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Miniaturization: speeding-up bioprocessdevelopment
Pedro Fernandes
Department of Bioengineering & Institute of Biotechnology and BioengineeringCentre for Biological and Chemical Engineering
Instituto Superior Tcnico
Universidade Tcnica de Lisboa
Portugal
Peniche, April 2013
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Overview
Miniaturized devices in bioprocesses
In-house case studies
Conclusions/remarks
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Overall costreduction
Fastened processdevelopment
Low volumes
Parallelization
Automation
ml
l
nl Increasedcompetitiveness of
bioprocesses
Fernandes et al. Recent Pat Biotechnol (2011) 5: 160173
Miniaturization in bioconversion
processes
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Screening for relevant metabolites from microbial strains
Metabolic flux analysis experiments
Measuring global gene expression
Screening for effect of environment on growth
Media development
Optimization of fermentation/(bio)conversion
Production stage
Microstructuredreactors
Process development
Non-structuredreactors
Microstructuredreactors
OperationBatch
Fed-batch
Continuous
Operation
Continuous
Miniaturization in bioconversionprocesses
Bolivar et al. Trends Biotechnol (2011) 29: 333342
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Process intensification
Large
Expensive
Energy-intensive
Process/EquipmentProcess/Equipment
Small
Less expensive
More efficient
Integration of multiple
operation in a single (or few)
devices
About 30% savings in raw materials, energy and operating costsAdapted from Chem. Eng. Technol. 2005, 28, 255-258
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Process intensificationMiniaturized devices in R&D
Typical pace of process development in drug discovery
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Tools of the trade
Non-structured vessels
MTP platforms
With sensor spots(monitoring)
200 l 4 ml reaction volume
With pH, DOT, T, shaking, monitoring and control
Cost
MTP
24-, 48-, 96-well
Shallow wellsDeep wells
xxx ~ 10,000
kLa 500 h -1
~ 100,000 / 200,000
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Non-structured vessels
Schpper et al. Anal BioanalChem (2009) 95:679695
50 150 l reaction volume
Stirred microreactors 10 ml working volume
Bareither and Pollard. Biotechnol Prog (2011) 27: 214
Stirred mini-reactors
AAB CA B
In-house reactors: 1 ml (A) and 25 ml (B) working volume
Nunes et al. Bioresource Technol (2013)http://dx.doi.org/10.1016/j.biortech.2013.02.057
pH, DOT, T, stirringmonitoring and control(optical fibers)
Stirring monitoringand control, (pHmonitoring), outer Tcontrol
Tools of the trade
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Non-structured vessels Stirred mini-reactors
Xtreme parallelization
Schematic of parallel (48), independent single usereactors (16 ml working volume), with microfluidicpumps for pH control
Prototype of the a microfluidic device mounted onthe bioreaction block. 144 micro-pumps arecontrolled
3.8 cm
12 cm
Gebhardt et al. Biotechnol. Prog., 27: 684690, 2011
Complexity and cost!
Tools of the trade
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Tools of the trade
Non-structured vessels Stirred mini-reactors
Complexity and cost!
Cell culture
Xtreme parallelization
pH and DOToptodes
Ambr TM from The Automation Partnership, TAP24 disposable reactors x 10 mL working volume
Bareither and Pollard. Biotechnol Prog (2011) 27: 214
6 microbioreactor per array x 300 to 700 L workingvolume
Microfluidic channels for feed(innoculation, pH adjustment)and sampling
Gas permeable membrane In-chambersensors,externalmonitoring
20 rpm
Low k La ~ 7 h -1Low k La ~ 30 h-1
~ 200,000 > 200,000
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Microchannel devices
Microchannel reactor & chip holder
Structured packing (viz. monoliths)
Tools of the tradeMicrostructured vessels
Microreactor operation with syringe pump
Coated-wallmicroreactor
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Exploded schematics of a nanospring reactor
Tools of the tradeNanosprings microreactor
Exploded schematics of a nanospring reactor
Morphology of silicon dioxide nanosprings beforeand after silanization with ATPES
Nanospring reactor operation
Substrate
QuencherSchilke et al. Biotechnol. Prog., 26: 15971605, 2010
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Tools of the tradeNovel packed bed reactor
Inflow
ScrewsInlet Outlet
Top view
Side viewTeflon sheet and channel cut inside the sheet
Biocatalyst packing
PMMA top layer
PMMA bottom layer
Inflow OutflowImmobilized enzyme particles
Pohar et al. Chem. Eng. J. (2012), doi:10.1016/j.cej.2012.02.035 Cvjetko et al. Proc Biochem (accepted)
Carvalho et al. (submitted)
Outflow
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Miniaturization and dspATPS
Meagher, Lab Chip 2008, 8, 527-532
SooHoo. Biomed. Microdevices 2009,11, 323-329
Marques & Fernandes, Molecules, 16, 8368-8401, 2011
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Miniaturization and dsp
Chromatography
96-well filter plates for screening of chromatographic resins, namely for ion exchange andantibody affinity chromatography
Pipette tips with chromatographic packings
Packed miniature column (< 10 mL)
Microfluidic column (1.5 L ion exchange chromatography column)
Fernandes et al. . Recent Pat. Biotechnol., 5, 160-173., 2011
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Organometallic reaction, Lonza, 700 kg of product in a few weeks Roberge et al. Org. Proc. Res. & Develop.2008, 12, 905910
15 tonnes per annum
Weiler et al. Chemistry Today, 27, 3 / May-June 2009
Synthesis of trinitroglycerin at a pharmaceutical grade with throughputs of 9 kg h-1
and further examples at Wiles C & Watts P. Green Chem. DOI: 10.1039/c1gc16022b
Microreactors: production scaleSome examples
25 tonnes of a nitration product under cGMP conditions in four weeks ( DSM )http://www.icis.com/Articles/2009/05/04/9211877/microreactors-gain-popularity-among-producers.html
SAFC, a Sigma Aldrichsubsidiary, Lonza,
BASF and Evonik IndustriesDSM
DuPont,Schering-Plough, Sanofi,
Aventis, Roche,GlaxoSmithKline, Novartis
and Astra Zeneca.
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Microreactors operate in continuous mode (flow chemistry), hence solving the apparentparadox of a microreactor producing on 10,000 tonnes/year of a specialty chemicals
Reactions fit for microreactor technology
Type A instantaneous, mixing controlled
Type B rapid (t~minutes), kinetically controlled Type C slow, with thermal hazard
Thus
very slow reactions, solids forming, are excluded
Why?
Technical notes: Lonza microreactor concept, www.lonza.com
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Significant potential for intensification if and only if the actual chemical transformation isrelatively fast the overall process occurs under heat and/or mass transfer limitations, yetbiotransformations are often slow, with 0.1 < k rate < 100 s 1 )
Selected cases of enzyme catalyzed reactions, particularly those involving multi-phasesystems (transport accross phase boundaries) may be adequate
Preferably coupled with easy reversible enzyme immobilization
Production scale
Very high loading (Fractogel)
Leaks easily under 2 M NaCl
Wiesbauer et al. ChemCatChem (2011) 3, 1299-1303 Marques & Fernandes, Molecules (2011) 16, 8368-8401
What about biological reactions?
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Fasten bioprocess development/shifting paradigms
where we stand
Laboratoryphase
Pilot phase Production phase
Typical batch development Typical (fed)batch production HTP batch development Typical (fed)batch production
Lack of reliablescaling criteriaScarce monitoring
Reliable scalingcriteriaMonitoring (andcontrol)
Pilot phase Production phaseLab phase
Production phase
yet
Micronit parallel multi-layeredreactor moduleMicronit mL
scalemicroreactorLab phase
Evolution!!
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New paradigms!!
Scaling-out rather than scaling-up
Pressure on analytical department
Focus in the development of on- and at-line monitoring tools
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In-House AchievementsOur path to success
HO
O
O
Mycobacterium sp. NRRL-3805
Sitosterol 4-Androstene-3,17-dione(AD)
Multi-step bioconversion
Production of intermediates (AD)for therapeutic steroids
Industrial use of mycobacteria (non-
pathogenic)(along with BCG, with an attenuatedM. bovis )
Model system
Typically performed in fermentative, fully aqueous processes, with growing cellsTime consuming (3 days +), volumetric productivity hampered by low solubility ofsubstrate and product (< 0.1 mM)
Mycobacterium sp. NRRL B-3805 have a lipophilic nature and tend to form aggregates
Fernandes, P., Cabral, J.M.S., Steroid bioconversions, in: Encyclopedia of Industrial Biotechnology, Vol. 7, John Wiley & Sons, New York, pp. 4610-4628, 2010
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Steroid side-chain cleavage
Can it be addressed with miniaturized devices (MTP)?
Marques et al. . J Chem Technol Biotechnol 82:856863 (2007)
Yes, it can!!
(but 2-phase systems canbe tricky)
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In-House AchievementsOur path to success
Effect of key parameters (k La, aeration,
medium composition) on productivity
Scale-up criteria
On-line: DOT, pH, T
Off-line analysis
Stero(l)ids, some nutrients: HPLC
Biomass, some nutrients:Spectrophotometry
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Synthetic medium selection: AD production from 2.4 mM sitosterol
20 gL -1 glycerol4 gL -1 NH4Cl
Complex medium
10 gL -1 glycerol10 gL -1 yeast extract
Effect of k La in AD productivity
Synthetic medium
Complex medium
Working with 24-well MTP
Marques et al. Biotechnol J (2010) 5: 402412
Unsealed
Sealed
Marques et al. J. Biotechnol (2009) 141: 174180
Marques et al. J. Biotechnol (2009) 141: 174180
Effect of sealing and ofshaking frequency in k La
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From MTP to bioreactor
Marques et al. Biotechnol J (2010) 5: 402412
kLa ~ 40 h -1 kLa ~ 160 h-1
24-well MTP 5 l bioreactor
Scaling-up from 24-well MTP to a 5 l bioreactor can be done!
If the adequate k La is chosen as scaling criterion
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Microfluidic environment
CholesterolHO
H H
HH
CholestenoneO
H H
HH
Cholesteroloxidase
Buffer + enzyme (4.4 l min -1)
n-heptane + cholesterol (1.0 gl -1; 9.6 l min -1)
Buffer + enzyme + H 2O2
O2+ H 2O2
n-heptane + cholestenone
STR - 200 rpm
STR - 300 rpm
STR - 400 rpm
PFR
0
10
20
30
40
50
60
70
80
90
100
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
X ( % )
Y - MR
0
10
20
30
40
50
60
70
80
90
100
0.0E+00 1.0E-04 2.0E-04 3.0E-04 4.0E-04 5.0E-04 6.0E -04 7.0E-04 8.0E-04
X ( % )
A B
STR 400 rpm
STR 300 rpm
STR 200 rpm
Microreactor
100
80
60
40
20
0
100
80
60
40
20
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
X 10 4
r
t
VtE
= FE
t=
STR: Residence time = 90 min
MR: Residence time = 1 min
Same X (%)
Marques et al. Chem Eng J (2010) 160:708714
X ( % )
X ( % )
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Continuous production ofcholestenone
Figure 1 . Scheme of the experimental setup
A
B
D
E
C
A. Packed-bed reactor
B. Y-microchannel reactor
C. Peristaltic pump
D. Spectrophotometer
E. Stirred aerated tank
F. Seringe pumps
F
Legend
Combining cholesterol oxidase (YMR) with catalase (PBR)
Cholestenone production andsimultaneous removal of hydrogenperoxide
0
5
10
15
20
25
30
35
40
45
0 50 100 150 200 250 300
Operational time (h)
P r o
d u c
t ( M )
36 M of cholestenone producedafter 300 h of continuous operation
Marques et al. New Biotechnol (2012) 29: 227-234
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Miniature bioreactorsCharacterization of immobilized biocatalysts
Activity vs pH, T, substrate concentration (3 days)
Operational stability of PVA-immobilized inulinase
Operational stability of sol-gel-immobilized inulinase
Fernandes et al. J ChemTechnol Biotechnol 2009; 84: 561564
Santa et al. Appl Biochem Biotechnol (2011) 165:112
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Miniature bioreactorsCharacterization of immobilized biocatalysts
Continuous operation with PVA-immobilized inulinase
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Productyield(%)
Time (days)
Continuous operation was performed during 20 days at 50 \ C in an inulinsolution of 50g/L pH 4.5, 0 .1M at a flow rate of 42 L/min.
Production of fructose syrup
Anes, J. et al Appl Biochem Biotechnol (submitted)
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Miniature bioreactorsCharacterization of immobilized biocatalysts
Continuous operation with immobilized invertase
Production of invert sugar syrupReactants Products
Immobilized biocatalyst (in CPC)
Length = 6 cm Hight = 1mm Width = 2 cm Volume = 120 LVoid volume = 20 L
Suport mass = 150 mg Carvalho, F.. et al J. Biotechnol. (submitted)
0
20
40
60
80
100
0 50 100 150 200 250 300 350 400
Conversion(%)
Flow rate (L/min)
[sucrose] 10 g/L
[Sucrose] 20 g/L
[Sucrose] 40 g/L
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Immobilizationsupportsscreened
Lentikat based beads: 3 mm sized particles (polyvinylalcohol derivative, PVA)
Activated Amberlite: micrometer (hundreds) sized particles
Sol-gel microparticles:p-nitrophenyl--glucoside p-nitrophenol
-glucosidase
Spectrophotometricreading
Representative
Simple
Easy to implement
Hydrolysis in acetate buffer
Lentikat lenses
Eupergit
Gelatine
Ca-alginate
Well-established, most proved feasible in large scale applications
Miniature bioreactorsCharacterization of immobilized biocatalysts
-glucosidase immobilization Cellulose hydrolysis
Operational stability
Figueira et al. Enzyme Res. 2011, Article ID 642460, 8 pages
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MTP designed for high throughput analysis ofreducing sugars
Nunes et al. Appl Biochem Biotechnol. (2010) 160:2129-2147
Naringin hydrolysis
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Naringin hydrolysis
A
C
Mechanical stirred reactorsOrbital shaken reactors Packed bed reactors
Microreactor 2000 L
B CD
Multibioreactor system10ml-600ml
Packed bed reactor 8ml
Thermostated microplate1000 L -2000 L
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