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CASE STUDY: Hyclone Single Use Bioreactor coupled to Finesse hardwareand DO probe evaluation for a Fed-batch production process

EMD Serono SA / Presented by Emmanuelle CAMEAU15 June 2010

Case Study Hyclone SUB | 15 June 20102

Presentation of the Company

� EMD Serono: Largest division of EMD KGaA

� Established: January 5, 2007

� Business: Innovative small molecules & biopharmaceuticals

� Employees: ~17,000

� President: Elmar Schnee

� Total revenues: € 5.3 billion in 2009

� Headquarters: Geneva, Switzerland

� Key growth drivers:


EMD-Serono Aubonne

Aubonne

�Infertility treatment �Neurology

�Biopharma devpre-clinical; phase I; phase II


Outline

� Goal of the study

� How ? With what process?

� Description of the Single Use Bioreactor

– Features of the Bioreactor

– Description of the SCADA

� Results

– Comparison of the disposable DO probe and stainless steel

– Description of the Experimental Scheme

– Results of the seed train bioreactors

– Results of the production bioreactors

� Conclusion and Perspectives


Goal of the Study

Test a flexible and innovative disposable bioreactor


Requirements

� System:

– Test a control system as flexible as possible

– Test a disposable bioreactor similar to conventional ones (geometry) in order to facilitate process developement

� Probes:

– Test disposable probes supposed to be as performant as conventional stainless steelprobes as no supplier had proposed before

– Test disposable probes in order to avoid sterilization and cleaning steps

� Process:

– Assess the possibility to replace a 50L stainless steel seed train bioreactor (5L smallscale model)


ATMI Lifesciences/Pierre Guérin NUCLEOSartorius BIOSTAT®

CULTIBAG STR

Xcellerex XDR-50

Sartorius BIOSTAT®

Cultibag

Hyclone SUB: Best choice for flexibility and process developement

� Test a 50L single use bioreactor: which one?

HYCLONE SUB

Limitations:

Cell density

Geometry

Heat overshooting

Limitation:

Geometry

Limitation:

Not commercialized yet

Limitations:

Lack of flexibility


How?


Fed-batch process, Mab-producing CHO cells

5L bioreactor

Seed train

(N-2, N-1)

ProductionAmplification

5L bioreactor

Fed Batch mode

T°, pH & DO : constant setpoints

Outputs: Mab titerIntegral Viable CellspCO2<60mmHg

50L bioreactorsmall scale model

pH control : CO2/NaOH, DO control: O2/air

50L or 300L bioreactorsmall scale model


The Hyclone Single Use Bioreactor coupled to Finesse…


A different approach of single use bioreactor technology

20µm


Finesse SCADA: flexibility with no rewiring necessary

� TruBioTM Software: based on DeltaV technology

� Configuration of control loops

� Possibility to use any output to control any other device

� Ex: DO output can be assigned to control the injection of Air or N2 instead of O2.


Results


TruFluor TM DO: A disposable DO probe comparable to conventional ones

30

35

40

45

50

55

60

65

70

00:0

0:00

00:5

0:00

01:4

0:00

02:3

0:00

03:2

0:00

04:1

0:00

05:0

0:00

05:5

0:00

06:4

0:00

07:3

0:00

08:2

0:00

09:1

0:00

10:0

0:00

10:5

0:00

11:4

0:00

12:3

0:00

13:2

0:00

14:1

0:00

15:0

0:00

15:5

0:00

16:4

0:00

17:3

0:00

18:2

0:00

19:1

0:00

20:0

0:00

20:5

0:00

21:4

0:00

22:3

0:00

23:2

0:00

Time

% p

O2

Inox DO probe

disposable DO probe

Bioreactor feeding

� DO regulation: Stainless steel probe

� Small constant difference between the probes

� Good regulation and oxygen transfer with soft PID

Possibly due to calibration (less than 2%)


Experimental scheme

Disposablebioreactor 50/100L

Disposablebioreactor 50/100L

Feed/harvest

2 X 5L bioreactors

2 X 5L bioreactors

2 X 5L bioreactors

Cultibag RM Optical 50L

Cell culture amplification T-flasks / Spinner

flasks

Cultibag RM Optical 2L

Seed train (N-2, N-1) Production

Amplification


Seed Train bioreactors

Dark blue: 5L bioreactor seed train

Light blue: SUB Seed train

� Comparable cell densities with 5L bioreactors or SUB

� SUB has the potential to reach higher cell densities than 5L bioreactors

0

1

2

3

4

5

6

20 21 22 23 24 25 26

WD

Via

ble

cells

(x1

06 /m

L)

Passage from N-2 to N-1 bioreactor


Production bioreactors

� Reproducible cell growth for all conditions (IVC)

� VCD at day 7 within the variability of the process developped in 50L and 5L bioreactors

� Mab titer has been proved to be linearly related to the IVC curve (The metabolic Pathway Engineering handbook, Christina C. Smolke)

All bioreactors have probably produced the same quantity of Mab

* All data are means of 2 to 3 runs

Viable Cell density at working day 7

0

0.5

1

1.5

2

2.5

33.5

4

4.5

5

mill

ions

cel

ls/m

L

SUB/SUB SUB/5L 5L/5L

Integral Viable Cells

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8

Working Day

IVC


Mab titer on day 7

0

100

200

300

400

500

600

700M

Ab

(mg/

L)

Production bioreactors

�SUB performance at least comparable to the 5L bioreactors

SUB/SUB SUB/5L 5L/5L

Potential of the SUB to be used as a seed train bioreactor and/or a production bioreactor



0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7

Working Day

pCO

2 m

mH

g

Sparger membrane: a good bubblecompromise

• White curves: Acid pH regulation

• Others: CO2 pH regulation

• Dark blue: 5L/5L

• Turquoise: SUB/5L

• Light blue: SUB/SUB

� pCO2 was kept under 60 mmHg during almost all runs

� SUB: lower pCO2 than some acid regulated runs

Performance of the sparger membrane in CO2 stripping:

� Bubbles small enough to avoid O2 limitation

� but big enough to strip efficiently CO2



How much??


A flexible investment….

� 50L Hyclone jacketed Single Use Bioreactor + Finesse Hardware and Software : $ 110 000

� Standard 50L bag with or without disposable probe: $ 3000


Conclusion and Perspectives


Hyclone SUB coupled to Finesse: a good combination

� SUB: very flexible (hardware to software)

� Finesse TruViuTM: Very powerful and user friendly

� Finesse TruFluorTM DO: can replace a conventional probe, no process calibration required

� Process feasability successful : same Mab titer and cell density

� SUB: Performant CO2 stripping during the culture

� Successful evaluation of the SUB coupled to Finesse for the use as a Mab production bioreactor or seed train in fed-batch

Simplicity of use of the SUB


Perspectives

� Clinical trial products: Rapid campain turnover

� Stainless steel Seed train bioreactors replacement:

– Usually lots of preparation for short use (<1 week)

– Possibility to reach as high cell densities as in conventional stainless steel bioreactors

� Alternative to commonly used wave bioreactors

– For processes demanding high seeding cell densities


Questions

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