1–10. Tusé D, et al . 2014. Manufacturing economics of  plant-made biologics: Case studies in therapeutic and industrial enzymes. Biomed Res. Int. 2014. Nandi S, et al . 2016. Techno-economic analysis of  a transient plant-based platform for monoclonal antibody production. MAbs 8 :0. http://mcdonald-nandi.ech.ucdavis.edu/ 7