Speeding up media design in cell culture

1. Speeding up media design: a novel high
throughput approach for rapid cell culture
media development
Cell Culture World Congress 2013, Munich, 26 February 2013
Authors: Arnaud Périlleux, Yolande Rouiller, Martin Jordan and Matthieu Stettler
Biotech Process Sciences, Upstream Development Group
Merck Serono S.A. Vevey, Switzerland

2. Merck Serono at a glance
 Merck Serono SA is the largest division
of Merck KGaA
– Established in January 2007,17’000 employees,
EUR 5.9 billion in 2011, headquarter in
Darmstadt, Germany
– In the United States and Canada, Merck Serono
operates under the name of EMD Serono (a
separately incorporated affiliate of Merck Serono)
 Merck Serono SA process development
and production site in Vevey,
Switzerland
– One of the largest and most technologically
advanced biotech centers in the world (4 x 5K and
8 x 15K production capacity)
– Production of Rebif® (INF beta-1a) since 1999
– Production of Erbitux® (Cetuximab) since 2011
Merck Serono’s headquarter in Darmstadt (top) and
development and production site in Vevey (bottom)
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Cell Culture World Congress | 26 February 2013

3. Presentation scope
 Overview of Merck Serono’s high throughput cell culture methods
 Case study #1: High throughput media blending
– How to obtain a new high performance medium in one single experiment?
 Case study #2: Product quality optimization
– How are quality analyses possible with micro-scale culture systems?
 Perspectives
– Strategies to quickly develop processes with strong quality requirements
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Cell Culture World Congress | 26 February 2013

4. Merck Serono’s HT cell culture methods
Overview
Integrated development approach
Cell line evaluation
Predictive, scalable and comprehensive set of tools
High throughput cell culture methods
for fed-batch processes assessing
24 to 400+ cultures in parallel
Medium development
High performance
cell lines
Media Blending
Feed Blending
DoE
Feed development
High performance
processes
CFD
Process development
Process validation
Quality by Design
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Cell Culture World Congress | 26 February 2013
Deliver the right
product quality

5. Merck Serono’s HT cell culture methods
High throughput evaluation of cell lines in fed-batch
Transfection and selection
A
B
C
D
Cloning in static 384 well plate
Adaptation in shaking 96DWP
A
Fed-batch in shaking 96DWP
Up to 500
clonal cell
lines
0.5 mL
0.5 mL
10 mL
Scale up to shake tubes
B
Fed-batch in shake tubes
C
5
Fed-batch in lab-scale bioreactors
Cell Culture World Congress | 26 February 2013
30 mL
15 mL
Fed-batch in micro-scale bioreactors
D
12 cell lines
4 candidate
cell lines
3 000 mL

6. Case study #1: High throughput media blending
A high-throughput media design approach for high
performance mammalian fed-batch cultures
Authors:
Yolande Rouiller, Arnaud Périlleux, Natacha Collet,
Martin Jordan, Matthieu Stettler, Hervé Broly
Manuscript accepted, to be published in mAbs

7. Case study #1: High throughput media blending
Workflow
47 components
16 media formulations
varying 43 out of 47 components
Blends automatically
mixed in 96DWP
Predicted vs. Actual
3 passages prior
a 14-days fed-batch
Titer (mg/L)
Data
analysis
376 media
blends tested
3000
2000
1000
0
0 2 4 6 8 101214
Elapsed time (days)
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Data
acquisition

8. Case study #1: High throughput media blending
 Protocol
– 3 passages prior fed-batch inoculation, an
antibody expressing cell line is diluted in
each of the 376 blends
• Guaranty to obtain a medium suitable for both
expansion and fed-batch process
Viable Cell Density
(x106 cells/mL)
Evaluation of 376 media blends
– Performance assessment
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20
15
10
Titer (mg/L)
2
4
6
8 10 12 14
2
4
6
8 10 12 14
2500
2000
1500
1000
500
• Cell count, Cell viability with a Guava Easycyte
• Titer quantification with an Octet
Fed-batch process
0
-6
3500 -8Ctrl 1-4 -2 0
3000 Ctrl 2
• Standardized and controlled experimental
setup
• Guaranty to obtain a medium adapted to the
feed system
Cell expansion
3 passages
5
– Each of the 376 cultures is diluted individually
targeting a specific cell density
– On day 2, 4, 7 and 10, a reference proprietary
feed is added
25
KQe
0
-8 -6 -4 -2 0
Time (days)

9. Case study #1: High throughput media blending
Data analysis opportunities
Data analysis using three approaches
1st approach: Excel spreadsheet
Ranking of various tested
conditions
Identification of key media
formulations
Identification of key components
Selection of promising
formulations
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2nd approach: DoE (Design
Expert)
Design of predicted best
formulation
List of key components to be
further optimized
Cell Culture World Congress | 26 February 2013
3rd approach: MVA (Simca P++)

10. Case study #1: High throughput media blending
Data analysis with Design of Experiment (DoE)
Titer at Day 14
Predicted values
 DoE analysis allows
– To identify the key formulations (out of the 16)
– To design new media formulations
 DoE analysis does not allow
– To understand why some media are better than others
3000
2000
1000
R2 = 0.88
Adj. R2 = 0.84
Pred. R2 = 0.76
0
0
10
Prediction 1
Prediction 4
Prediction 2
Prediction 5
Predicted Values
Prediction 3
Average
Prediction PDL
F16
F15
F14
F13
F12
F11
F10
F9
F8
F7
F6
F5
10.47 238
3933
10.42 226
3908
10.45 230
3902
5th
F4
3960
4th
F3
10.43 226
3rd
F2
Titer
2nd
Cell Culture World Congress | 26 February 2013
IVC
1st
35%
30%
25%
20%
15%
10%
5%
0%
F1
% of each formulation
in the predicted medium
Compositions of best predicted media based on the 16 formulations
1000 2000 3000
Observed values
10.39 224
3885
Average
10.43 228
3910

11. Case study #1: High throughput media blending
Data analysis with MultiVariate Analysis (MVA)
 MVA analysis allows
– To rank the 43 components in terms of influence on performance
– To identify the key components that could be interesting for further optimization (in orange and yellow)
 MVA analysis does not allow
– To have a strong confidence in the conclusions due to the relatively low number of conditions
tested compared to the high number of factors evaluated
Components with strong influence in MVA
Components that correlate by design with key components
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Titer Day 14 (mg/L)
0.4
0.3
0.2
0.1
0
-0.1
-0.2
-0.3
L-Serine
D-Biotin
L-Arginine
Thymidine
L-Aspartic acid
L-Leucine
L-Glutamic acid
Hypoxanthine
Zinc Sulfate
myo-Inositol
NaH2PO4
L-Histidine
Sodium Selenite
Putrescine
L-Tyrosine
Riboflavin
Choline Chloride
Pyridoxine
L-Lysine x HCl
L-Phenylalanine
L-Isoleucine
Calcium Chloride
Folic acid
Vitamin B12
Thiamine
Pluronic
Ethanolamine
L-Aspargine
Cupric sulfate
L-Cysteine
Niacinamide (B3)
Glycine
L-Threonine
L-Tryptophan
L-Proline
Magnesium Sulfate
L-Alanine
L-Methionine
Sodium pyruvate
Potassium Chloride
L-Valine
D-Pantothenic acid x 1/2Ca
Ferric ammonium citrate
Coefficients scaled & centered
(components 1 to 3 of PLS model)
Influence of increased levels of the tested components in the PLS regression of titers at day 14
Ferric Ammonium Citrate (mg/L)

12. Case study #1: High throughput media blending
Scale-up, confirmation and conclusions
Reference New
medium medium
Cell line 1
Cell line 2
Cell line 3
 8 media from the ranking approach and 1
from the DoE approach were scaled up in
shake tubes
 1 medium was selected for scale up in
bioreactor and evaluated on several cell lines
– The 3 cell lines showed from 30 to 60% titer
improvement
Titer (mg/L)
– data not shown
6000
5000
4000
3000
2000
1000
0
 Conclusions
0
2
4
6 8 10 12 14 16 18
Time (days)
– Media blending allows in one single experiment to identify new media formulations with high
potential for performance improvement
– Same approach can be made with feeds
– An approach combining medium and feed blending can be designed
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Cell Culture World Congress | 26 February 2013

13. Case study #2: Product quality optimization
using high-throughput cell culture methods

14. Case study #2: Product quality optimization
Are quality analyses possible with micro-scale cultures?
 Context
– Cell culture process development required to optimize a large number
of critical quality attributes (CQAs)
• Isoforms (deamidation, oxydation, …), glycoforms, (galactosylation,
mannosylation, fucosylation, sialylation), product integrity, process
impurities
– Most of the CQAs are linked to the recombinant cell line, the medium
and feeds, and the process (physical parameters)
– To ensure a successful process development (e.g. next generation
process), it is important that the new process provides a product with
the same quality
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Cell Culture World Congress | 26 February 2013

15. Case study #2: Product quality optimization
Are quality analyses possible with micro-scale cultures?
 Challenges
– High throughput cell culture methods (i.e.
96DWP) provide small amounts of product
Fed batch process in
96DWP
400-500 µL
225-1500 µg
– Analytical lab should be able to analyze 400+
samples with 5+ analytical methods
• Capture, glycan analysis, charge profile,
integrity, …
 Achievements
– A combination of media blending and DoE
generated 400 samples of about 400 µL
Capture on Phytips®
Charge profile
iCE280
Glycan analysis
CGE-LIF
– Samples were captured using Phytips®
– Samples were analyzed in 2.5 weeks on 5
analytical methods
– 8800 results were generated
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Integrity
SE-HPLC
Caliper NR

16. Case study #2: Product quality optimization
Experiment design
Factors tested in DoE
 Combine media blending and DoE design
Zn
N-acetylcystein
Cu
pH  HCl
Fe
NaCl
Se
NaButyrate
Mn
Hydrocortisone
Galactose
Spermine
Ascorbic acid + Retinol
+ 4-aminobenzoic acid
+ a-tocopherol
Lipids
– 5 new Basal Media (BM) mixed to obtain 11 media
– 17 factors tested in DoE by addition on day 5
– Standard platform feeding strategy
– Analytics performed on day 14
• Biacore
• CGE-LIF, iCE280
• Caliper NR, SE HPLC
Fucose
Uridine
ManNAc
Additional factors
2ndary feed
Main Feed
Glucose
0
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3
5
7
10
14

17. Case study #2: Product quality optimization
Results
– After optimization, the confirmation and
scale up of a new manufacturing process
version was performed
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Cell Culture World Congress | 26 February 2013
Model: R2 = 74.9% / R2 adj. = 71.2%
BM4
• to select 4 factors and 2 media for further
optimization
32 DoE conditions
Mix1
• to fix the levels of 4 factors
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
Spermine
– Models allow
Std. Coef. (Cluster 4)
– For most of the CQAs, at least some
conditions were able to match the TPP
Control (n=4)
BM3
 Conclusions
Mix5
10
Mix4
– Lists of key media and factors were
established from models
15
NaCl
– Models were defined
20
All data
BM1
BM2
BM3
BM4
BM5
Mix1
Mix2
Mix3
Mix4
Mix5
Mix6
BM1
BM2
BM3
BM4
BM5
– Distribution of results was compared to
Target Product Profile (TPP)
25
Cu
 For each CQA
Charge Profile
Cluster 4 (%)
30

18. HT cell culture – Speeding up media design
Conclusions
 High throughput tools are now available for cell culture, but also
for purification and analytics
 Designing the right experiments allows to get the maximum of
these tools and to reduce development timelines
 Their use has been mainly directed to media design, but their
application to feed design could even provide more interesting
results
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Cell Culture World Congress | 26 February 2013

19. Acknowledgements
 Biotech Process Sciences
(BPS), Merck Serono SA
Vevey, Switzerland
– Hervé Broly - Head of BPS
– Upstream Development Group
– Flavie Robert - Head of Analytical
Group
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20. Questions
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Cell Culture World Congress | 26 February 2013