© 2016 Centre for Process Innovation Limited. All Rights Reserved.

Scale up/scale down strategies and

devices16/11/2016

Valentina Mangiafridda MEngGas Lab and Fermentation Manager

CPI mission

We use applied knowledge in science and engineering combined with

state of the art facilities to enable our clients to develop, prove,

prototype and scale-up the next generation of products and

processes.

Industrial Biotechnology and Biorefining

How is it possible to give solid and reliable

answers to scale up challenges at early

research stage?

The instrument we need should help us

• ... to forecast process results in Demo scale

• ... to mimic and predict the client manufacturing scale processes

• ... to easily increase the consistency in process performance through process characterisation

• ... to evaluate the scalability of our process in development

Challenges covered with Scale Down Model (SDM) Approach

750L

5/10/20L 1L1mL

Scale Down Model Design

• Bioprocesses performances are multiphase systems: gas-liquid, or three-phase;

• Mass transfer between the phases controls process kinetics.

Scale Down Model Design

• The approach is based on the mass transfer uniformity among the scales

• Its aim is to have the same kLa during the process to transfer

• This must accurately reproduce the event of a process (pH drifting, media sterilisation

peculiarities, parameters control inefficiencies, etc..)

Scale Down Model Design

The data collected and analyses are:

�Bioreactor design & Mass Transfer performances

�Process parameters (setpoint & controls)pO2, Air Flow, stirrer speed, pH, Head pressure, Temperature, ...

�Fermentation batchDynamic behaviour of the process

�Growth curves

�OutputProduct, Metabolites, Impurities (with a Kinetic study during the process)

INP

UT

OU

TP

UT

Bibl. Villadsen at al., Bioreaction Engineering Principles, 2011

Scale Down Model Design

Impeller performances with gas dispersion retrofit comparison

Bakker A. 2006 Fermenter Specific Modeling Issues www.bakker.org

SDM step-by-step

Collect the Bioreactor design and

KLa data

Analysis on the

geometrical

parameters

Bioreactor set up and re-

design

Mixing

Characterization

Comparison of kLa method

determination and values

Culture conditions to apply in the

new bioreactor

• A rigorous approach need to be

followed to de-risk a bioprocess

technical transfer

Example – rProtein production in E. coli

The SDM aim is to have the same kLa across scales

Scale (L) Gas velocity(m/s)

Maximum impeller speed (RPM)

Harvest OD600 Protein Yield (g/L)

kLa (s-1)

2 2.6 x 10-3 1200 75 4.2 ± 0.1 0.12

20 5.6 x 10-3 1000 66 4.6 ± 0.5 0.10

200 1.2 x 10-2 500 51 1.3 ± 0.1 0.06

P. Farrell et al. / Vaccine 30 (2012) 5695– 5698

Scale (L) Gas velocity(m/s)

Maximum impeller speed (RPM)

Harvest OD600 Protein Yield (g/L)

kLa (s-1)

2 2.6 x 10-3 880 50 1.8 ± 0.4 0.06

20 5.6 x 10-3 580 46 1.2 ± 0.1 0.06

200 1.2 x 10-2 500 51 1.3 ± 0.1 0.06

CPI case study (750L → 20L)

750L20L

750L batch fermentation to replicate on the 20L SDM

GC of 750L vs 20L SDM

The most used method is the Dynamic Gassing-out method

How measure the kLa?

( ) XqCCakdt

dCoALL

ALAL 2*

−−=Slope: kLa

Process values to to apply during the ABSORPTION:

- Stirrer Speed

- Inlet gasses flow

- Temperature

- Pressure

kLa= f(stirrer speed, inlet gasses flow)

Question

Tech- Transfer based Project for the scale-up of aerobic process for enzyme production

at 750L.

• The client performed in 1L scale the process at maximum stirrer speed and Air flow with

remarkable results in terms of Oxygen supply during the C-source feeding

• The KLa in these condition is 240 h-1

• The maximum KLa achievable in 750L is 175 h-1.

Which Issue can we forecast?

The direct scale up will show a severe Oxygen limitation, with

• production of side-products

• Riduction of productivity in large scale

Run Lost!!

Question

Aim: To define scalable Fed-batch aerobic process for enzyme production at 750L.

• The maximum KLa achievable in 750L is 175 h-1.

Study will be focus on Glucose profile and the effect to DOT control at defined mass transfer in

5L bioreactor.

• The mass transfer condition will be scaled down to 5L

• Which conditions will give a KLa of 175 h-1?

5L Mixing Characterisation Results

80

400

800

0

50

100

150

200

250

0.15

0.5

1

Stirr

er

Speed (

rpm

)

KLa (

h-1

)

Air Flow (vvm)

Internal CPI dataKLa in 5LWV @0.2 bar 37degC

0-50 50-100 100-150 150-200 200-250

Gas-liquid flow regimes

Bakker A. 2006 Fermenter Specific Modeling Issues www.bakker.org

Time for your calculations

0

50

100

150

200

250

300

0 200 400 600 800 1000

KLa (

h-1

)

Stirrer Speed (rpm)

KLa dep. Stirrer Speed

0.1 vvm

0.5 vvm

1 vvm

0

50

100

150

200

250

300

0 0.2 0.4 0.6 0.8 1 1.2

KLa (

h-1

)

Air Flow (vvm)

KLa dep. Air Flow

80 rpm

400 rpm

800 rpm

Time for your calculations

y = 0.323x - 4.5344R² = 0.9775

0

50

100

150

200

250

300

0 100 200 300 400 500 600 700 800 900

KLa (

h-1

)

Stirrer Speed (rpm)

KLa dep. Stirrer Speed

0.1 vvm

0.5 vvm

1 vvm

• The maximum Air flow

in 750L is 1vvm

• 175h-1 can be reached

with 556rpm

Solution

– The Scale Down Model limit the stirrer speed and air flow cascade ranges to 556rpm

and 1vvm

– The C-source feeding will be reduced once reached the DOT set point

– The C-source feeding will be scaled up as compromise for a continuous enzyme

production in presence of sufficient Oxygen

Other features….

• SDM information to be integrated with the CFD analysis, in order to fully

understand the overall mass transfer distribution in the bioreactor.

Bakker A. 2006 Fermenter Specific Modeling Issues www.bakker.org

Other features….

• In SDM scale the variability of your model &

process can be determinate.

• Process optimisation and Process

Characterisation can be done with DoE studies in

that scale.

1L1mL

Summary

• Bioprocesses are prone to mixing related performance problems:

– Gas dispersions can never be homogenous.

– Mass transfer will limit productivity or affect process control.

• A rigours model needs to be establish to forecast common issues typical of large scale

bioreactor

• Scale down model (SDM) kLa based can help to

– to evaluate the scalability of our process in development

– to forecast process results in Demo scale

• Coupled with CFD and DoE Model, represent the next step for a completer understanding

of Mid-stream steps in bioprocesses

Demonstration in the lab

KLa

• Validate results of the previous question: 556rpm and 1vvm will give a kLa of 175h-1?

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