cell disruption technologies for bioprocess

cell disruption technologies for bioprocess

2. Intact cell
Totally disrupted cell
Permeabilized cell
Cell disruption and permeabilization

3. Methods for Cell Disruption
Heat shock Detergents
Sequestrants

4. Physical methods-decompression
Italsoknownasexplosivedecompressionorcellbombmethod.
Inthisprocess,cellsareplacedunderhighpressure(usuallynitrogenor
otherinertgasuptoabout25,000(psi)andthepressureisrapidlyreleased.
Therapidpressuredropcausesthe
dissolvedgastobereleasedas
bubblesthatultimatelylysesthecell
Largequantitiesofnitrogenarefirst
dissolvedinthecellunderhighpressure
withinasuitablepressurevessel.
Then,whenthegaspressureissuddenly
released,thenitrogencomesoutof
thesolutionasexpandingbubbles
thatstretchthemembranesofeach
celluntiltheyruptureandrelease
thecontentsofthecell.
Cell Disruption Bombs (Parr)

5. Itcanbeconductedatlowtemperaturebypre-chillingorbyoperatingthe
bombinanicebath.
Thereisnooxidation:
Meansitoffersprotectionagainstoxidationofcellcomponents.
Althoughothergases:carbondioxide,nitrousoxide,carbonmonoxideand
compressedairhavebeenusedinthistechnique,nitrogenispreferred
becauseofitsnon-reactivenatureandbecauseitdoesnotalterthepHofthe
suspendingmedium.
Inaddition,nitrogenispreferredbecauseofit’slowcost.
Itisagentlemethod:
Claimedtobemoreprotectiveforenzymesandorganellesthanultrasonic
andmechanicalhomogenizingmethods

6. Anysuspendingmediumcanbeused.
Thesuspendingmediumcanbechosenforitscomparabilitywiththeend
useofthehomogenateandwithoutregardforitsadaptabilitytothe
disruptiveprocess.
Thisoffersgreatflexibilityinthepreparationofcellsuspensionsand
producesacleanhomogenatethatwillnotrequireintermediatetreatmentto
removecontaminateswhichmightbeintroducedwhenusingother
disruptionmethods.
Eachcellisexposedonce:
Heresubstancesarenotexposedtocontinuedattritionthatmightdenature
thesampleorproduceunwanteddamage.

7. Useanysamplesize.
Celldisruptionbythismethodisindependentofsamplesizeor
concentration
Easy to control.
Can be easily controlled by adjusting the nitrogen pressure
Theproductisuniform:
Sincenitrogenbubblesaregeneratedwithineachcell,thesamedisruptive
forceisapplieduniformlythroughoutthesample,thusensuringunusual
uniformityintheproduct
Thereisnoheatdamage:
Unlikeothermethodnitrogendecompressionprocedureisaccompanied
byanadiabaticexpansionthatcoolsthesampleinsteadofheatingit.

8. .
Thetechniqueisusedto:
Homogenizecellsandtissues
Releaseintactorganellesandtopreparecellmembranes
Releaselabilebiochemicals
Produceuniformandrepeatablehomogenates
Well suited for treating mammalianand other membrane bound cells.
Used successfully for treating plant cells, for releasing virus from fertilized
eggs and for treating fragile bacteria.
Not recommended for untreated bacterial cells. Yeast, fungus, sporesand
other materials with tough cell walls do not respond well to this method

9. Disadvantagesincludes:
Onlyeasilydisruptedcellscanbeeffectivelydisrupted(stationaryphaseE.
coli,yeast,fungi,andsporesdonotdisruptwellbythismethod).
Largescaleprocessingisnotpractical.
Highgaspressureshaveasmallriskofpersonalhazardifnothandled
carefully

10. Physical methods-Ultrasonic cell disruption
Acommonlaboratory-scalemethodforcelldisruptionappliesultrasound
(typically20-50KHz)tothesample(sonication).
Thetreatmentofmicrobialcellsinsuspensionwithinaudibleultrasound
(greaterthanabout18kHz)resultsintheirinactivationanddisruption.
Ultrasonicationutilisestherapidsinusoidalmovementofaprobewithinthe
liquid.
Ultrasoundischaracterisedby:
highfrequency(18kHz-1MHz),
Smalldisplacements(lessthan
about50µm),
moderatevelocities(afewms
-1
),
steeptransversevelocity
gradients(upto4,000s
-1
)
andveryhighacceleration
(uptoabout80,000g).
Cell suspension
Ultrasound tip
Ultrasound
generator

11. Ultrasonicationproducescavitationphenomenawhenacousticpowerinputs
aresufficientlyhightoallowthemultipleproductionofmicrobubblesat
nucleationsitesinthefluid.
Thebubblesgrowduringtherarefyingphaseofthesound
wave,thenarecollapsedduringthecompressionphase.
Oncollapse,aviolentshockwavepassesthroughthemedium.
Thewholeprocessofgasbubblenucleation,growthand
collapseduetotheactionofintensesoundwavesiscalled
cavitation.
Thecollapseofthebubblesconvertssonicenergyinto
mechanicalenergyintheformofshockwavesequivalentto
severalthousandatmospheres(300MPa)pressure.

12. Reasonsforthismaybetheconformationalliabilityofsome(perhapsmost)
enzymestosonicationandthedamagethattheymayrealisethoughoxidation
bythefreeradicals,singletoxygenandhydrogenperoxidethatmaybe
concomitantlyproduced.
Damagebyoxidativefreeradicalscanbeminimizedbyflushingthesolution
withnitrogenand/orincludingscavengerslike
-cysteine,dithiothreitolorother-SHcompoundsinthemedia.
UseofradicalscavengerN
2O,havebeenshowntoreducethisinactivation.
Aswithmostcellbreakagemethods,veryfinecelldebrisparticlesmaybe
producedwhichcanhinderfurtherprocessing.
Sonicationremains,however,apopular,usefulandsimplesmall-scale
methodforcelldisruption

13. Disadvantagesinclude:
Heatgeneratedbytheultrasoundprocessmustbedissipated.
Highnoiselevels(mostsystemsrequirehearingprotectionandsonic
enclosures)
Yieldvariability
Freeradicalsaregeneratedthatcanreactwithothermolecules.
Forbacterialcells,suchasE.Coli,30to60secondsmaybesufficientfor
smallsamples
Foryeastcells,thedurationis2to10min.
Ultrasonicvibrationisfrequentlyusedinconjunctionwithchemicalcell
disruptionmethods–reducedenergyrequiredforcelldisruption

14. Physical methods-Osmotic shock
Cytolysis,orosmoticlysis,occurswhenacellburstsduetoanosmotic
imbalancethathascausedexcesswatertomoveintothecell.
Itoccursinahypotonicenvironment,wherewaterdiffusesintothecelland
causesitsvolumetoincrease.Ifthevolumeofwaterexceedsthecell
membrane'scapacitythenthecellwillburst

15. Cytolysisdoesnotoccurinplantcellsbecauseplantcellshaveastrongcell
wallthatcontainstheosmoticpressure,orturgorpressure,
Contrarytoorganismswithoutacellwall,plantcellsmustbeinahypotonic
environmentinordertohavethisturgorpressure,whichprovidesthecells
morestructuralsupport,preventingtheplantfromwilting.
Inahypertonicenvironment,plasmolysisoccurs,whichisnearlythe
completeoppositeofcytolysis:Insteadofexpanding,thecytoplasmofthe
plantcellretractsfromthecellwall,causingtheplanttowilt
Plasmolysisisthetermwhichdescribesplantcellswhenthecytoplasm
shrinksfromthecellwallinahypertonicenvironment.
Crenationisthecontractionorformationof
abnormalnotchingsaroundtheedgesofacell
afterexposuretoahypertonicsolution,due
tothelossofwaterthroughosmosis

16. Osmoticshockisusuallyusedtolysemammaliancells
Withbacterialorfungalcells,thecellwallneedstobeweakenedbefore
applicationofosmoticshock
Osmoticshockisusedtoremoveperiplasmicproducts(mainlyproteins)
fromcellswithoutcelldisruption
Incaseofrecombinantaswellasnon-recombinantgramnegativebacteria,
proteinssecretedintoperiplasmicspace,suchcellstransferredtohypotonic
buffer,cellimbibewaterthroughosmosisandvolumeconfinedincell
membraneincreasessignificantly
Thecellwall,whichisrigidnotexpandlikecellmembraneandmaterial
presentinperiplasmicspaceisexpelledintoliquidmedium
Cytorrhysisisthecompletecollapseofaplantcell'scellwallwithinplants
duetothelossofwaterthroughosmosis.Thisusuallyfollowsplasmolysis

17. Other Physical methods
Freezeandthaw
Repeatedfreezingandthawingofbacterialcellsdisruptsthembecauseofthe
repeatedformationofsharpicecrystals-manybeginningtogrowinthe
insideofthecells.
“Sortoflikeburstinballoonbyusingneedlesfromtheinside”
e.g.chillinga37°ccultureto4°cbyaddingiceandbackto37°c

18. Mechanical Methods –Homogenization
Bead mill homogenizer
Cascading
beads
Cells being
disrupted
Rolling
beads
Itconsistsoftubular
vesselmadeofmetalorthick
glasswithinwhichacell
suspensionisplacedalong
withsmallmetalorglassbeads(typicalbeadsloadingisabout80-90%)
Thetubularvesselisrotatedaboutitsaxisandasaresultofthisthebeads
startsrollingawayfromthedirectionofvesselrotation
Athigherspeed,somebeadsmoveupalongwiththecurvedwallandthen
cascadebackonthemassofbeadsandcellsbelow
Thedisruptiontakesplaceddueto–thegrindingactionoftherollingbeads
aswellastheimpactresultingfromcascadingbeads
Wearresistantbeads–zirconiumoxide/silicate,titaniumcarbide,glass,
aluminaceramic

19. Disruptionoccursbythecrushingactionofthe
glassbeadsastheycollidewiththecells.
Comparedtohigh-pressuremethodsofcell
disruptionwetbeadmillingislowinshearing
forces.
Membranesandintracellularorganellescanoften
beisolatedintact.
Itisthemethodofchoicefor
disruptionforspores,yeastandfungiandworks
successfullywithtough-to-disruptcellslikecyanobacteria,mycobacteria,
sporesandmicroalgae.
Morerecently,ithasbeenappliedtosoilsamplesandtoplantandanimal
tissue.IfPCRtechniquesaretobeused,thishomogenizationmethodisone
ofthefewthattotallyavoidspossiblecross-contaminationbetweensamples
becausebothvialsandbeadsaredisposable.

20. Highlights:
•Beadmillingcangenerateenormousamountsofheat
•Cryogenicbeadmilling:Liquidnitrogenorglycolcooledunit
•Application:Yeast,animalandplanttissue
•Smallscale:Fewkilogramsofyeastcellsperhour
•Largescale:Hundredsofkilogramsperhour.
Withbeadmill–celldisruptioninvolvessizereduction–similarlylike
grinding

21. •First Order
•k is a function of
–Rate of agitation (1500-2250 rpm)
–Cell concentration (30-60% wet solids)
–Bead diameter (0.2 -1.0 mm)
–Temperature)exp1(
)(kt
mrRR

 )exp1(
)(max kt
rrCC


or
Where,
R
r–concentration of released product (Kg/m
3
)
R
m–maximum concentration of released product (Kg/m
3
)
t –time (s)
K –time constant (s)
ThevalueofR
mandkcanbedeterminedexperimentally
ThevalueofK,stronglydependson-typeofimpeller,beadsize,beadload,
speed,andtemperature

22. Forcontinuousmode,meanresidencetime,andtheresidencetime
distributionornumberofcontinuousCSTRinseriesshouldbetakeninto
accounttopredictrelease
Optimalconditionsforreleaseofintracellularproductare
a)Themicro-organismused
-Cellwallthicknessandcomposition
-Size
b)Locationoftheproduct
-incytoplasm
-incellorganelles
-inperiplasmicspace
c)Typeofbeadmill
-beaddiameterandtypeofimpeller
-beadloading
-tipspeedofimpeller
d)Residencetimeofcellsuspension
e)Cellconcentration
f)Temperature(rise)
Bead mill performance

23. Bead mill performance

24. Bead mill performance

25. Scale up Bead mill
Removalofenergydissipatedinthebroth–probleminscaleup
Apowerinputisdissipatedinheatandisneedstoberemovedbycylinder
wall.Sotheratioofheattransferareatothebeadmillvolumeis:T
LT
TL
VvolumeMill
Aareasurface
L
4
4
)(,
)(,
2



Where
T –Cylinder diameter (m) and L –length of bead mill (m)
On scaling up, the cylinder diameter will increase and the ratio 4/T will
decrease53
,, DcpNpINPUTPOWER 
Where
C –dimensionless constant
ρ–suspension density (Kg/m
3
)
N –rotational speed of impeller (S
-1
)
D –impeller diameter (m)
C depends on type of flow (turbulent or laminar) and type of impeller

26. Homogenizationisthewidelyusedmethodforlargescaleoperationsaswell
aslabscale.
ThismethodemploysequipmentcalledHomogenizerorCelldisruptor
adaptedfromdairyindustrywhichoperatesatextremelyhighpressures
(upto400-2500bars).
Celldisruptorsandhomogenizersarebothpositivedisplacementpumps
eachdiffersinthewaythattheycreatepressureonthesampleandtransfer
itfrompressurizedchambertoanotherchamberwhichisatlowerpressure.
Homogenizerspressurizethesampleinachamberwhichisthenreleased
intoachamberoflowerpressurethroughahomogenizingvalve.
Celldisruptorsuseahydraulicforcetoacceleratethesampletohigh
pressureandforcingthemthroughaminuteorificetohitonadisruption
headwhichisatalowerpressure.
Homogenization

27. Pressurecanbecontrolledbyadjustingtheforceimpartedonthevalve,
whichiscontrolledeitherpneumaticallyorhydraulically.
Asthecellsuspensionispumpedthroughaminuteorificeathighpressure
itcausesashearonthecellmembranes.Thisisfollowedbythesudden
releaseofthesuspensionwithinstantexpansion.
Disruptionofthecellisaccomplishedatthreestagescausingtheexplosion
therebyreleasingitscontents.
1. Impingement on the homogenizing valve
2. High turbulence and shear combined with compression produced in the
minute gap
3. Sudden pressure drop upon release

28. Themaindisruptivefactorinthisprocessisthepressureappliedonthe
sampleandconsequentpressuredropacrossthevalve.
Thiscausestheimpactandshearstressonthecellsmakingthemtobreak
whichareproportionaltotheoperatingpressure.
Theoperatingparameterswhichaffectthe
cellbreakingefficiencyofhigh-pressure
homogenizersareasfollows:
-Operating Pressure
-Process Temperature
-Number of passes
-Valve/Orifice design
-Flow rate of the sample

30. There are certain variables to be considered while designing a
homogenizer/cell disruptor. They are:
-type of homogenizing
valve/orifice
-operating pressure
-stages of disruption
-viscosity of the sample
-temperature
-type of the surfactant
Inthecommonly-usedoperatingrangewithpressuresbelowabout75MPa,
thereleaseconstant(k)hasbeenfoundtobeproportionaltothepressure
raisedtoanexponentdependentontheorganismanditsgrowthhistory
-(e.g.k=k'P
2.9
inSaccharomycescerevesiaeandk=k'P
2.2
in
Escherichiacoli,wherePrepresentstheoperatingpressureandk'
isarateconstant).
Thehighertheoperatingpressure,themoreefficientisthedisruption
process

31. Theproteinreleaserateconstant(k)istemperaturedependent,disruption
beingmorerapidathighertemperatures.
Inpractice,thisadvantagecannotbeusedsincethetemperaturerisedueto
adiabaticcompressionisverysignificantsosamplesmustbepre-cooled
andcooledagainbetweenmultiplepasses.
Atanoperatingpressureof50MPa,thetemperatureriseeachpassisabout
12deg.C.
Inadditiontothefragilityofthecells,enzymes/proteinsarereleasedat
variousratesdependingontheircellularlocation.
Proteinslocatedintheperiplasmarereleasedfasterwhereastheproteins
locatedwithinthecellularcomponentsarereleasedataslowerrate.
Unboundintracellularproteinsmaybereleasedinasinglepasswhereas
membraneboundenzymesorproteinsmayrequireseveralpassesfor
reasonableyieldstobeobtained

32. Multiplepassesareundesirablebecause,ofcourse,theydecreasethe
throughputproductivityrateandbecausethefurtherpassageofalready
brokencellsresultsinfinedebriswhichisexcessivelydifficulttoremove
furtherdownstream.
Consequently,homogeniserswillbeusedatthehighestpressures
compatiblewiththereliabilityandsafetyoftheequipmentandthe
temperaturestabilityoftheenzyme(s)released.
Highpressurehomogenisersareacceptablygoodforthedisruptionof
unicellularorganismsprovidedthe
-enzymesneededarenotheatlabileand
-theshearforcesproducedarenotcapableofdamagingenzymes
freeinsolution.
Thevalveunitispronetoerosionandmustbeprecisionmadeandwell
maintained.

33. ROTOR-STATORHOMOGENIZERS (alsocalledcolloidmillsorWillems
homogenizers)
Cell
suspension
Rotor
Stator
Disrupted
cells
Thesearewellsuitedforplantandanimaltissue
andoutperformcutting-bladetypeBenders.
Comparedtoablender,foaming,swirling
andaerationareminimizedand
smallersamplevolumesareaccommodated.
Mechanismofcelldisruption:
-Highshearandturbulence
Thecellularmaterialisdrawnintotheapparatus
byarotorsitedwithinastatictubeorstator.
Thematerialisthencentrifugallythrownoutwardtoexitthroughslotsor
holesonthetipofthestator.
Becausetherotoristurningatveryhighspeed,thetissueisrapidlyreduced
insizebyacombinationofturbulenceandscissor-likemechanicalshearing
occurringwithinthegapoftherotorandstator.
Theprocessisquitefastand,dependinguponthetoughnessofthetissue
sample,desiredresultsareusuallybeobtainedin10-60seconds.

34. Fortherecoveryofintracellularorganellesorreceptorsitecomplexes,
shortertimesand/orreducedrotorspeedsareused.
Samplesoftenmustbepre-choppedor-fragmentedwithascissors,single-
edgerazorbladeorcryopulverizer(adevicethatquicklypowderstissueat
liquidnitrogentemperatures).
Unlikemanyothertypesofcelldisrupters,rotor-statorshomogenizers
generatenegligibleheatduringoperation.
Mostlaboratoryrotor-statorhomogenizersaretopdrivenwithacompact,
highspeedelectricmotorwhichturnsat8,000to60,000rpmandfunction
properlywithviscosityrangeof<10,000cps
thesizeoftherotor-statorprobecanvaryfromthediameterfor0.5-50mL
samplevolumestomuchlargerunitshandling10litersormore.
Foaming and aerosols are the problems with rotor-stator homogenizers
Bottom-driven laboratory rotor-stator homogenizers
SINGLE OR MULTIPASS OPERATION

35. 







t
C
C
exp1
max Single pass
Multi passN
t
C
C
















exp1
max
Where,
N–isthenumberofpasses
ɵ-timeconstsant

36. FRENCHPRESS
Plunger
CylinderCell
suspension
Impact
plate
Jet
Orifice
•Application: Small-scale recovery of intracellular proteins
and DNA from bacterial and plant cells
•Primary mechanism: High shear rates within the orifice
•Secondary mechanism: Impingement
•Operating pressure: 10,000 to 50,000 psig

37. FREEZE-FRACTURING
Bothmicrobialpastesandplantandanimaltissuecanbefrozeninliquid
nitrogenandthengroundwithamortarandpestleatlowtemperature.
Presumablythehardfrozencellsarefracturedunderthemortarbecauseof
theirbrittlenature.Also,icecrystalsattheselowtemperaturesmayactas
anabrasive
Afreeze-fracturingdevicecalledtheBessmantissuepulverizerisusefulfor
fragmenting10mgto10gquantitiesoffibroustissuesuchasskinor
cartilagetothesizeofgrainsofsalt.(materialistheneasilyhomogenizedby
othermethods)
Lookingsomewhatlikeatabletpress,thepulverizerconsistsofahole
machinedintoastainlesssteelbaseintowhichfitsapiston.Thebaseand
pistonarepre-cooledtoliquidnitrogentemperatures.
Tenmgtotengramsofhardfrozenanimalorplanttissueisplacedinthe
hole.Thepistonisplacedintheholeandgivenasharpblowwitha
hammer.
Theresultingfrozen,powder-likematerialcanbefurtherprocessedbyPestle
andTube,BeadMillorRotor-statorhomogenizers

38. Disadvantages include:
Not well suited for larger volume processing.
Awkward to manipulate and clean due to the weight of the
assembly (about 30 lbs/14 Kg).

39. Fixed-Geometry Fluid Processors
ThepatentedtechnologyofMicrofluidics'fixed-geometryfluidprocessors
aremarketedunderthenameofMicrofluidizer®processors.
Theprocessorsdisruptcellsbyforcingthemediawiththecellsathigh
pressure(typically20,000-30,000psi)throughaproprietaryinteraction
chambercontaininganarrowchannelthatgeneratesthehighestshearrates
Theultra-highshearratesallowfor:
Processing of more difficult samples
Fewer repeat passes to ensure optimum sample processing
Microfluidizer®systemsprovideahighlyreproducible,convenient,and
efficientmethodforcelllysis.
Thesystemspermitcontrolledcellbreakagewithouttheneedtoadd
detergentortoaltertheionicstrengthofthemedia.

40. The fixed geometry of the interaction chamber ensures:
-Day-to-day reproducibility
-Machine-to-machine reproducibility
-Direct scalability from laboratory scale (20 ml to several liters) to
production scale (10s of liters per minute)
Disadvantagesincluded:
Inmanycircumstances,especiallywhensamplesareprocessedmultiple
times,theMicrofluidizer®processorsdorequiresamplecooling

41. Optimalconditionsforreleaseofintracellularproductdependson
a)Themicro-organismused
Cellwallthicknessandcomposition
-Size
-b)Locationoftheproduct
-incytoplasm
-incellorganelles
-inperiplasmicspace
c)Typeofhomogenizer
-pressure
-typeofvalveandseat
-temperature(rise)
-numberofpassages(N)
d)Residencetimeofcellsuspension
e)Cellconcentration
Homogenizer

42. Homogenizer –kinetics of release NpfK
CC
C
h
rr
r
)(ln
max
max










Where,
f–isfunctionofpressuredifference,(Δp)
N–numberofpassages)exp1(
))((max Npfk
rr
h
CC


Formanycasesf(Δp)=Δp
β
Theexponentisoforder1.5to3.Forbakersyeastexponentis2.9,
therefore)exp1(
)(max
9.2
Npk
rr
h
CC



43. Homogenizer –kinetics of release
Scaleupofhomogenizerinvolvesinstallingbiggerplungerpumpand
dischargevalve
Thepowerinputisproportionaltohomogenizationpressure,Δpandthe
volumetricflowprocessed 
pvv
cpp
Where,
ϕ
v–volumetricflowrate(m
3
/s)
ρ–density(Kg/m
3
)
Cp–specificheat(JKg
-1
K
-1
)
Δɵ-Temperaturedifference(K)

44. SonicAgitationLiquid
Pressing
Freeze pressing
Animal cells 7 7 7 7
Gram –ve bacilli &
cocci
6 5 6 6
Gram +ve bacilli 5 4 5 4
Yeast 3.5 3 4 2.5
Gram +ve cocci 3.5 2 3 2.5
Spores 2 1 2 1
Mycelia 1 6 1 5
Sensitivity of cells to disruption

45. Heat
All mechanical methods require a large input of energy, generating
heat. Cooling is essential for most enzymes. The presence of
substrates, substrate analogues or polyolsmay also help stabilise the
enzyme.
Shear
Shear forces are needed to disrupt cells and may damage enzymes,
particularly in the presence of heavy metal ions and/or an air
interface.]
Hazards likely to damage enzymes during cell disruption
Proteases
Disruption of cells will inevitably release degradativeenzymes which
may cause serious loss of enzyme activity. Such action may be
minimised by increased speed of processing with as much cooling as
possible. This may be improved by the presence of an excess of
alternative substrates (e.g. inexpensive protein) or inhibitors in the
extraction medium.