1. 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,”
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