BIOMAN2011 Downstream Fino

8/9/2019 BIOMAN2011 Downstream Fino

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BIOMAN 2011CHO-tPA Production System

Downstream Processing

Mie !ino

MiraCosta Co""ege


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Unit Operations

Many decisions to #e made ateac$ ste% in t$e %rocess

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Downstream &'am%"e

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Har)est Se%aration*C"ari+cation,

• .$ere are two tec$no"ogies /or remo)ingt$e ce"" mass /rom t$e so"ution containingt$e target %rotein %rior to "oading ontoco"umns – Centri/ugation *eg dis stac, – !i"tration

• Dead-ended +"tration *aa norma" ow mem#rane3 de%t$,

• Crossow mem#rane +"tration *aa tangentia"ow,• Crossow mem#ranes are %re/erred /or

"arge sca"e o%erations and $a)e manyad)antages


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Media and Cells In, ClarifedMedia Out

SLUDGE

CLARIFIEDBROTH

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NORMAL FLOW FILTRATION (NFF):

Traps contaminants larger than the pore size on the top surface of the membrane.

Contaminants smaller than the specified pore size pass through the membrane.

Used for critical applications such as sterilizing and final filtration.

5

MEMBRAE

DE!TH


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Steri"i6ing !i"tersIndustry78egu"atory standard

• Ca%a#"e o/ ac$ie)ing an 98: ;< /or a Bdiminuta c$a""enge using AS.Mmet$odo"ogy *%er !DA =uide"ines,

– ; < 98: means >1 micro#e 7 10


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Tan"ential Flo#Filtration

Clarif$ation%!urif$ation Con$entration Bu&er E'$(an"e

Uses Crossflow to reduce build upof retained components on the

membrane surface

Allows filtration of high foulingstreams or high resolution


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Dierent Si6e Pores in .!!

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)(at is Me*+raneInte"rit-

Integral Membrane Non-Integral Membrane

Contaminants

larger than

pores upstream

Downstream

contamination

No downstream

contamination

Contaminants

larger than expected

pores upstream

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!rin$iples o. Inte"ritTestin"

A benefit of membrane filters is the ability to

perform a non-destructie integrity test!

"esting ensures filtration #$#"%M integrity before&

during& or after filtration!

Membrane prefilters and depth filters cannot beintegrity tested with precision or accuracy because

of wide pore distribution!

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Reasons to Inte"rit Test

Confirms manufacturers specifications Assures integrity after steaming or autoclaing Assures integrity before sterili'ation

Detects system lea(s due to o-rings& gas(ets&faulty seals

Assures the correct pore si'e filter )art of corporate standard operating procedure

*M) re+uirement Audit re+uirement

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T#o Basi$ Tpes o. Inte"rit Test

Destructie )roided as a manufacturers

assurance of microbial retention! ,acterial Challenge

Non-Destructie )roided to allow in-situ testing

)ressure hold ,ubble )oint Diffusion

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Basi$ Ele*ents o. aBa$terial Retention Test

Saline lactose

media w/

B. Diminuta

"est ilter .!// or .!0 µm disc or

filter cartridge

Assay ilter 123mm M%C

disc4

23mm disc

on "#A

M&C mi'ed esters o/ ce""u"ose

.SA try%tic soy agar

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Non-Destructive Integrity Test

Bubble Point

ully wetted membrane filters

hold li+uid in their pores by

surface tension and capillary

forces!

,ubble point pressure is

inersely related to largest

pore diameter

O%en%ores%ace

ater$e"dwit$sur/ac

etension

View of the membrane cross-section

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)(at is !ressure Hold%Bu++le!oint-

5ater 5et

Integral Membrane

5ater 5et

Non-Integral Membrane

Air pressure

upstream

greater than

specification

*as will flow through

large opening and is

easily obsered downstream

5ater in pores is a

barrier to gas flow6

No gas flow obsered

downstream until

upstream pressure

exceeds critical alue

Air pressure

upstream

less than

specification

psi psi

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In)erse 8e"ations$i%Pore si6e ) Bu##"e Point

• Asteri"i6ing

+"ter $asa "ogreduction)a"ue o/

greatert$an < Decreasing%ore si6e

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.!! System

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!eed !"owIn"et Pressure

Permeat

e !"ow

8etentate!"owOut"et

Pressure

Ho""ow !i#er


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2(

PERISTATI! P"#P$ Creates a gentle s+uee'ingaction to moe fluid through

flexible tubing!


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Introduction .!! 9ayout EO%eration

2

• O%erating Ste%s – !"us$ – C"ean ater

!"u'

– Pum% cur)e – Integrity .est – Buer !"us$ – Micro+"ter

– Or Concentrate – Or Dia+"ter

%ermeate

&eed%um%

&ilter

&eed

retentate

dia&iltrate

%roductrecovery

initial

feed

reservoir


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Fey Parameters

• !eed !"ow rate – !"ow rate "ea)ing t$e %um% – Set #y %um% s%eed

• .ransmem#rane %ressure *.MP, – A)erage o/ in"et7out"et %ressures – Set #y #ac%ressure *retentate,

• Permeate contro" – !"ow rate t$roug$ t$e +#ers – Set #y #ac%ressure *%ermeate, – e don?t use t$is contro" in t$is c""ass

• Mem#rane area – Sca"es "inear"y

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.ransmem#rane Pressure*.MP,

25

)in 7 8.psi )out 7 /.psi

Inlet eed )ressure 9etentate )ressure

)ermeate)ressure

.MP *Pin 3 Pout,72 -P%erm

.MP *(0 3 20,72 - 0 24 PSI

)perm 7 .psi

We leave this

line

unrestricted ilter membrane


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System O%eration

Ste%s

• C"ean water u'• Pum% Cur)e• Integrity .est

• !i"tration

2<

Diafiltration Buffer

Initial Feed

Retentate

Permeate

Feed

Tank

PumpMemrane

Flu!"


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O%eration Micro+"tration

2@

.ras$

Co""ect

and Fee%


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2

.ras$

Co""ect

and Fee%


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(0

.ras$

Co""ect

and Fee%


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(1

.ras$

Co""ect

and Fee%


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(2

.ras$

Co""ect

and Fee%


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((

.ras$

Co""ect

and Fee%


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O%eration Concentration

• Dewater t$eretained so"utes

• Procedures – !i"" tan wit$

%rocess uid – Start %um% and

adGust system torecommended

ows7%ressures – 8emo)e %ermeate


Initial Feed

Retentate

ermeate

Feed

Tan!

ump"embrane

Flush

(


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(4


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(5


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(<


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(@


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0


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2


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O%eration Dia+"tration

• as$ out%ermea#"eso"utes- %roductor contaminants

• Procedure – Add

dia+"tration#uer to t$e/eed tan att$e same ratet$at %ermeateis #eingremo)ed /romt$e system


Initial Feed

Retentate

ermeate

Feed

Tan!

ump"embrane

Flush

(


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d i /


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Bacground :irus Sa/ety&ecti)e C"earance Ste%s

• :irus !i"tration – 9arge *en)e"o%ed, E sma"" *non-en)e"o%ed,

)iruses – Sma""est %ar)o)irus is a#out 40J #igger t$an

an anti#ody

• Inacti)ation – 9ow %H or So")ent detergent *en)e"o%ed,

• C$romatogra%$y – Protein A AKnity /or MA#s *en)e"o%ed E non-

en)e"o%ed, – Anion &'c$ange !"ow t$roug$ /or MA#s

*en)e"o%ed E non-en)e"o%ed, 42


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.y%es o/ C$romatogra%$y

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Co"umn C$romatogra%$y

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Common"y em%"oyed downstream %rocessing met$ods

Processing#et'od Attributes Bene&its imitations

!lari&ication6

#edimentation based

clarification

Continuous centrifugation Capable of handling ery large

harest olumes

:pen process- contamination and

safety issues

Normal flow iltration Microporous ;olume and throughput limited

Charged filter media

Cellulose pads

"angential f low f il tration Contained systems Capable of handl ing large harest

olumes

!a%ture$

Chromatography )rotein A Affinity


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.y%ica" contaminant c"earance )a"ues /rom eac$c$romatogra%$y stage

!ontaminantA&&inityload

Intermediate%uri&icationload

Polis'ing load

ml4 0.? 0.8 0.

%ndotoxin 1%@>ml4 0. 0. B0

DNA 1pg>ml4 0. 0.8 0./

4<

C tit t d t$ d / "


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Common %rocess constituents and met$ods o/ remo)a" or%uri+cation

!om%onent

!ulture 'arvest

level

(inal %roduct

level

!onventional

met'od

"herapeutic Antibody .!0-0!? g>l 0-0. g>l @>Cromatography

Isoforms ;arious Monomer Chromatography

#erum and host proteins .!0-8!. g>l B .!0-0. mg>l Chromatography

Cell debris and colloids 0.

>ml None M

,acterial pathogens ;arious B0.->dose M

;irus pathogens ;arious B0.->dose 10/=9;4

irus filtration

DNA 0 mg>l 0. ng>dose Chromatography

%ndotoxins ;arious B.!/? %@>ml Chromatography

=ipids& surfactants .-0 g>l B.!0-0. mg>l Chromatography

,uffer *rowth media #tability media @

%xtractables>leachables ;arious B.!0-0. mg>l @>Chromatography

)urification reagents ;arious B.!0-0.mg>l @

4@


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Downstream Design

? yield>step

. yield>step

E? yield>step

4

Ion &'c$ange


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Ion &'c$angeC$romatogra%$y

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• I/ t$e c$arge on t$e #ead is %ositi)eLit wi"" #ind negati)e"y c$arged

mo"ecu"es – .$is tec$niue is ca""ed anion e'c$ange

• I/ t$e #eads are negati)e"y c$argedLt$ey #ind %ositi)e"y c$argedmo"ecu"es – .$is tec$niue is ca""ed cation

e'c$an e


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Ion &'c$angers

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• Ion e'c$ange c$romatogra%$y is#ased on adsor%tion and re)ersi#"e#inding o/ c$arged sam%"e mo"ecu"es

to o%%osite"y c$arged grou%s attac$edto an inso"u#"e resin• .$e %H )a"ue at w$ic$ a #iomo"ecu"e

carries no net c$arge is ca""ed t$eisoelectric point *%I,


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I& *cont?d,

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• $en e'%osed to a %H #e"ow its %IL t$e#iomo"ecu"e wi"" carry a %ositi)e c$arge andwi"" #ind to a cation e'c$anger

• At a %H a#o)e its %IL t$e %rotein wi"" carry anegati)e c$arge and wi"" to #ind to an anione'c$anger

• De%ending on w$at %H t$e #iomo"ecu"e is

more sta#"e at wi"" decide w$et$er an anionor cation e'c$anger is used


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• .$e main %roteins in t$e media usedto grow tPA are tPAL Bo)ine seruma"#umin *BSA,L insu"inL and trans/errin

&ac$ %rotein $as a s%eci+c isoe"ectric%oint ca""ed t$e %I – BSA $as a %I o/

– tPA is


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• e are a#"e to se"ecti)e"y #ind t$e tPA to t$e resin#y contro""ing t$e %H and ionic strengt$ o/ t$eeui"i#ration #uer *aa Buer A,

• At a %H o/ 50L tPA is more cationic *%ositi)e"yc$arged, t$an eit$er BSA or .rans/errin

• .$ere/oreL t$e more %ositi)e c$arged tPA wi"" #indto t$e resin and t$e ot$ers wi"" ow t$roug$ t$eco"umn and out to waste

• tPA is t$en remo)ed /rom t$e co"umn using a $ig$

concentration o/ sa"tL w$ic$ com%etiti)e"y #um%st$e %rotein o t$e resin as t$e sodium ions #ind


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Ste%s in C$romatogra%$y

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• Prime and de-#u##"e t$e system• Condition t$e co"umn resin wit$ a so"ution t$at promotes t$e #inding

o/ your %rotein – Ca""ed Equilibration

• Pum% your sam%"e so"ution o)er t$e co"umn resinL w$ic$ s$ou"d #indas muc$ o/ your %rotein as %ossi#"e – Ca""ed Applying Sample

• &)eryt$ing t$at doesn?t #ind goes to t$e drain• At t$is %ointL your %rotein wi"" stay #ound to t$e resin inde+nite"y• Now %um% a so"ution o)er t$e resin t$at competes /or #inding on t$e

resin wit$ t$e %roteins /rom your so"ution – Ca""ed Elution

• At some %ointL t$e com%eting so"ution wi"" #eat out t$e )arious

%roteins /or %osition on t$e resin and t$ey wi"" "et go o/ t$e resin• ou wi"" co""ect /ractions a"ong t$e way t$at can #e /ro6en andana"y6ed "ater


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5@


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5

QtaPrime 9iuid C$romotogra%$y System


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QtaPrime 9iuid C$romotogra%$y System


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QtaPrime !"ow Pat$

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BIOMAN2011 Downstream Fino