Mixing, mass transfer and bioprocess scalingup and down in new generation single-useg g

bioreactors

Nico Oosterhuis CELLution Biotech

Questions of today

Mixing and mass transfer in single use bioreactor: g gcan we design a multipurpose single-usebioreactor?

Can it be scaleable?

Why Single use?

STERILITY Reduced cross contamination Pre-sterilized (validated) Easy sterile connections

COSTS No cleaning Simple validation Simple infrastructure Lower maintenance Flexibility turn-aroundy

Reasons to apply single-use

What is the pharma-industry telling us today?

Source: Paul Cook, 2010

Many types of single use bioreactors(Eibl, 2008)

Applicability todays single-use bioreactors?

Rocking systems Stirred systems

Mass transfer, klamax = 40 50 hr-1Restricted heat transfer

Scaleability present (rocking) systems questionableScaleability present (rocking) systems questionable

Only applicable for mammalian cell cultureOnly applicable for mammalian cell culture

Single-use bioreactors applicability?

WORKINGkla

REACTOR TYPEWORKING VOLUME TYPE OF BAG TYPE OF MIXING SUPPLIER (hr-1)

Wave Bioreactor 1 200 L pillow shape rocking GE Healthcare < 30Wave Bioreactor 1 200 L pillow shape rocking GE Healthcare < 30Cultibag RM 1 100 L pillow shape rocking Sartorius Stedim Biotech < 30AppliFlex 1 25 L pillow shape rocking Applikon Biotechnology < 40C ltibag STR200 50 200 L t kli ti d S t i St di Bi t h < 40Cultibag STR200 50 200 L tankliner stirred Sartorius Stedim Biotech < 40

SUB 50 1000 L tankliner stirredThermo-Fischer (Hyclone) < 40

XDR 40 2000 L t kli ti d XC ll < 20XDR 40 2000 L tankliner stirred XCellerex < 20Nucleo 50 100 L square 3D Paddle agitation ATMI / Pierre Guerin < 20Shaking bioreactor < 200 L tankliner orbital shaker Khner/ExcellGene < 40CellMaker Regular 1 50 L bubble column rotating sparger Cellexus < 10

=> All single-use systems limited applicable=> All single-use systems limited applicable

Mammalian vs microbial in biopharma?MAb Therapeutic proteinsEnzymes InsulinsHormones/GF Protein vaccinesToxin-conjugates PEG-Conjugates

300

350 Scaffolds MAb Mimetics

41

1510

49

295200

250

71192

46

100

150

90

034126210617

0

50

Microbial MammalianMicrobial Mammalian

44% 56%Phase III projects in 2009 (source: Boehringer 2011)( g )

Single use bioreactors today

Rocking type: mainly applied on small scale (< 150Ltr)g yp y pp ( ) Stirred type: up to 2000 Ltr All: All:

Mixing, mass- and heat transfer restrictedO l li d f li Only applied for mammalian processes

Need for high-performance and microbial processes

Regime analysis (Kossen & Oosterhuis, 1985)

R i l i i f t i l ti t tRegime analysis: comparison of typical time constants

0,0001 0,01 1 100 10000(SEC)

(MIN) (HR)MASSACTION

ENZYMEINDUCTION ORGANISMSSELECTION

ALLOSTERICINTERACTION

MIXING REACTOR/ENVIRONMENTMASSTRANSFER

REACTORDYNAMICS

Regime analysis microbial

STR< 10L

STR> 100L

Wave CTr10L

CTr100L< 10L > 100L 10L 100L

TRANSPORT

Oxygen transfer < 10 (s) < 15 (s) > 90 (s) 10 15 (s) 12 20 (s)( ) ( ) ( ) ( ) ( )Mixing < 5 (s) 10 40 (s) > 30 (s) < 10 (s) 40 100 (s)Heat transfer 100 200 (s) 300 600 (s) n.a. 30 60 (s) ?CONVERSION

O2 consumption 6 15 (s)Heat production 300 400 ( )Heat production 300 400 (s)Substrate conv. > 1000 (s)Growth > 2000 (s) 2000 (s)

Regime analysis cell-culture

STR< 10L

STR> 100L

Wave CTr10L

CTr100L< 10L > 100L 10L 100L

TRANSPORT

Oxygen transfer 100 200 (s) > 200 (s) > 150 (s) 30 50 (s) 40 60 (s)( ) ( ) ( ) ( ) ( )CO2 transfer > 500 (s) > 500 (s) > 250 (s) 40 75 (s) 60 90 (s)Mixing 50 200 (s) 200 300 (s) > 60 (s) 50 - 120 (s) 80 - 120 (s)Heat transfer 100 200 (s) 300 600 (s) n.a. 30 60 (s) ?CONVERSION

O2 consumption > 200 ( )O2 consumption > 200 (s)CO2 production < 200 (s)Heat production > 800 (s)p 800 (s)Substrate conv. hoursGrowth days

Required mass- and heat transfer

Cell cultureOUR: 0 2 2 mmol/106 c/hr

Microbial culture (E.coli)OUR: 4 mmol O /g hrOUR: 0,2 2 mmol/106 c/hr

When OUR = OTR 20 x 106 c/ml => 4 mmol/l.hr

1

OUR: 4 mmol O2/g.hrWhen OUR = OTR

50 g/L => 200 mmol/l.hr1 4 mmol / l.hr => kla 20 hr-1 (air) 200 mmol/l.hr => kla 1000 hr-1 (air)

200 mmol/l.hr => kla 400 hr-1 (50% O2)

Cooling: 469 KJ/mol O2 consumed At 10L => 8 W cooling power needed

Cooling : 469 KJ/mol O2 consumed At 10L => 260 W cooling power needed

MASS TRANSFER might be an ISSUE

g p

MASS and HEAT TRANSFER is an ISSUE

2-Dimensional movementin rocking motiong

Increase of mass transfer due to additional translational movementdue to additional translational movement

Stirring versus 2-D shaking ?

The microenvironment of the cell determines its physiological behavior.

Conditions have to be compared: local concentrations of O and CO nutrients local concentrations of O2 and CO2 nutrients

energy dissipation scale (shear)ti t t time constants

gradients

Mixing and mass transfer

Shape/geometry of the bioreactor or power / volume?

Stirring versus 2-D shaking

GAS phase

O2Mass

CO2

phase

LIQUID phase

O2 CO2

transfer GRADIENTS

Nutrients

CELL pH Temperaturemixing

Side-productsProd ct

Membrane transportSHEAR FORCES

Product

Design using an engineering view

MASS-transfer & mixingR l SURF ti (t di i l ki ) i i i d Real SURF motion (two dimensional rocking) improves mixing and oxygen transfer

Proper and accurate temperature control Heating by convection (incubator) Direct cooling (heat exchanger under the bag) Short mixing times => no gradientsShort mixing times > no gradientsEASY handling & real single use Bags flexible in volume

R b t b i t ( t k bl ) Robust one box equipment (stackable) In-line measurements non-invasive, fully disposable (pH, DO,), bottom

mounted, others (glucose/lactate)Scaleable Same process performance at different scales

RESULTS

Mass transferMass transfer E.coli

H t t hi l Heterotrophic algae Pre-culture Corynebacterium (lysine) Mammalian cells (PER.C6 - CHO) ScaleabilityScaleability

Mass-transfer CELL-tainer

Mass transfer results (dynamic, water, 20 oC)

800

At higher rocking rate:decrease of kla due to inertia of the liquid

600

700

CT'r (10L)

CTr (15L)

inertia of the liquid

400

500

kla

(hr-

1)

CTr (15L)

Wave (20 - 10L)

Range microbial

100

200

300 Range microbial(35 55 rpm; kla >300 hr-1)

020 25 30 35 40 45 50

rocking speed (rpm) / at maximum circle (angle)rocking speed (rpm) / at maximum circle (angle)

Range cell culture operation(15 25 rpm; kla 50 - 100 hr-1)

Comparison to STR

0,25

0,2

0,15

(s-1

)

0,1Kla

CELL-tainerSTR

0,05

00 1 2 3 4

P/V (W/Ltr)

Mass transfer volume rocking speed

Interdependence kla volume rocking speed

Mass transfer - rocking speed

Linear evolution kla rocking speed

Comparison mass transfer

CELL-culture shake flask STR(SUB)

wave CELL-tainer

P/V (W/L) 0 02 0 1 0 1 0 5 0 1 1 0P/V (W/L) - 0,02 0,1 0,1 0,5 0,1 1,0

Gasflow (vvh) - < 1 0,5 - 1 < 1kla (hr-1) < 50 2 - 20 < 30 50 - 150kla (hr ) 50 2 20 30 50 150

Microbial culture shake STR wave CELL-flask (standard) (sparger) tainer

P/V (W/L) - 2 - 6 < 0,5 2 - 3G fl ( ) 1 0 1 0 5Gasflow (vvm) - 1 < 0,1 0,5kla (hr-1) < 100 > 400 40 100 > 300

Mixing times

200CT10 5L

CELL-tainer 15L

100

150

time

(s) CT10-5L

CT10-15LW10-2L

wave 2L

50

100

mix

ing

010 15

20

CELL-tainer 5L

2025

rocking speed (rpm) Angle: 10 degrees

Batch and Fed-batch E.coli

Operational stability

Growth of heterotrophicpalgea (TU Berlin)

Runs > 1000 h Stable pH measurementp No issues with bags Control of DO by shakingy g

speed

Comparison100L shaker

CELL-tainer Shaker

Inoculation of a (large-scale) bioreactor?

Step1 Step 2 n Final processStep1Step 2-n.Final process

Many steps involved

Corynebacterium inoculation

Experiment 1

Copy of shakeCopy of shake-flask conditions

Experiment 2Experiment 2

Controlled startControlled start

Result: simplification seed train

Reduction of steps

Also suitable for cell-culture? YES!

CELL tainer(Mab g/L)

CELL-tainer

wavewave

(days)

Productivity with PER.C6 fed-batch

CHO subclone 2 Bioceros

8040006Viable cell density x 106 Titer (g/L) Q values (pcd)

60

70

3000

35005

40

50

2000

2500

3

4

30

40

1500

2000

2

3

10

20

500

1000

1

0

10

0 2 4 6 8 10 12 14 160

500

0 2 4 6 8 10 12 14 160

0 2 4 6 8 10 12 14 16

Culture Time (days)