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
Synthetic Biology
•Closed-loop control over temperature,
pH, dissolved oxygen, and cell optical
density.
•Scaled-down and economic version of a
traditional large-scale fermenter.
•Programmable and flexible microbial
manufacturing enabled by synthetic biology.
•Host cell: Pichia Pastoris
Various modes enabled
with turn-key operation
• 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
Microbioreactor Components
Integrated
Hose Barbs
8 Pressure Regulators
Bioreactor Culture Chip Layout
A 16-Module System
Microbioreactor Volume Control
Device Bonding and Fabrication
Full membrane deflection allows volume control
Device fabrication
•CNC machining and polishing
- Arbitrary 3D channel profiles
- Optical clarity
C
Peristaltic Pump
•Batch
•Fed-batch
•Chemostat
•Turbidostat
•Perfusion
• 3 chambers with 500 uL
volume each.
• Mixing has an optimum
period.
• Tradeoff between turbulent
flow and total flow rate.
• Repeatable Inoculation.
•Plastic to PDMS bonding
- Plastic for dimensional stability
- PDMS for active microfluidics
Plastic to PDMS Bonding Process
Full Deflection Membrane Mixer
Bonding between Polycarbonate and PDMS
Disposable Devices
2 Inches
Growth Experiment with Microbioreactor
OD
pH
Oxygen
Flow
Glucose Feed
Microscale Continuous Cell Culture with Escherichia Coli.
Continuous Culture Sequential Induction
Performance Comparison
Monitoring: Confocal Raman Spectroscopy
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
k La
<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
pH, DO, Feed,
T, DCO2
Online
DO, pH
DO, OD, pH
pH, DO
pH, DO
pH, DO, DCO2,
T, OD
pH, DO, DCO2,
T
Incubator
Yes
Yes
Water Bath
Water Lines
No
No
Agitation
Shaker
Rotator
Rotator
Propeller
Membrane
Deflection
Propeller
Bench
Validation
Yes
Yes
Yes
-
-
-
Comments
24 Deep Well
Plate
Cassettes on a
Rotator
Miniature
Bioreactors
Miniature
Bioreactors
Microfluidic
Chip
Bench Scale
Bioreactors
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 NIR Raman Spectroscopy Schematic
•Confocal Raman spectroscopy
integrated with microbioreactor can
enable real-time nutrient and
metabolite monitoring and lead to new
control strategy.
• Microbioreactor is suitable for small-volume (~ mL) biopharmaceutical production with
tightly controlled fermentation environment.
Lactate and Glucose Concentration Monitoring
Current Development: Optical Induction
Zone of Raman signal collection
~ 0.5 mm
~ 1.6 mm
Acknowledgement
Ram Lab
Professor Rajeev Ram and Dr. Gajendra Singh
Baseline
Cu Inducer
Lu Lab
Professor Tim Lu, Dr. Pablo Perez-Pinera, and Fahim Farzadfard
aTc Inducer
Love Lab
Professor Christopher Love, Dr. Kerry Love, Dr. Kartik Shah, and Nicholas
Mozdzierz
Schematic for the System Configuration
Turbidostat OD Control
Microbioreactor with Light Input
Pharyx Inc.
Dr. Harry Lee and Dr. Kevin Lee
Sequential Induction
• 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.
• Using light as signaling input for gene regulation.
MIT Center for Biomedical Innovation
BioMOD Team
• Advantage: Fast dynamic response compared to signaling
molecules such aTc.
• Periodic optical illumination through microbioreactor chip.
• Real-time fluorescence measurement with flow cells.
Flow Cell for
Fluorescence
Measurement
Funding Agent: DARPA