Continuous cultures in shake flasks March 28 2012 ... cultures in shake flasks March 28 2012...

Continuous cultures in shake flasks March 28 2012

Continuous cultures in shake flasks Nordics Bioprocess Improvement Seminar Innovation in cell culture process development & production

Stockholm, March 28 2012

Bjarne Rask Poulsen, Novo Nordisk A/S

Continuous cultures in shake flasks Slide no 2 March 28 2012

R&D facilities Manufacturing Global/regional headquarter China Denmark India Japan Switzerland US

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China Denmark US


Production of difficult-to-express proteins

• Low titers: mg/L range

• Unstable and fragile => low residence times preferred

Our choice of solution:

Perfusion cultures


From the cells point of view

These are the conditions we want to mimic!

Attached to other cells

Surrounded by a constant flow of blood filtrate • bringing substrates like sugar and oxygen • taking away waste products like lactate, ammonia and CO2

Consider the original environment of a mammalian cell

Our choice of scaling factor:

Supply of medium per cell


Patient focus

We have to give our patients consistent quality

• Perfusion cultures give the option to produce in steady-state

• Steady-state gives high probability of producing consistent quality


Perfusion culture

• Cell retention or immobilization

• Exchange of medium

• Low waste product concentration

• Low product residence times

• High volumetric productivity

Fresh

Medium

Harvest

Bleed

Balance


Perfusion cultures

• Requires a lot of equipment:

• retention device

• in-flow

• out-flow

• bleed-flow

• method for constant volume

• Requires a lot of resources:

• time-consuming

• expensive

Fresh

Medium

Harvest

Bleed

Balance


• We want to optimize medium and conditions by DOE

• require high number of experiments

• We want to screen a high number of cell line candidates in the relevant steady-state conditions

• Possible number of experiments is inversely proportional to their complexity

Simplified down-scale models for Perfusion cultures are needed


1st simplification

Perfusion cultures -> continuous cultures

Simplified down-scale models for Perfusion cultures are needed

Fresh

Medium

Harvest

Balance

Fresh

Medium

Harvest

Bleed

Balance


Simplified down-scale models for Perfusion cultures

2nd simplification

• Continuous cultures -> continuous cultures in shake flasks

• only in-flow

• no outflow

• using flow of medium per cell as scaling factor


Continuous cultures only in-flow in shake flasks

teVtV 0)(

Simple: only in-flow no out-flow

Flow =

Volume =

teVdt

dVtF 0)(


Continuous shake flask culture (theoretical)

Doubling time=50 h

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time [days]

Vo

lum

e [

mL]

0

1

2

3

Flo

w [

mL/

h]

VC

D [

E6

/m

L]

Volume

Flow

VCD

Continuous culture only in-flow


Continuous cultures

Definition of continuous cultures: constant dilution rate Flow Volume

Traditional continuous culture: Flow constant Volume constant

Continuous cultures only in-flow:

Flow exponentially increasing with a rate constant of µ Volume exponentially increasing with a rate constant of µ

= constant dilution rate

= constant dilution rate

= dilution rate


Chemostat in steady-states (theoretical)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.00 0.01 0.02 0.03 0.04 0.05

Dilution rate [1/h]

VC

D [

E6

cells/m

L]

Flo

w p

er c

ell

[n

L/cell/d

ay]

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Su

bstr

ate

[m

M]

VCD

Flow per cell

Substrate

All well-known theory of continuous cultures applies

14

DTmin = 16 h

(µmax = 0.043 h-1)



Setup



Installation of tubing



Sampling


Experimental conditions

Continuous cultures only in-flow in shake flasks applied to:

• In-house cell lines producing difficult-to-express proteins

• Commercial CD-media with additions

• 250 rpm (throw 2.5 cm diameter)

• Pulse 1 min + pause 59 min

• Experiment 1: 4 repeats

• Experiment 2: DOE – addition of two components


Growth

0 5 10 15 200

1

2

3

4

5

90

92

94

96

98

100

Shaker 1

Shaker 2

Shaker 3

Shaker 4

Cultivation time [days]

V

iab

le c

ell

den

sit

y [

E6 c

ell

s/m

L]

V

iab

ility [%

]


Metabolism

0 5 10 15 200

10

20

30

0

2

4

6

8

10

Shaker 1

Shaker 2

Shaker 3

Shaker 4


G

lc,

Lac c

on

cen

trati

on

[m

M]

G

ln c

on

cen

tratio

n [m

M]


API production

0 5 10 15 200.0

0.2

0.4

0.6

0.8

1.0Shaker 1

Shaker 2

Shaker 3

Shaker 4


Pro

du

ct

co

ncen

trati

on

(n

orm

alised

) [-

]


0 5 10 15 200.0

0.2

0.4

0.6

0.8

0

100

200

300Shaker 1

Shaker 2

Shaker 3

Shaker 4


D

ilu

tio

n r

ate

[1/d

ay]

V

olu

me [m

L]

Culture dilution


Balanced-controlled pumps for accurate flow


DOE: +/- addition of two components plus centerpoints in double determination

0 5 10 15 200

1

2

3

4

5

50

60

70

80

90

100

+,-

o,o

-,+

-,-

o,o

+,+


V

iab

le c

ell

den

sit

y [

E6 c

ell

s/m

L]

V

iab

ility [%

]


DOE: +/- addition of two components plus centerpoints in double determination

Term Prob>|t|

Component 1 0.0010 *

Component 2 0.0142 *

Component 1 * Component 2 0.0691

-,- -,+ o,o +,- +,+30

35

40

45

50

Inte

gra

l o

f V

CD

[E

6 c

ells*d

ay/m

L]


Alternative system for continuous cultures only in-flow:

ambr, Automated bioreactor system (15 mL)

• Automated cell culture bioreactor system ambr

• 15 mL working volume

• 24 parallel single-use reactors

• Integrated monitoring of cell number

• pH control

• DO control


Conclusions

• Easy to implement technology

• Simple

• Inexpensive

• Using standard laboratory equipment

• Physiological studies at steady-state!

• High reproducibility

• Suitable tool for screening and DOE


Thanks to:

Martin Schalén for performing initial experimental studies

Martin Heitmann for performing most experimental studies

My email: [email protected]

Acknowledgements


Continuous cultures only in-flow Definition of continuous cultures = constant dilution rate (D)

=> dV/dt = D·V, V is volume and t is time

=> V(t) = V0·exp(D·t)

From steady-state mass balances:

D = µ (cell specific growth rate)

D = F/V, F is flow => F = µ·V

µ = ln2/T2, T2 is doubling time

F(t) = µ·V0·exp(µ·t)

F(t) = ln2/T2·V0·exp(ln2/T2·t)


Doubling time and volume

0 5 10 15 200

20

40

60

80

0

50

100

150

200Shaker 1

Shaker 2

Shaker 3

Shaker 4

Cultivation time [d]

D

ou

bli

ng

tim

e [

h]

V

olu

me (m

L)


4 repeats

0 5 10 15 200

2

4

6

8

Shaker 1

Shaker 2

Shaker 3

Shaker 4

Cultivation time [d]

G

ln,

NH

4+

co

ncen

trati

on

[m

M]


Diabetes

Haemophilia

Growth disorders

Inflammation

Insulin and GLP-1

Coagulation factors

Human growth hormone

Monoclonal antibodies

Expand leadership

Establish presence

Expand portfolio

Achieve leadership

Establish presence

Pro

tein

engin

eering a

nd f

orm

ula

tion

Pro

tein

dru

g d

elivery

Larg

e-s

cale

bio

logic

s m

anufa

ctu

ring

Glo

bal com

merc

ial in

frastr

uctu

re

Therapeutic area Compounds and capabilities Strategic focus

Novo Nordisk Way

Obesity GLP-1

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