Microfluidics for Biopharmaceutical Production Ningren Han, Gajendra Singh, and Rajeev Ram 4.13 On-demand Biopharmaceutical Production Approach Microscale Bioreactors Integrated unification for upstream, downstream and analytics subunits. Microscale fermentation and production approach. Rapid release of single-dose biopharmaceuticals (within 24 h). Microbioreactor control capability PID temperature control (+/- 0.1 ºC) PID oxygen control (+/- 5%) Threshold pH control (+/- 0.07) Threshold optical density control (+/- 0.03) Upstream Biopharmaceutical Production “Hardware” “Software” Miniaturized Microbioreactors Closed-loop control over temperature, pH, dissolved oxygen, and cell optical density. Scaled-down and economic version of a traditional large-scale fermenter. Synthetic Biology Programmable and flexible microbial manufacturing enabled by synthetic biology. Host cell: Pichia Pastoris Bioreactor Culture Chip Layout Various modes enabled with turn-key operation Batch Fed-batch Chemostat Turbidostat Perfusion Motivation: Integrated and Scalable Cyto-Technology (InSCyT) biomanufacturing platform. Goal: On-demand programmable biopharmaceutical protein production in small scale. Bioreactor Culture Chip Image adapted from MIT News, “Cell circuits remember their history,” http://web.mit.edu/newsoffice/2013/cell-circuits-remember-their-history-0210.html A 16-Module System Microbioreactor Components Microbioreactor Volume Control Device Bonding and Fabrication Integrated Hose Barbs C 8 Pressure Regulators Full membrane deflection allows volume control Device fabrication CNC machining and polishing Arbitrary 3D channel profiles Optical clarity Plastic to PDMS bonding Plastic for dimensional stability PDMS for active microfluidics 3 chambers with 500 uL volume each. Mixing has an optimum period. Tradeoff between turbulent flow and total flow rate. Repeatable Inoculation. Peristaltic Pump Plastic to PDMS Bonding Process Full Deflection Membrane Mixer Bonding between Polycarbonate and PDMS Disposable Devices 2 Inches Growth Experiment with Microbioreactor Performance Comparison Monitoring: Confocal Raman Spectroscopy In situ bioprocess monitoring Confocal Raman spectroscopy for cell culture metabolite monitoring in a microbioreactor. Prediction of lactate and glucose concentration in Chinese hamster ovary (CHO) cell culture with confocal Raman spectroscopy. Confocal Raman spectroscopy integrated with microbioreactor can enable real-time nutrient and metabolite monitoring and lead to new control strategy. Type Applikon 24 SIM Cell HTBR Ambr (TAP) This Work Bench Scale Source Chen 2009 Legmann 2009, Amanullah 2010 Kondragunta 2010 - Lee 2006 Lee 2011 Bareither 2010 Max Cell Density 2.6 x 106 cells/mL 12 x 106 cells/mL 2.3 x 106 cells/mL 3 x 107 cells/mL kLa <30 h-1 7 h-1 0.9 h-1 <500 h-1 1 – 15 h-1 Working Volume 5 – 6 mL 300 – 700 µL 35 mL 10 – 15 mL 1 – 3 mL 1 – 30 L Parallel 24 6 12 24 or 48 16 1 Control pH pH, Feed pH, DO, Feed, T, DCO2 pH, DO, Feed, T, DCO2, OD Online DO, pH DO, OD, pH pH, DO pH, DO, DCO2, T, OD pH, DO, DCO2, T Incubator Yes Water Bath Water Lines No Agitation Shaker Rotator Propeller Membrane Deflection Bench Validation Comments 24 Deep Well Plate Cassettes on a Rotator Miniature Bioreactors Microfluidic Chip Bench Scale Bioreactors OD pH Oxygen Confocal NIR Raman Spectroscopy Schematic Flow Glucose Feed Microbioreactor is suitable for small-volume (~ mL) biopharmaceutical production with tightly controlled fermentation environment. Zone of Raman signal collection ~ 0.5 mm ~ 1.6 mm Microscale Continuous Cell Culture with Escherichia Coli. Lactate and Glucose Concentration Monitoring Continuous Culture Sequential Induction Current Development: Optical Induction Acknowledgement Ram Lab Professor Rajeev Ram and Dr. Gajendra Singh Lu Lab Professor Tim Lu, Dr. Pablo Perez-Pinera, and Fahim Farzadfard Love Lab Professor Christopher Love, Dr. Kerry Love, Dr. Kartik Shah, and Nicholas Mozdzierz Pharyx Inc. Dr. Harry Lee and Dr. Kevin Lee MIT Center for Biomedical Innovation BioMOD Team Funding Agent: DARPA Baseline Cu Inducer aTc Inducer Schematic for the System Configuration Microbioreactor with Light Input Turbidostat OD Control Sequential Induction Using light as signaling input for gene regulation. Advantage: Fast dynamic response compared to signaling molecules such aTc. Periodic optical illumination through microbioreactor chip. Real-time fluorescence measurement with flow cells. Saccharomyces Cerevisiae with duel inducible reporter proteins (RFP and YFP) cultivated inside microbioreactor in continuous cell culture mode. Turbidostat control maintaining OD600 at 1. Sequential induction with Cu and aTc inducers. Each inducer promotes the expression of the corresponding reporter protein. Flow Cell for Fluorescence Measurement