fernandes 2013 april 16 miniaturization speeding-up bioprocess development

7/27/2019 Fernandes 2013 April 16 Miniaturization Speeding-up Bioprocess Development

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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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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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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


-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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fernandes 2013 april 16 miniaturization speeding-up bioprocess development

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