Cell line development: Reducing timelines and increasing titres by identification of host cell lines with improved characteristics. To develop a mammalian expression platform which rapidly leads to efficient, robust and high quality biomanufacturing processes
Cell Line Development: Reducing timelines and increasing titres
1. Cell line development: Reducing timelines and increasing titres by identification of host cell lines with improved characteristics
Dr Alison Porter, FUJIFILM Diosynth Biotechnologies
IBC Cell Line Development and Engineering, Berkeley, CA September 9, 2014
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2. FUJIFILM Diosynth Biotechnologies
EU and USA operations
Track record
~950 staff
Over 1000 cGMP batches manufactured
5 Commercial Products
FDA & MHRA Inspection history
Extensive LSS application
Contract Manufacturing
&
Process Development
Billingham (UK)
RTP (NC, USA)
3. Scope
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Aims and Objectives
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Host cell line development (CLD)
•
Directed evolution approaches
•
Results
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CLD run-through
•
Experimental design
•
Results
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Summary
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4. Aims and Objectives
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To develop a mammalian expression platform which rapidly leads to efficient, robust and high quality biomanufacturing processes
►
New host cell line
►
New vector
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Optimised cell line development process
►
Complementary medium and feed platform
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6. Desirable features/attributes for a host cell line
Adapted to CD media
Adapted to chemically defined (CD) media
Short doubling time
1 to 5 g/L titre for a model mAb
Suitable product quality
Rapid adaptation to production media
CHO-based
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7. How do we improve host cell functional capability?
CELL LINE ENGINEERING?
DIRECTED EVOLUTION?
•
Despite decades of research and a variety of different strategies to engineer CHO cells, outcomes remained mixed
•
May be a need to engineer multiple targets
•
May require a systems-based analysis for a tailor-made response
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Utilise functional heterogeneity within CHO cell populations to identify better host cell lines
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We already utilise this approach
•
Identify high producing recombinant clones during CLD
•
Adaptation to serum-free chemically defined media
•
Potential ability to manipulate multiple phenotypic outputs
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8. Isolate a cell line with more desirable attributes from a heterogeneous cell population
Approaches taken to obtain a host cell line with improved features/attributes
Adaptation to chemically defined conditions
Chemostat
Subculture regime
Cloning
FACS
Multiple rounds of sorting for cells with extended viability during batch shake flask screening
Allows for evolution of rare mutants with desirable growth properties within a large population
Continuous subculture (different regimes) and ability to grow in low [Gln] for lower [Amm] production
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10. 0.000.250.500.751.00050100150200 Growth rate (day-1)Days in culture
The long and winding road to CD media adaptation: The C2 story
120 rpm
110 rpm
140 rpm
Static
100% MEMα
+10%FBS
75% OptiCHO
80% OptiCHO
85% OptiCHO
90% OptiCHO
100% CD DG44
Clumping issues
3-day sub
3/4-day sub
0.2 x 106 seeding
0.3 x 106 seeding
0.2 x 106 seeding
1:100 anti-clump
1:700 anti-clump
No anti-clump
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Desirable growth rate for a recombinant cell line assuming:
•
Seed = 0.2 x 106/mL
•
VCD obtained = 2.0 x 106/mL
•
Culture duration = 4 days
•
Allows a 1:10 split ratio during seed train
11. ►
Assess growth and metabolite profiles during an 8-day batch shake- flask screen (at which point viability < 50%)
•
Indication of a cell line’s potential ability to grow during extended culture
•
Ideally have a limited accumulation of waste metabolites (e.g. ammonium and lactate)
Batch shake-flask screening of CD variants
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051015C1C2C32B-12B-2 IVC (x 106cell∙day/mL) 0102030C1C2C32B-12B-2 Metabolite Concentration (mM) AmmLac
12. 020406080C1C2C32B-12B-2 Transfection efficiency (%)
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Assess transfectability (using GFP) and overall expression/secretion capacity (using a model mAb)
•
Comparison of percentage GFP positive cells 24 hours after transfection
•
Comparison of secreted mAb concentration 5 days post-transfection
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Higher transfection efficiency and transient titre obtained with cell line C2
C2 progressed to directed evolution stages due to superior performance during evaluation
Higher transfection efficiency and transient titre obtained with cell line C2
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01234C1C2C32B-12B-2 Transient mAb expression (mg/L)
DG44-2B in ACF
13. Comparison of host cell lines obtained by directed evolution
Adapted to CD media
Growth rate during subculture
IVC obtained in batch shake- flask screen
Colony survival in semi-solid medium
Waste metabolite (Amm and Lac) production in batch shake- flask screen
Transient expression of mAb 1 and mAb 2
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18. 024681012141618 Clone 11Sort 5Sort 41 mMClone 272 mMStat 6Clone 56Sort 66 mMClone 26Stat 2Stat 5Stat 1Clone 59DG44-2B4 mMClone 78 mMC2Stat 4Stat 3Clone 46Clone 36Average Ranking
Average ranking used to select host cell lines to progress
Top 5 cell lines progressed
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19. Simultaneous comparison of host cell lines: Summary
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For each screen, cell lines were ranked according to their performance
•
The average ranking from all experiments gave an overall score for each host cell line
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Top ranked host cell line isolated from cloning of parental C2 host
•
Clone 11
►
Not all cell lines identified performed better than parental C2 host
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2 cell lines from the chemostat and cloning approaches ranked lower
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Performance of cell lines varied between the screens:
•
The chemostat derived cell lines ranked towards the top when measuring growth properties in shake-flasks
•
In contrast, these cell lines performed poorly when assessing growth in semi-solid medium, with only low numbers of colonies being produced
•
Demonstrates importance of assessing multiple characteristics
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Top 5 host cell lines, Clone-11, Sort-5, Sort-4, 1mM and Clone-27, were chosen to evaluate further in a ‘mini-pool’ assessment (representative of early stage stable cell line generation)
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20. Mini-pool evaluation of candidate host cell lines
1. Transfection
mAb 3
mAb 1
mAb 2
2. Removal of HT
3. Addition of selective pressure and plating into 96-well plates at 5000 cells/well
4. Screening of confluent colonies by OctetTM
5. Progression to 24- well plates
6. Screening by OctetTM
7. Confirmation of the top host cell line(s) for progression
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Experiment biased towards mAb 1 as mAb 2 and mAb 3 have previously proven to be “difficult-to-express”
21. 051015202530 mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3mAb 1mAb 2mAb 3 DG44-2BClone 11Clone 27Sort 4Sort 51 mM (in1mM GLN) 1 mM (in8mM GLN) 24wp titre (mg/L)
►
Rank results from mini-pool evaluation combined with previous ranking results to select the top 2 host cell lines
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Decision made to ensure cell lines from different directed evolution routes progressed to mitigate risk:
•
Sort-5 and Clone-27
•
DG44-2B included as a control
24-well plate screen titre
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23. Process map of the CLD run-though
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Transfection & Selection
96-well plate screen
Fed-batch shake-flask screen
ClonePix™ screen
24-well plate screen
Batch shake-flask screen
Stability study
ambr15™ & Bioreactor evaluation
Titre assessment used a generic antibody standard curve and Octet™ assay
Titre assessment used Octet™ or ProA HPLC & a specific antibody standard curve
•
Clone 27 and Sort 5 assessed in CLD run-through
•
New FDB expression vector used with the new host cell lines
•
Original host/vector system included as a comparator up to the RCB stage
•
Original system also uses different medium/feed system
Model mAb 1
Host cell line
Research cell bank
24. 24
CLD run-through: Transfection to recovery
Pools from Clone-27 host
recovery
Pools from Sort-5 host
Pools from DG44-2B host
recovery
recovery
Transfection
Host cell line
2
4
7
5
3
6
8
9
12
10
11
14
13
15
16
Day 0
1
Pools from the original host cell line (DG44-2B) had the longest recovery period
-HT
+ Selection reagent
25. CLD run-through: ClonePix™2 screening
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FITC1000 Exterior Mean Intensity
400003000020000100000
Clone-27
Sort-5
DG44-2B
Exemplar colony image
►
The fluorescence intensity and colony size were lower for the original host/vector system (DG44-2B)
26. CLD run-through: 96-well plate screen
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►
The highest titres achieved were from colonies originating from the Clone-27 host
►
A greater number of colonies from the original system had expression below the detection level; the max titre was considerably lower
>150 top colonies progressed
Clone-27
Sort-5
DG44-2B
0501001502002500100200300400500600700800 Titre (mg/L) 0501001502002500100200300400500600700800 Titre (mg/L) 0501001502002500100200300400500600700800 Titre (mg/L) Colony Number
27. 050100150200050100150200250 Titre (mg/L)
CLD run-through: 24-well plate screen
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Titre (mg/L)
>30 top colonies progressed
►
The highest titres achieved were from colonies originating from the Clone-27 host
►
Colonies from the original host/vector system had much lower titres
Clone-27
Sort-5
DG44-2B
050100150200050100150200250 Titre (mg/L)050100150200050100150200250Colony number
28. 28
CLD run-through: Suspension culture
Clone-27
Sort-5
DG44-2B
43210 1009080706050
VCD (x 106 cells/mL)
Viability (%)
Clone-27
Sort-5
DG44-2B
Viable cell density (VCD) and viabilities achieved during a routine subculture of the top 36 producers
Cell lines from DG44-2B host (original system):
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Difficulty in adaptation to suspension
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Lower VCD during routine subculture
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Several top ten producers in batch screen not suitable for progression to fed-batch screen due to poor growth
29. 29
CLD run-through: Batch shake-flask screen
Suspension adapted cell lines
*
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All recombinant cell lines from Clone-27 in the batch screen achieved titres above 300 mg/L
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One cell line from DG44-2B produced >300 mg/L; the majority produced <100 mg/L
Clone-27
Sort-5
DG44-2B
0100200300400500600 Titre (mg/L) 0100200300400500600 Titre (mg/L) 0100200300400500600 Titre (mg/L)***** * cell growth was not acceptable for progression
30. CLD run-through: Growth profiles from the fed-batch shake-flask screen
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►
Non-optimised process for Clone-27 and Sort-5
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A greater number of cell lines from Clone-27 host grew to >10 x 106 cells/mL; all cell lines were harvested on day 14
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Only one cell line from Sort-5 host achieved high VCD; several cell lines were harvested before day 14
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Cell lines from DG44-2B generally had poor growth profile and some were harvested as early as day 5
DG44-2B
Clone-27
Sort-5
Note: different medium/feed system required for DG44-2B
051015202502468101214 VCD (x 106cells/mL) Culture time (days) 051015202502468101214Culture time (days) 051015202502468101214Culture time (days)
31. ►
Lactate:
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Clone-27 and Sort-5 typically back-metabolise lactate
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Cell lines from DG44-2B generally reached higher lactate concentrations throughout culture
►
Ammonium:
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Harvest ammonium concentration for all cell lines from Clone-27 and Sort-5 were below 15 mM
•
Cell lines from DG44-2B generally had higher ammonium concentration throughout the culture and at harvest 31
CLD run-through: Further observations from the fed-batch shake-flask screen
32. 32
CLD run-through: Product concentration achieved from the fed-batch shake-flask screen
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A higher proportion of cell lines from Clone-27 had product titres ~2 g/L and above
►
Most cell lines from Sort-5 produced between 1 and 1.5 g/L
►
Productivity of cell lines from DG44-2B were considerably lower at <1 g/L
Cell lines
Clone-27
Sort-5
DG44-2B
0.00.51.01.52.02.53.0 Titre (g/L) 0.00.51.01.52.02.53.0 Titre (g/L) 0.00.51.01.52.02.53.0 Titre (g/L)
33. 33
CLD Run-through: Specific productivity from the fed-batch shake-flask screen
DG44-2B
Clone-27
Sort-5
►Despite lower titres, cell lines from Sort-5 had higher specific productivity (QP) - due to lower growth and IVC
►With the exception of one cell line, all cell lines from DG44-2B had QP less than 12 pg/cell·day
01020304050 Qp (pg/cell.day) 01020304050 Qp (pg/cell.day) 01020304050 Qp (pg/cell.day)
34. No substantial difference in product characteristics between cell lines from different hosts
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Size exclusion chromatography
Capillary electrophoresis-SDS (reduced)
N-linked glycans
Cation-exchange chromatography
Exemplar Overlay graph
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The majority of the product from all recombinant cell lines in monomeric form (≥98%)
Exemplar electropherogram
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Major peaks identified as LC and HC
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Very low percentage of non-glycosylated (~0.5%) and other variants.
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Low percentage of acidic and basic variants
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Lower percentage of main peak and additional basic peaks for DG44-2B* cell lines * Different medium/feed system
Basic peaks overlays
Cell lines from new hosts
Cell lines from DG44-2B
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Predominant glycan species did not change between host cell lines
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Man5 varied from 2 to 15% between cell lines
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Some minor differences with DG44-2B but this had different medium / feed system
•
Adapting all cell lines to this system removed the minor differences
Clone-27 derived cell line
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No considerable aggregation detected
CONFIDENTIAL
35. 35
Comparison of top producers in the ambr15TM screen
point size: Qp
►
The highest titre was achieved by a Clone-27 derived cell line
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3 g/L
►
Cell lines from Sort-5 generally had higher Qp but lower IVC
Sort-5
Clone-27 0.00.51.01.52.02.53.03.50500100015002000250030003500400045005000 Harvest Titre(g/L) IVC (x 106cells.h/mL)
36. 3.02.52.01.51.0 Product Concentration (g/L)
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Decision on lead host cell line
Box plot summary of key data for cell lines derived from Clone-27 and Sort-5
Clone-27
Fed-batch shake-flask
ambr15TM
Sort-5
Sort-5
200150100500
Qp (pcd)
4540353025201510
►
Clone-27 selected as lead host cell line:
►
Clone-27 cell lines achieved the highest titres and IVCs in both shake-flask and ambr15TM screens
►
Difference in QP reduced between Sort-5 and Clone-27 cell lines in ambr15TM
Clone-27
Fed-batch shake flask
ambr15TM
Fed-batch shake flask
ambr15TM
IVC (x 106 cells.d/mL)
Clone-27
Sort-5
Sort-5
Clone-27
Clone-27
Sort-5
Sort-5
Clone-27
37. 37
New hosts/vector system
Original host/vector system
Suitable PQ
Better growth
Higher Titres
More undesirable waste metabolites
Poor growth & productivity
CLD run-through: Summary
The new host/vector system performed better than the original system at every screening stage
Vector
Host
+
Suitable PQ
CLD run-through
38. ►
Four directed evolution approaches used to develop new host cell lines
•
FACS; cloning; chemostat; subculture regime
►
Multiple host cell lines with improved characteristics identified
•
Growth rate and expression capability
•
Need to look at multiple characteristics when assessing potential new host cell lines demonstrated
►
Top two potential host cell lines assessed in full CLD process
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Performance of both superior to original system, which had been included as a comparator
►
A final new host cell line was selected
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Product concentrations up to 3 g/L observed in ambr15™ using a non- optimised process
•
Improved growth characteristics
•
Acceptable product characteristics
Summary
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39. Summary - continued
►
Aim: To develop a mammalian expression platform which rapidly leads to efficient, robust and high quality biomanufacturing processes
►
New host cell line
►
New vector
►
Optimised cell line development process
►
Complementary medium and feed platform – under development
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40. ►
Adeline Bayard
►
Naz Dadehbeigi
►
Clare Lovelady
►
Leon Pybus
►
Fay Saunders
►
Alison Young
►
All members of the Mammalian Cell Culture R&D team – UK and USA
►
Analytical Development, FDB
Acknowledgements
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