Post on 28-Mar-2018
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
Algeria Brazil China Denmark France Japan US
China Denmark US
Continuous cultures in shake flasks Slide no 3 March 28 2012
Production of difficult-to-express proteins
• Low titers: mg/L range
• Unstable and fragile => low residence times preferred
Our choice of solution:
Perfusion cultures
Continuous cultures in shake flasks Slide no 4 March 28 2012
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
Continuous cultures in shake flasks Slide no 5 March 28 2012
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
Continuous cultures in shake flasks Slide no 6 March 28 2012
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
Continuous cultures in shake flasks Slide no 7 March 28 2012
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
Continuous cultures in shake flasks Slide no 8 March 28 2012
• 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
Continuous cultures in shake flasks Slide no 9 March 28 2012
1st simplification
Perfusion cultures -> continuous cultures
Simplified down-scale models for Perfusion cultures are needed
Fresh
Medium
Harvest
Balance
Fresh
Medium
Harvest
Bleed
Balance
Continuous cultures in shake flasks Slide no 10 March 28 2012
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 in shake flasks Slide no 11 March 28 2012
Continuous cultures only in-flow in shake flasks
teVtV 0)(
Simple: only in-flow no out-flow
Flow =
Volume =
teVdt
dVtF 0)(
Continuous cultures in shake flasks Slide no 12 March 28 2012
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 in shake flasks Slide no 13 March 28 2012
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
Continuous cultures in shake flasks Slide no 14 March 28 2012
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)
Continuous cultures in shake flasks Slide no 15 March 28 2012
Continuous cultures only in-flow in shake flasks
Setup
Continuous cultures in shake flasks Slide no 16 March 28 2012
Continuous cultures only in-flow in shake flasks
Installation of tubing
Continuous cultures in shake flasks Slide no 17 March 28 2012
Continuous cultures only in-flow in shake flasks
Sampling
Continuous cultures in shake flasks Slide no 18 March 28 2012
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
Continuous cultures in shake flasks Slide no 19 March 28 2012
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 [%
]
Continuous cultures in shake flasks Slide no 20 March 28 2012
Metabolism
0 5 10 15 200
10
20
30
0
2
4
6
8
10
Shaker 1
Shaker 2
Shaker 3
Shaker 4
Cultivation time [days]
G
lc,
Lac c
on
cen
trati
on
[m
M]
G
ln c
on
cen
tratio
n [m
M]
Continuous cultures in shake flasks Slide no 21 March 28 2012
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
Cultivation time [days]
Pro
du
ct
co
ncen
trati
on
(n
orm
alised
) [-
]
Continuous cultures in shake flasks Slide no 22 March 28 2012
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
Cultivation time [days]
D
ilu
tio
n r
ate
[1/d
ay]
V
olu
me [m
L]
Culture dilution
Continuous cultures in shake flasks Slide no 23 March 28 2012
Balanced-controlled pumps for accurate flow
Continuous cultures in shake flasks Slide no 24 March 28 2012
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
+,+
Cultivation time [days]
V
iab
le c
ell
den
sit
y [
E6 c
ell
s/m
L]
V
iab
ility [%
]
Continuous cultures in shake flasks Slide no 25 March 28 2012
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]
Continuous cultures in shake flasks Slide no 26 March 28 2012
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
Continuous cultures in shake flasks Slide no 27 March 28 2012
Conclusions
• Easy to implement technology
• Simple
• Inexpensive
• Using standard laboratory equipment
• Physiological studies at steady-state!
• High reproducibility
• Suitable tool for screening and DOE
Continuous cultures in shake flasks Slide no 28 March 28 2012
Thanks to:
Martin Schalén for performing initial experimental studies
Martin Heitmann for performing most experimental studies
My email: bjrp@novonordisk.com
Acknowledgements
Continuous cultures in shake flasks Slide no 29 March 28 2012
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)
Continuous cultures in shake flasks Slide no 30 March 28 2012
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)
Continuous cultures in shake flasks Slide no 31 March 28 2012
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]
Continuous cultures in shake flasks Slide no 32 March 28 2012
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