Application of HTP microfluidic
culture systems to media and
process optimisation
Steven C. Peppers, Ph.D., MBA
Principal Scientist, R&D
Reg Joseph, B.Sc., MBA
Business Area Manager, BioProduction
BioProduction Systems and Services
Invitrogen Corporation
Complexity of Cell’s Needs
Metabolic pathways chart from
Boehrringer-Mannheim
Invitrogen Proprietary and Confidential
Complexity of culture systems
Interactions within:
Uptake of nutrients
Receptor signaling
Metabolic pathways
Physicochemical qualities
Dynamic states
No tools to fully probe this complexity
Build up of components at inappropriate concentrations
Special technologies for managing complexity:
1. Statistical designs and strategy
2. Scaled down HTP tools
3. Expertise in cell requirements and media components
DOE (Design of Experiment):
Statistically sound means of planning and analyzing efficient experiments:
Examples: 2-level factorials and fractional factorials, central composite designs,
minimum run designs, mixtures, steepest ascent, Box-Behnken, D-optimal
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Multi-factor central composite designs
+1 (8)
(Glutamine, nM)
Component B
Run chart showing coded levels
0 (6)
-1 (4)
-1 (2)
0 (3)
Component A
(Glucose, g/L)
+1 (4)
Run
#
A
Gluc.
B
L-Gln
C
Osm
1
-1
-1
+1
2
+1
-1
-1
3
-1
+1
-1
4
+1
+1
+1
5
0
0
0
6
0
0
0
7
-1.4
0
0
8
+1.4
0
0
9
0
-1.4
0
10
0
+1.4
0
11
0
0
-1.4
12
0
0
+1.4
Half-factorial central composite for 3 factors
Invitrogen Proprietary and Confidential
What’s The Right Tool For The Right Job?
Effective bioprocess development tool:
1.
High throughput—100’s of different conditions
2.
Scalable—Predict performance in ST bioreactors
3.
Reliable—High precision, accuracy and reproducibility
4.
Efficient—Process in time and labor, cost effective
Currently Used Platforms
Requirement
New Tool
Well Plates
Shake Flasks
Stirred Tank
Bioreactors
BioProcessors
SimCell
HTP
Yes
Rather limited
No
Yes
Scalable
No
Not generally
Yes
Yes
Reliable
Yes
Yes
Yes
Yes
Efficient
No
No
No
Yes
Invitrogen Proprietary and Confidential
BioProcessors SimCell System
Fluidic Module
Sampling Module
Incubation Modules
Micro-Bioreactor Array
(MBA)
6 Chambers
600 uL Working Vol.
Independent Loading
Stirring by Bubble
Central Robot
Invitrogen working
with BioProcessors
for 2 yr
Invitrogen Proprietary and Confidential
Loading Cell
Optical Sensing Module
Scalability of SimCell Results
SimCell MicroBioreactors with CHO Cells
SimCell at Day 6
Mean +/- SD (n=6)
Total Cells/mL x10 6
(by Optical Density)
6
pH 6.9
pH 7.2
5
4
pH 6.9
4.45 x106 TC/mL
pH 7.2
3.07 x106 TC/mL
3
Difference/Average = 36.7%
2
1
0
0
1
2
3
4
5
6
7
8
Days
Stirred Tank Bioreactors with CHO Cells
Mean +/- SD (n=2)
6
ST Bioreactors at Day 6
pH 6.9
pH 7.2
6
Total Cells/mL x10
5
4
3
2
pH 6.9
4.57 x106 TC/mL
pH 7.2
3.13 x106 TC/mL
Difference/Average = 37.4%
1
0
0
1
2
3
4
Days
Invitrogen Proprietary and Confidential
5
6
7
8
Ratio of “Dif/Avg” values = 0.98
Complex Factorials in SimCell
Goal: Confirm SimCell capability in complex factorials
Fractional Central Composite Design, N=192
Coded Level
Level
-1.68
-1
0
1
1.68
Starting Media and pH Conditons
pH
Osmo Target Glucose
6.9
246.2
0.64
6.9
270
2
7.1
305
4
7.3
340
6
7.3
363.8
7.36
Invitrogen Proprietary and Confidential
Glutamine Pluronic
0.32
0.8
1
0.8
2
1.3
3
1.8
3.68
2.14
Feed Additions, Day 4
Gluc Feed Glutamate Feed
Aspartate
0
0
0
0
0
0
2
2
0.1665
4
4
0.3330
5.36
5.36
0.4462
Response Surface from Analysis
T o ta l C e ll D e n s ity
A v g . D a ys 6 -8
5E+006
X 1 = C : G lu cos e in
S ta rting M e d ium
C e lls / m L
4E+006
X 2 = F : G luc ose F ee d
Day 4
R e s p o n se S u rfa c e C :F
W ith O th er F ac to rs a t:
3E+006
2E+006
A : p H = 6.9
B : O s m o = 2 70 m O s m
D: G LN = 3 m M
E : P lu ron ic = 1 .8 g/L
G : G LU Feed = 4 m M
H : A S P F e ed = 0.33 fo ld
1E+006
5
4
X 2 : G lu c .F e e d
a t d a y4 (g /L )
D e s ign -E xp e rt® S oftw a re
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7
3
6
4
1
3
0
1
X 1 : G lu c o s e
a t S ta rt (g /L )
Interaction in Late Log Phase Growth
Design-Expert® Software
Interaction
Glucose at Start (g/L)
OD Avg 5-6.5 days
4.10E+06
Design Points
C- 2.000
C+ 6.000
Actual Factors
B: Osmo = 305.00
D: Glutamine = 2.00
E: Pluronic = 1.30
F: Gluc Feed = 2.00
G: Glutamate = 2.00
H: Aspartate = 0.167
OD Avg 5-6.5 days
X1 = A: pH
X2 = C: Glucose
3.25E+06
2
2.40E+06
1.55E+06
7.00E+05
6.90
7.00
7.10
A: pH
Invitrogen Proprietary and Confidential
7.20
7.30
Interaction in Final tPA Productivity
Design-Expert® Software
Interaction
Glucose at Start (g/L)
tPA at Day 8
210
Design Points
C- 2.000
C+ 6.000
Actual Factors
B: Osmo = 305.00
D: Glutamine = 2.00
E: Pluronic = 1.30
F: Gluc Feed = 2.00
G: Glutamate = 2.00
H: Aspartate = 0.167
tPA at Day 8
X1 = A: pH
X2 = C: Glucose
157.5
105
52.5
2
0
6.90
7.00
7.10
A: pH
Invitrogen Proprietary and Confidential
7.20
7.30
Optimizing both Cell Density and Productivity
Selected Outcome
Set to
Weight
Cells/mL, days 5-6.5 Maximum
Cells/mL, days 6.5-8 Maximum
Prod’n of tPA, day8 Maximum
3
3
5
Prediction
Desirability
Design-Expert® Software
6.00
0.819
Desirability
1
0
0.748
5.00
Actual Factors
B: Osmo = 270.01
D: Glutamine = 2.80
E: Pluronic = 0.84
F: Gluc Feed = 4.00
G: Glutamate = 3.73
H: Aspartate = 0.333
Glucose at Start (g/L)
X1 = A: pH
X2 = C: Glucose
0.675
4.00
0.603
0.458
0.530
3.00
2.00
6.90
7.00
7.10
A: pH
Invitrogen Proprietary and Confidential
7.20
7.30
SimCell™ at Invitrogen
Purchased model
with 4 incubators
1008 chambers
possible
24-factor 2-Level
factorials possible
Recently installed
at Grand Island
(GIBCO) site
Currently in OQ
phase
PQ and early
implementation
scheduled for 3rd
and 4th quarter
Invitrogen Proprietary and Confidential
General Workflow Plans
Compose 100’s of variations of a medium
B
B
Hamilton STARplus
C
A
C
B
B
E
A
A
C
A
C
D
Database and
Firewall
DOE design
SimCell™
System
Next
Optimization
Cycle
HTP Assays and
Data analysis
Occasional
scaled-up
verification
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Improved economics for services
Development
program:
11 factors - CCD
Std Project
cost
Time
*Cost per data
point
Value per data
point
SimCell
Bioreactor
Combination
plates/shaker
w/Bioreactor
Optimized
SimCell
X
7.5X
1.2X
½X
11 weeks
56 weeks
33 weeks
11 weeks
$146
$1085
$465
$167
High
High
Low/Med
?
*Not a consumable cost; calculated by dividing the full project cost by number of data points
Invitrogen Proprietary and Confidential
Improving media design & manufacturing
½
Reduce
components
80-100
X
¼
components
– many
replicates at
varying
conc.
¼
Traditional
Mixtures
Experiment
Preserve or
increase
performance
Let’s get smarter around media components:
a) Multiple Salt forms* – calcium nitrate + calcium chloride
b) Hydration levels - L-Histidine vs L-Histidine HCl H2O
c) Differing forms of the same amino acid - cysteine vs cystine
*May have opposite effects
Invitrogen Proprietary and Confidential
Benefits
 Reduce COGS
 # of weighs/product
 raw material mgmt,
incoming QC
 Decrease variability &
formulation errors
 Eliminate redundancies &
counter effects
Conclusions
• SimCell system installed at Invitrogen
- Currently going through validation
- Beta programs with key cell lines underway
- Formal service offering in 2007
•
•
•
•
Demonstrated ability to perform complex factorial designs
Developed and validated a fed-batch model using SimCell
Optimize media, process, and feed simultaneously
Cost models enables high value services at reasonable prices
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