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Mammalian Cell Culture: Current Status, Future Prospects
Cell Culture and Upstream Processing, Berlin, September 2004
John BirchLonza
slide 2Author / Date
Cell Culture: an Industrial Process
Much of the early process development driven by vaccines ( human and veterinary ) – Polio vaccine 1954
FMD vaccine in deep tank suspension culture
Since late 1980s (tPA 1987), large scale use driven particularly by recombinant therapeutic proteins
slide 3Author / Date
Significance of Mammalian Cells
Used especially for large complex proteins especially those requiring posttranslational modifications – in practice a large proportion of the totalApprox. 73 licensed proteins ( USA ) of which ca. :35 produced in micro organisms : E.coli ( 28 ) , S.cerevisiae ( 7 )38 ( 52% ) produced in mammalian cells
CHO 27 ( 10 Mabs )NS0, SP2/0,hybridomas etc 8 ( 8 Mabs )BHK 2C127 1
slide 4Author / Date
Biopharmaceutical Proteins
Strong demand – sales of biopharmaceuticals represent 10-30% of all new pharmaceuticals in USA in recent years(1)Large number ( hundreds ) of proteins in developmentSales of ca.$ 30bn in 2003 (2)expected to grow to $59bn by 2010 ( Datamonitor )Mammalian cell products account for ca. 60% of market (1)Mabs are the fastest growing category ( from 1% of biopharmaceutical market in 1995 to 14% by 2001 )(1)
1. Polastro & Tulcinski, Scrip magazine Sept. 2002 2. Walsh, Nature Biotechnology 2003, 21, 865 – 870
slide 5Author / Date
Key Issues in Manufacturing (1)
Speed in development (especially for early phase clinical material)
Rapid cell line creation – avoid amplification and multiple rounds of single cell cloningUse of cell lines (CHO) preadapted to suspension cultureUse of generic technologyPredictive scale up systems
slide 6Author / Date
Key Issues in Manufacturing (2)
High dose requirements, particularly for antibodies, leads to large volume demand (10’s to 100’s kg/year )Estimated 2004 protein demand > 2000kg (mostly Mabs and fusion proteins )( UBS )This is a driver for –
Increased capacity Increased reactor size – up to 20000 litres (economy of scale )Improved technology to increase cost efficiency ( taking note of downstream implications )
slide 7Author / Date
Mammalian Cell Capacity
Long lead times (3-5 years ) and high costs ($200m - $500m)($3+ Million / m3)
510 ( 26%)19702006
190 ( 23%)8402002
Contract Manufacturers
Total
Estimated capacity
( thousands of litres )
slide 8Author / Date
20,000L Bioreactor & Add TanksPortsmouth, New Hampshire
slide 9Author / Date
Improved Upstream ProcessEfficiency
Parameters TargetedReactor throughputSpecific production rateMaximum viable cell concentrationProlongation of culture at high cell viabilityGrowth rate
Routes to optimisationDesign of gene vectorProperties of host cell lineCell line screeningOptimisation of fermentation process
slide 10Author / Date
Design of Gene Vector to Maximize Transcription
Strong promoter to drive expression of product gene(s)
Amplification of gene copy number
Vectors with elements that create genomic environment for high transcriptional activity (positional independence)
Targeting of expression vector to transcriptionally active site in genome by homologous recombination
slide 11Author / Date
Glutamine synthetase (GS) gene expressionsystem
Expression vector encoding product gene plus GS gene, allowing glutamine synthesisGS is inhibited by methionine sulphoximine (MSX)Selection in glutamine-free medium for GS minus cell types (e.g. NS0)Selection in the presence of MSX for GS positive cell types (e.g. CHO)Only cells with GS gene (and hence product gene) surviveIncrease selection stringency - use weak promoter on GS gene - selects for rare integration into transcriptionally efficient sites in genomeExpression of linked product gene, driven by strong promoter, enhanced by favourable integration site
slide 12Author / Date
Improved Upstream ProcessEfficiency
Parameters TargetedReactor throughputSpecific production rateMaximum viable cell concentrationProlongation of culture at high cell viabilityGrowth rate
Routes to optimisationDesign of gene vectorProperties of host cell lineCell line screeningOptimisation of fermentation process
slide 13Author / Date
Properties of The Host Cell Line
Significant opportunities to create cell lines with improved growth and productivity characteristics
Metabolic engineering
Variant SelectionCholesterol independent NS0 variantSuspension variants of CHO
slide 14Author / Date
Improved Upstream ProcessEfficiency
Parameters TargetedReactor throughputSpecific production rateMaximum viable cell concentrationProlongation of culture at high cell viabilityGrowth rate
Routes to optimisationDesign of gene vectorProperties of host cell lineCell line screeningOptimisation of fermentation process
slide 15Author / Date
Cell Line Screening
Highly productive transfectants are rare even with a good selection system
Various approaches to improve screening process to find these rare events
Increase transfection efficiency ( larger pool to select from )Improve stringency of selectable marker to eliminate lowproducersHigh throughput methods (FACS + cell surface product capture)Early screening needs to predict cell specific production capability AND growth characterisitics at production scale
slide 16Author / Date
Cell line selection
Transfection and selection conditions for GS-CHO cell lines expressing cB72.3 antibody
1973
1242
681
Numbers of stable
transfectants
Electroporation condition
2.5 x 106 cells electroporated
slide 17Author / Date
Cell Line Selection
Influence of Selection Conditions for GS-CHO Cell Lines making cB72.3 Antibody
Cell lines have not been amplified.
0
2
4
6
8
10
12
14
16
40 80 120 160 200 240 280 320
Antibody (mg/L)
25 µM MSX
50 µM MSX
slide 18Author / Date
secreted antibody
fluorochrome-labelled detection antibody
neutravidinbridge
biotinylated Protein A
biotinylated-cell surface
Affinity-matrix surface capture
slide 19Author / Date
Flow cytometric analysis of AMSC-labelled GS-CHO cells
slide 20Author / Date
Clonal Variation – GS-NS0 (non-optimised culture )
60
31
24
19
19
17
Specific prod.rate (pg/cell/day)
320
573
518
212
209
379
Cumulative cell time
(109 cell·h/L)
79014
75019
53522
17019
1658
26017
Product concn.(mg/l)
Max.viable cell concn. (105 /ml)
slide 21Author / Date
Selecting high producing cell lines
Transfect 107 host cells with vector
30-60 transfectants
30-60 transfectants
productivity assessment(quantitative)
Preliminary productivity assessment (quantitative)
STATIC CULTURE
SUSPENSION(shake flask culture)
Select 3 cell lines for further analysis
200-300 transfectants
Adapt to protein-free medium
6 week
4 weeks
8 weeks
3 weeks
5-10 transfectantsFed-batch assessment of
growth, productivity, product quality3 weeks
Single colonies per well
slide 22Author / Date
Prediction of bioreactor behaviour from shake-flask model
0.0
0.4
0.8
1.2
1.6
2.0
antibody Qp
Parameter
Valu
e in
reac
tor r
elat
ive
to s
hake
-flas
k
slide 23Author / Date
Improved Upstream ProcessEfficiency
Parameters TargetedReactor throughputSpecific production rateMaximum viable cell concentrationProlongation of culture at high cell viabilityGrowth rate
Routes to optimisationDesign of gene vectorProperties of host cell lineCell line screeningOptimisation of fermentation process
slide 24Author / Date
Improving the Fermentation Process
Significant potential to improve processes
Physicochemical environment
Medium design and feeding strategies ( most processes are fed-batch, some perfusion culture )
Use of chemically defined media
slide 25Author / Date
The Physicochemical Environment
Control pH, temperature, dissolved oxygen concentrationSmall changes in pH can have profound effect on cell growth and productivity
Responses are cell line specific and can impact:
Maximum cell concentrationIntegral viable cell hoursSpecific production rateMetabolism: lactate accumulation
slide 26Author / Date
1
10
100
0 50 100 150 200 250 300 350 400
Time (hours)
Viab
le c
ell c
once
ntra
tion
(105 /m
L)
0
0.3
0.6
0.9
1.2
1.5
Lact
ate
conc
entr
atio
n (g
/L)
pH 7.3 pH 7.0
slide 27Author / Date
Chemically Defined Media
Increasing use of chemically defined media free of animal derived raw materials
Reduced risk of introducing adventitious agentsImproved process consistency and robustness (avoids potential variability of raw materials such as serum proteins and hydrolysates)Chemical definition assists process optimisationBenefits purification (reduced contaminant load)
slide 28Author / Date
Downstream Benefits of Chemically Defined Medium for GS-NS0 Cell Line
Purity of MAb at harvest
Optimised Protein containing <30%culture
Optimised protein free culture 62%-76%
slide 29Author / Date
Medium Design and Feeding Strategies
Optimise basal medium
Optimise feeds
Maintain nutrient sufficiency
Minimise waste metabolite formation
Use of GS system avoids accumulation of ammonium ions from metabolism of glutamine
slide 30Author / Date
Optimisation of a GS-CHO Process
Culture conditions for a GS-CHO making cB72.3 antibody were optimisedSuspension variant of CHO-K1 isolated:
grows in chemically defined medium without need for adaptation (can take several months)
Efficiency and stringency of transfection conditions increased to improve selection of highly productive clonesGrowth conditions further optimised
slide 31Author / Date
Process optimisation for a GS-CHO cell line
4301
3560
2829
1917
585
334
139
Antibody(mg/L)
26Iteration 4
31
20
14
4
2
Fold increase
Iteration 5
Iteration 3
Original cell line
New cell line
Iteration 2
Iteration 1
Process
slide 32Author / Date
GS-CHO: antibody production
P rocess developm ent s tage22H 11 orig22H 11 v1 22H 11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5
Antib
ody
(mg/
L)
0
1000
2000
3000
4000
5000
22H 11 orig22H 11 v1 22H 11 v2 LB01 v2 LB 01 v3 LB01 v4 LB01 v5Spec
ific
prod
uctio
n ra
te (p
g /(c
ell.h
))
0.0
0.5
1.0
1.5
2.0
slide 33Author / Date
GS-CHO growth
P r o c e s s d e v e lo p m e n t s t a g e2 2 H 1 1 o r ig 2 2 H 1 1 v 1 2 2 H 1 1 v 2 L B 0 1 v 2 L B 0 1 v 3 L B 0 1 v 4 L B 0 1 v 5
Viab
le c
ell c
once
ntra
tion
(106 /m
L)
0
2
4
6
8
1 0
1 2
1 4
1 62 2 H 1 1 o r ig 2 2 H 1 1 v 1 2 2 H 1 1 v 2 L B 0 1 v 2 L B 0 1 v 3 L B 0 1 v 4 L B 0 1 v 5
IVC
(106 c
ells
.h/m
L)
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 02 2 H 1 1 o r ig 2 2 H 1 1 v 1 2 2 H 1 1 v 2 L B 0 1 v 2 L B 0 1 v 3 L B 0 1 v 4 L B 0 1 v 5
Proc
ess
dura
tion
(day
)0
5
1 0
1 5
2 0
2 5
slide 34Author / Date
Growth comparison: “old” vs. “new” GS-CHO cell lines
0
40
80
120
0 100 200 300 400
Time (h)
Viab
le c
ell c
once
ntra
tion
(10
5 /mL)
22H11LB01
slide 35Author / Date
Process optimisation for a model GS-NS0
1405
1427
1239
1026
772
640
Cumulative cell time
(109 cell·h/L)
1422
1035
807
589
293
476
cB72.3 antibody(mg/L)
0.97Iteration 4
0.71Iteration 3
0.64Iteration 2
0.60Iteration 1
0.36Chemically defined
0.74Serum-free
Qp
pg/(cell·h)Process
slide 36Author / Date
Future Opportunities ( 1 )
Continued process improvements ( 10g/l achievable ?)
Continued improvements in selection procedures for finding highly productive clones
Improved cell lines e.g. engineered for improved growth, product synthesis, energy metabolism , glycosylation characteristics
Use of genomic and proteomic approachesto increase our knowledge of cell physiology and to inform design of cells and processes
slide 37Author / Date
Future Opportunities (2)
Improved product potency – impact on quantities required
Alternative manufacturing routes – microbial production of e.g. antibody fragments or whole antibodies, transgenic production for very large quantities ?
Mammalian cell culture likely to be key technology for foreseeable future
slide 38Author / Date
Summary
Increasing number of mammalian cell products in development is a driver for improved and faster process development technologiesHigh volume demands are driving both capacity and improvements in process efficiencyGreat potential to use advances in basic science to inform process improvements