Microfluidics for Biopharmaceutical Production

Ningren Han, Gajendra Singh, and Rajeev Ram4.13

On-demand Biopharmaceutical Production

AcknowledgementCurrent Development: Optical InductionContinuous Culture Sequential Induction

Monitoring: Confocal Raman SpectroscopyPerformance ComparisonGrowth Experiment with Microbioreactor

Device Bonding and FabricationMicrobioreactor Volume ControlMicrobioreactor Components

Microscale BioreactorsApproach

Ram LabProfessor Rajeev Ram and Dr. Gajendra Singh

Lu LabProfessor Tim Lu, Dr. Pablo Perez-Pinera, and Fahim Farzadfard

Love LabProfessor Christopher Love, Dr. Kerry Love, Dr. Kartik Shah, and Nicholas Mozdzierz

Pharyx Inc.Dr. Harry Lee and Dr. Kevin Lee

MIT Center for Biomedical InnovationBioMOD Team

Funding Agent: DARPA

• Motivation: Integrated and Scalable Cyto-Technology (InSCyT) biomanufacturing platform.

• Goal: On-demand programmable biopharmaceutical protein production in small scale.

• Integrated unification for upstream, downstream and analytics subunits.• Microscale fermentation and production approach.• Rapid release of single-dose biopharmaceuticals (within 24 h).

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.

“Hardware”

Upstream Biopharmaceutical Production

Synthetic Biology

•Programmable and flexible microbial manufacturing enabled by synthetic biology.•Host cell: Pichia Pastoris

“Software”

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

Bioreactor Culture Chip Layout

Bioreactor Culture Chip

Microbioreactor control capability

•PID temperature control• (+/- 0.1 ºC)

•PID oxygen control• (+/- 5%)

•Threshold pH control• (+/- 0.07)

•Threshold optical density control• (+/- 0.03)

Various modes enabled with turn-key operation

•Batch•Fed-batch•Chemostat•Turbidostat•Perfusion

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.

Confocal NIR Raman Spectroscopy Schematic

Lactate and Glucose Concentration Monitoring

Zone of Raman signal collection~ 0.5 mm~ 1.6 mm

Device fabrication

•CNC machining and polishing- Arbitrary 3D channel profiles- Optical clarity

•Plastic to PDMS bonding- Plastic for dimensional stability- PDMS for active microfluidics

Plastic to PDMS Bonding Process

Bonding between Polycarbonate and PDMS Disposable Devices

C

8 Pressure Regulators

Peristaltic Pump

Full Deflection Membrane Mixer

Integrated Hose Barbs

2 Inches

• 3 chambers with 500 uL volume each.

• Mixing has an optimum period.• Tradeoff between turbulent

flow and total flow rate.• Repeatable Inoculation.

Full membrane deflection allows volume control

OD

pH

Oxygen

Flow

Glucose Feed

Microscale Continuous Cell Culture with Escherichia Coli.

• 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.

Schematic for the System Configuration Microbioreactor with Light Input

Flow Cell for Fluorescence Measurement

Type Applikon 24 SIM Cell HTBR Ambr (TAP) This Work Bench Scale

Source Chen 2009Legmann 2009,Amanullah 2010

Kondragunta 2010

-Lee 2006Lee 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

pH, DO, Feed, T, DCO2

Online DO, pH DO, OD, pH pH, DO pH, DOpH, DO, DCO2,

T, ODpH, DO, DCO2,

T

Incubator Yes Yes Water Bath Water Lines No No

Agitation Shaker Rotator Rotator PropellerMembrane Deflection

Propeller

Bench Validation

Yes Yes Yes - - -

Comments24 Deep Well

PlateCassettes on a

RotatorMiniature

BioreactorsMiniature

BioreactorsMicrofluidic

ChipBench Scale Bioreactors

• Microbioreactor is suitable for small-volume (~ mL) biopharmaceutical production with tightly controlled fermentation environment.

• 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.

Turbidostat OD Control

Sequential Induction

Baseline Cu Inducer aTc Inducer