Development of novel chemically-defined media for CHO cell applications
Dr. Dipl-Ing. Jörg von Hagen
Head of Process Development & Launch
Management Merck Millipore, Darmstadt, Germany
Cell Culture World Congress, Munich 2013
2
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
2
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
3
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100
0 3 4 5 6 7 8 9 10 11 12 13 14
Elapsed Time (Day)
Cell
Via
bil
ity (
% V
iab
le C
ell
s)
0
0.5
1
1.5
2
2.5
3
3.5
IgG
Tit
er
(g/L
)
Competitor A Competitor B Cellvento CHO-200
Competitor A Competitor B Cellvento CHO-200
A Comparison of IgG Production in CVC-200 and Two Competitor Media and Feed Supplements.
IgG Production in Cellvento CHO-200 was 2-3 fold higher than two competitor
products over a 14 day fed batch culture.
4
Cell Growth (VCD) - Competitive Evaluation in Fed Batch
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Elapsed Time (Day)
VC
D (
10e6 c
/ml)
Competitor A Competitor B Cellvento CHO-200
Cell growth in Cellvento CHO-200 achieved above 1x107 cells /mL in a 14 day
fed batch culture.
A Comparison of Cell Growth in CVC-200 and Two
Competitor Media and Feed Supplements
5
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
5
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
6
0.00
5.00
10.00
15.00
20.00
25.00
3 4 5 6
Elapsed Time (Days)
Inte
gra
l V
iab
le C
ell
Den
sit
y
(10e6 c
ell
day/m
l)
Lot 006_1
Lot 006_2
Lot 006_3
Comparison of Average Cell Growth in Batch Culture
Cell growth in Cellvento CHO-200 was consistent in batch culture across three
lots of media
7
Comparison of Cell Growth in Fed Batch Culture
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2,0
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12,0
14,0
16,0
18,0
0 5 10 15
Via
ble
Ce
ll D
en
sit
y (
10
e6
c/m
l)
Elapsed Time (Day)
Three Lots of Feed tested
Feed Lot 007_1
Feed Lot 007_2
Feed Lot 007_3
Cell growth in Cellvento CHO-200 was consistent in fed batch culture across
three lots of feed
8
Comparison of Volumetric Titers Across Three Lots of Feed
0.0
0.2
0.4
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1.0
1.2
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1.6
1.8
2.0
7 10
Sample Day
IgG
(g
/L) Feed Lot 007_1
Feed Lot 007_2
Feed Lot 007_3
Comparison of IgG Production in Fed Batch Culture
IgG production in Cellvento CHO-200 was consistent in fed batch culture
across three lots of feed
PSD Media
9
10
PSD Feed
Presentation title in footer | 00 Month 0000 11
0
20
40
60
80
100
120
His Asn Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Met Val Ile Leu Phe
Am
ino
aci
d c
on
cen
trat
ion
(in
% o
f th
e
theo
reti
cal v
alu
e)
Cellvento CHO 200 media
Batch 1
Batch 2
Batch 3
Reproducibility amino acid content
12
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
12
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
13
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14
Via
ble
Ce
ll D
en
sit
y (
10
e6
c/m
l)
Elapsed Time (Day)
3L Cellready Bioreactor
50 ml Spin Tubes
A Comparison of Cell Growth in Spin Tubes and 3L Benchtop Bioreactors
Similar growth performance was achieved with Cellvento CHO-200 in spin tubes
and benchtop bioreactors over a 14 day fed batch culture.
14
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
7 10
IgG
(g
/L)
Sample Day
50 ml Spin Tubes
3L Cellready Bioreactor
A Comparison of IgG Production in Spin Tubes and 3L Benchtop Bioreactors
IgG titers averaged 1.5 g/L after 10 days of fed bath culture using
Cellvento CHO-200 in spin tubes and benchtop bioreactors.
15
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
15
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
NIR - Near Infrared Spectroscopy
Measurement conditions
Integrating sphere, PbS-detector, diffuse reflexion
Instrument: BrukerVector 22/N
Number of scans: 16, Resolution: 8 cm-1, Measurement Range: 12000 – 4000 cm-1
PCA-Possibility for clustering good and poor performing media batches
A fingerprint method for media performance prediction
low performance
medium
performance
high performance
EMD Millipore
Supplier C-1
Supplier C-2
Supplier C-3
Supplier B
Supplier A
Supplier S
Principle component analysis (PCA) Correlation of cellular performance and NIR analysis
PCA of DMEM-F12. Analysis by NIR-Spectroscopy (Bruker, Vektor MPA)
Quality Testing of Media …
0
5
10
15
20
25
0 days 3 days 5 days 7 days 10 days 12 days 14 days 17 days
cu
mu
lati
ve P
DL Mill Tech 1-1
Mill Tech 1-2
Mill Tech 1-3
Mill Tech 2-1
Mill Tech 2-2
Mill Tech 2-3
Impact of the milling technology on CHO-S cells grown in chemically defined media.
No significant differences are observed in the basic cell culture assay analyzing the
cumulative population doubling levels (PDL) using different produced mammalian
cell culture media.
…depends on the appropriate test system
Sensitive Cell Culture Assays
0
10
20
30
40
50
60
70
Supplier A Mill Tech 1-1 Mill Tech 1-2 Mill Tech 1-3 Mill Tech 2-1 Mill Tech 2-2 Mill Tech 2-3
pla
tin
g e
ffic
acy (
%)
Impact of the milling technology on CHO-S plating efficacy in chemically defined media.
CHO-S cells were diluted in the corresponding media and analyzed after 2 weeks on
plating efficacy.
… underline differences in cell culture media performance
Impact of Processing Technologies
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
Supplier A Mill Tech 2
Tit
er
( %
to
in
tern
al
refe
ren
ce)
The media were tested in the production of a monoclonal antibody.
Titer was measured by Surface Plasmon Resonance Spectroscopy.
Red line indicates the minimum achievable titer according to the specifications.
Impact of the milling technology on mAb titer
Milling Process Impact on final NBE Attribute
0
5
10
15
20
25
30
35
Supplier A Mill Tech 2
Cri
tical
Pep
tid
es (
%)
Final molecules after capture were analyzed by HPLC to determine the amount (%) of
critical peptides that are known to be correlated with lower half life of the drug substance
in patient serum samples. The amounts are directly linked with the raw material quality
and the production process.
Red line indicates the max. value given by the authorities. Dotted red line represents the internal specification limit.
Range between dotted blue lines show the target space for good media performance.
Impact of the milling technology on critical antibody attributes
22
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
22
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
Humidity and impact on powder flowability
After opening the package the first time the CDM II powder is clumpy. The DMEM-
F12 and CDM I media are opened several times and are free flowing powders.
CDM I
DMEM-F12
CDM II
CDM II: particle size distribution
CDM II particle size analysis is
technically not feasible by air-jet
sieving. After 3 min all material
gets like chewing gum, whereas
for CDM I and DMEM-F12
particle size distribution analysis
went without clumping.
CDM composition comparison
The CDM I and II composition have the following critical hygroscopic
ingredients:
– Calcium Chloride 2H2O
– Choline Chloride
– Ferric Nitrate 9 H2O
– Ferrous Sulfate 7 H2O
– Sodium phosphate, monobasic, monohydrate
the above listed ingredients are present with a concentration in sum of 1.5% in
the CDM I and 9.7% in the CDM II formulation.
6.3-fold enrichment of hygroscopic compounds resulted in a final H2O titer
of 4.05% measured by Karl Fischer Titration in the CDM II powder
(compared to 1.05 to 1.2 in DMEM-F12 and CDM I.)
Humidity effects solubility of DMEM F12
6 h / 28 °C / 90 % rH. 2 – 8 °C 4 fold accelerated (5min real)
17° increase in avalanche angle after DMEM F12 exposure to 90% rH
DMEM F12 before and after flowability measurement (3h/25°C at 90% rH)
DMEM-F12 compacted
uncompressed 20KN 30KN 40KN 50KN 60KN
R = Rosa = pinkish
DMEM-F12 compacted
uncompressed 20KN 30KN
40KN 50KN 60KN 1-3 mm particle sizes
1 mm 1 mm 1 mm
1 mm 1 mm 1 mm
Solubility of DMEM F12 powder vs. granules
Filterability of Powder vs. Granules of DMEM F12
Medium Compaction
(kN)
Filter
Family
Filter
Name
Filter Pore
Size
(µm)
Vmax
(L/m2)
Amin
(m2)
DMEM
F-12
0
0
Express SHR 0,1 9 305 0,28
Express SHR-2L 0.5/0.1 20 555 0,29
30
30
Express SHR 0.1 11 221 0,29
Express SHR-2L 0.5/0.1 37 911 0,31
60 Express SHR 0.1 10 596 0,30
60 Express SHR-2L 0.5/0.1 44 219 0,31
The Amin represents the minimal surface of membrane needed to process 500L
within the defined time.
32
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
32
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
Tyrosine solubility in feed with >110 g/L amino acid & vitamin load
1 1 50 70
g/L @ pH 7.0 +/- 0.2
L-Tyr L-Tyr 2Na mod L-Tyr I mod L-Tyr II
CHO-S growth profile comparing L-Tyrosine Disodium salt vs. modified Tyrosine
time (days)
VC
D (
x1
05 v
c/m
L)
Medium 0 1 4 5 6 7 8
1 1 29,6333333 753,033333 1264,53333 1541,13333 988,936665 333,58 0
2 1 33,2 681,6 1218,13333 1470,4 1108,24002 548,75
3 1 11,5333333 61,0666667 73,0666667 64,8999992 46,4400007 63,1033333 1,92
4 1 24,15 528,6 1318,4 1409,82503 1393,7 1324,422 2,07
5 1 24,6666667 462,9 1197,26667 1441,11333 1578,69001 1502,80333 2,49
6 1 19 85,2666667 157,4 266,206664 375,935004 415,776667 0,29546573
7 1 16,4666667 116,266667 135,9 117,206667 78,376667 71,34
18,33
0,31
0
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1 3 5 7 9 11
time
Leb
en
sze
llzah
l [vc
/ml]
x1
0^
5
4,5mM 2Na-Tyr
4,5mM 2Na-P-Tyr
9mM 2Na-P-Tyr
4.5 mM L-Tyr 2Na
4.5 mM mod L-Tyr II
9.0 mM mod L-Tyr II
0
20
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1 3 5 7 9 11
time
Leb
en
sze
llzah
l [vc
/ml]
x1
0^
5
4,5mM 2Na-Tyr
4,5mM 2Na-P-Tyr
9mM 2Na-P-Tyr
0
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120
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180
200
1 3 5 7 9 11
time
Leb
en
sze
llzah
l [vc
/ml]
x1
0^
5
4,5mM 2Na-Tyr
4,5mM 2Na-P-Tyr
9mM 2Na-P-Tyr
0
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100
120
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1 3 5 7 9 11
time
Leb
en
sze
llzah
l [vc
/ml]
x1
0^
5
4,5mM 2Na-Tyr
4,5mM 2Na-P-Tyr
9mM 2Na-P-Tyr
35
Presentation Outline
CHO Cell Media & Feed – Performance evaluation 1
2 Lot to Lot Consistency
Platform Consistency 3
35
5 Powder handling improvements
Solubility Toolbox for high performing media 6
Perspectives 7
4 Media Performance Prediction
Perspectives in media preparation • The raw material quality is key for optimal
media performance on cellular level
• Raw material selection
• Raw material pre-processing
• Novel raw material development
• Supply chain
• The production process is directly linked with
batch to batch consistency and cellular
performance
• Milling process
• Mixing process
• Packaging
• The combination of raw materials and the
production process is mandatory for excellent dry
powder media performance
Raw material quality
Production process
Cellular performance
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