Reducing Timelines & Increasing Titres by Identification of Host Cell Lines with Improved Characteristics

1. Cell line development: Reducing timelines
and increasing titres by identification of
host cell lines with improved characteristics
Dr Alison Porter, Fujifilm Diosynth Biotechnologies, UK
Cell Line Development and Engineering,
09 September 2014, Berkeley CA
1

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
RTP (NC, USA)
Contract Manufacturing
&
Process Development
Billingham (UK)

3. Scope
►Aims and Objectives
►Host cell line development (CLD)
• Directed evolution approaches
• Results
►CLD run-through
• Experimental design
• Results
►Summary
3

4. Aims and Objectives
►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
4

5. 5
Host cell line development

6. Desirable features/attributes for a host cell line
Adapted to
chemically
defined (CD)
media
Adapted to
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
6

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
• Utilise functional heterogeneity
within CHO cell populations to
identify better host cell lines
• 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
7

8. Approaches taken to obtain a host cell line
with improved features/attributes
Isolate a cell line
with more desirable
attributes from a
heterogeneous cell
population
Adaptation
to
chemically
defined
conditions
Chemostat
Subculture
regime
FACS Cloning
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
8

9. Derivation of multiple DG44 variants able to grow
in chemically defined (CD) culture medium
Static culture
100% MEM-α
(+10% FBS)
Suspension
100% MEM-α
(+10% FBS)
50% CD-DG44
50% MEM-α
(+10% FBS)
75% CD-DG44
25% MEM-α
(+10% FBS)
90% CD-DG44
10% MEM-α
(+10% FBS)
C1
100% CD-DG44
Suspension
75% OPTI-CHO
25% MEM-α
(+10% FBS)
80% OPTI-CHO
20% MEM-α
(+10% FBS)
90% OPTI-CHO
10% MEM-α
(+10% FBS)
C2
100% CD-DG44
C3
100% CD-DG44
75% CD-DG44
25% OPTI-CHO
50% CD-DG44
50% OPTI-CHO
9

10. The long and winding road to CD media
adaptation: The C2 story
1:100 anti-clump 1:700 anti-clump No anti-clump
0.2 x 106 seeding 0.3 x 106 seeding 0.2 x 106 seeding
3/4-day sub 3-day sub
85% OptiCHO
Clumping issues
Static 110 rpm 120 rpm 140 rpm
1.00
0.75
0.50
0.25
0.00
0 50 100 150 200
Growth rate (day-1)
Days in culture
100% MEMα
+10%FBS
75% OptiCHO
80% OptiCHO
90% OptiCHO 100% CD DG44
Desirable growth rate
for a recombinant cell
line assuming:
• Seed = 0.2 x 106/mL
• VCD obtained = 2.0 x
10
106/mL
• Culture duration = 4
days
• Allows a 1:10 split
ratio during seed
train

11. Batch shake-flask screening of CD variants
15
10
5
30
20
10
► 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)
11
0
C1 C2 C3 2B-1 2B-2
IVC
(x 106 cell∙day/mL)
0
C1 C2 C3 2B-12B-2
Metabolite
Concentration
(mM)
Amm
Lac

12. Higher transfection efficiency and transient
titre obtained with cell line C2
80
60
40
20
0
C1 C2 C3 2B-1 2B-2
Transfection
efficiency (%)
4
3
2
1
► 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
► Higher transfection efficiency and transient titre obtained with cell line C2
C2 progressed to directed evolution stages due to superior
performance during evaluation
12
0
C1 C2 C3 2B-1 2B-2
Transient mAb
expression (mg/L)
DG44-2B in ACF

13. Comparison of host cell lines obtained by
directed evolution
Growth rate
Adapted to
CD media
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
13

14. 0
5
10
15
20
25
30
DG44-2B
C2
Sort-1
Sort-2
Sort-3
Sort-4
Sort-5
Sort-6
Clone-7
Clone-11
Clone-26
Clone-27
Clone-36
Clone-46
Clone-56
Clone-59
Stat-1
Stat-2
Stat-3
Stat-4
Stat-5
Stat-6
8 mM
6 mM
4 mM
2 mM
1 mM
Doubling time (h)
Doubling time during subculture
14
► Improved doubling time with C2 compared to DG44-2B: ~27hrs to ~21hrs
► Directed evolution approaches had little effect on doubling time

15. 0
2
4
6
8
10
12
14
DG44-2B
C2
Sort-1
Sort-2
Sort-3
Sort-4
Sort-5
Sort-6
Clone-7
Clone-11
Clone-26
Clone-27
Clone-36
Clone-46
Clone-56
Clone-59
Stat-1
Stat-2
Stat-3
Stat-4
Stat-5
Stat-6
8 mM
6 mM
4 mM
2 mM
1 mM
Batch shake-flask screen harvest IVC
(x 106 cell∙day/mL)
IVC obtained during batch shake-flask screening
15
► Potential to increase IVC from directed evolution approaches
► Higher IVC for DG44-2B may be media dependent (presence of hydrolysate)

16. 0
1
2
3
DG44-2B
C2
Sort-1
Sort-2
Sort-3
Sort-4
Sort-5
Sort-6
Clone-7
Clone-11
Clone-26
Clone-27
Clone-36
Clone-46
Clone-56
Clone-59
Stat-1
Stat-2
Stat-3
Stat-4
Stat-5
Stat-6
8 mM
6 mM
4 mM
2 mM
1 mM
mAb 2 transient
expression (mg/L)
mAb 1 transient
expression (mg/L)
0
2
4
6
8
10
12
DG44-2B
C2
Sort-1
Sort-2
Sort-3
Sort-4
Sort-5
Sort-6
Clone-7
Clone-11
Clone-26
Clone-27
Clone-36
Clone-46
Clone-56
Clone-59
Stat-1
Stat-2
Stat-3
Stat-4
Stat-5
Stat-6
8 mM
6 mM
4 mM
2 mM
1 mM
Transient expression
16
► Improved titre obtained
from C2 compared to
DG44-2B
► Directed evolution had
variable effect: up to 3-fold
increase compared to
DG44-2B

17. % Colony survival in semi-solid media
0
20
40
60
80
100
120
DG44-2B
C2
Sort-4
Sort-5
Sort-6
Clone-7
Clone-11
Clone-26
Clone-27
Clone-36
Clone-46
Clone-56
Clone-59
Stat-1
Stat-2
Stat-3
Stat-4
Stat-5
Stat-6
8 mM
6 mM
4 mM
2 mM
1 mM
Growth in semi-solid media
17
► Improved colony survival obtained from C2 compared to DG44-2B
► Directed evolution approaches have a variable effect on colony survival

18. 0
2
4
6
8
10
12
14
16
18
Clone 11
Sort 5
Sort 4
1 mM
Clone 27
2 mM
Stat 6
Clone 56
Sort 6
6 mM
Clone 26
Stat 2
Stat 5
Stat 1
Clone 59
DG44-2B
4 mM
Clone 7
8 mM
C2
Stat 4
Stat 3
Clone 46
Clone 36
Average Ranking
Average ranking used to select host cell lines
to progress
Top 5 cell
lines
progressed
18

19. Simultaneous comparison of host cell lines:
Summary
► 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
► 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
• 2 cell lines from the chemostat and cloning approaches ranked lower
► 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
► 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)
19

20. Mini-pool evaluation of candidate host cell lines
1. Transfection
mAb 1
mAb 2
mAb 3
2. Removal of HT
3. Addition of selective pressure
and plating into 96-well plates at
5000 cells/well
5. Progression to 24-
well plates
4. Screening of
confluent colonies
by OctetTM
6. Screening
by OctetTM
7. Confirmation of
the top host cell
line(s) for
progression
20
Experiment biased towards
mAb 1 as mAb 2 and mAb 3
have previously proven to be
“difficult-to-express”

21. 24-well plate screen titre
30
25
20
15
10
5
0
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
mAb 1
mAb 2
mAb 3
DG44-2B Clone 11 Clone 27 Sort 4 Sort 5 1 mM (in
1mM GLN)
1 mM (in
8mM GLN)
24wp titre (mg/L)
► Rank results from mini-pool evaluation combined with previous ranking results
to select the top 2 host cell lines
► 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
21

22. Cell Line Development
Run-Through
22

23. Process map of the CLD run-though
23
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
Transfection
recovery
Pools from
Sort-5 host
Pools from
DG44-2B host
recovery
recovery
Host cell line
Day 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Pools from the original host
cell line (DG44-2B) had the
longest recovery period
-HT
+ Selection
reagent

25. CLD run-through: ClonePix™2 screening
25
FITC1000 Exterior Mean Intensity
Apollo Alternative Host Cell Line DG44-2B
40000
30000
20000
1 0000
0
FITC1 000 Exterior Mean Intensity
Clone-27 Sort-5 D G44-2B
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
26
► 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
DG44-2B Sort-5 Clone-27
250
200
150
100
50
0
0 100 200 300 400 500 600 700 800
Titre (mg/L)
250
200
150
100
50
0
0 100 200 300 400 500 600 700 800
Titre (mg/L)
250
200
150
100
50
0
0 100 200 300 400 500 600 700 800
Titre (mg/L)
Colony Number

27. CLD run-through: 24-well plate screen
200
150
100
50
200
150
100
50
0
0 50 100 150 200 250
Titre (mg/L)
27
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
DG44-2B Sort-5 Clone-27
0
0 50 100 150 200 250
Titre (mg/L)
200
150
100
50
0
0 50 100 150 200 250
Colony number

28. 28
CLD run-through: Suspension culture
Viable cell density (VCD) and viabilities achieved during a routine
subculture of the top 36 producers
Clone-27 Sort-5 DG44-2B
4
3
2
1
0
VCD (x1E6)
100
90
80
70
60
50
Viability (%)
Viability (%)
VCD (x 106 cells/mL)
Clone-27 Sort-5 DG44-2B
Cell lines from DG44-2B host (original system):
► Difficulty in adaptation to suspension
► Lower VCD during routine subculture
► 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
► All recombinant cell
lines from Clone-27 in
the batch screen
achieved titres above
300 mg/L
► One cell line from
DG44-2B produced
>300 mg/L; the
majority produced
<100 mg/L
DG44-2B Sort-5 Clone-27
600
500
400
300
200
100
0
Titre (mg/L)
600
500
400
300
200
100
0
Titre (mg/L)
600
500
400
300
200
100
0
Titre (mg/L)
* * * * *
* cell growth was not
acceptable for progression

30. CLD run-through: Growth profiles from the
fed-batch shake-flask screen
30
Clone-27 Sort-5 DG44-2B
25
20
15
10
5
25
20
15
10
5
25
20
15
10
5
► Non-optimised process for Clone-27 and Sort-5
► 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
► Only one cell line from Sort-5 host achieved high VCD; several cell lines were
harvested before day 14
► Cell lines from DG44-2B generally had poor growth profile and some were
harvested as early as day 5
Note: different
medium/feed system
required for DG44-2B
0
0 2 4 6 8 10 12 14
VCD (x 106 cells/mL)
Culture time (days)
0
0 2 4 6 8 10 12 14
Culture time (days)
0
0 2 4 6 8 10 12 14
Culture time (days)

31. ►Lactate:
• Clone-27 and Sort-5 typically back-metabolise lactate
• Cell lines from DG44-2B generally reached higher
lactate concentrations throughout culture
►Ammonium:
• 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
► 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
DG44-2B Sort-5 Clone-27
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Titre (g/L)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Titre (g/L)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Titre (g/L)

33. 33
CLD Run-through: Specific productivity
from the fed-batch shake-flask screen
DG44-2B Sort-5 Clone-27
► 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
50
40
30
20
10
0
Qp (pg/cell.day)
50
40
30
20
10
0
Qp (pg/cell.day)
50
40
30
20
10
0
Qp (pg/cell.day)

34. No substantial difference in product characteristics
between cell lines from different hosts
34
Size exclusion
chromatography
Capillary
electrophoresis-SDS
(reduced)
•The majority of the
product from all
recombinant cell lines in
Exemplar Overlay
monomeric form (≥98%) Exemplar
graph
Cation-exchange N-linked glycans
chromatography
electropherogram
•Major peaks identified as
LC and HC
•Very low percentage of
non-glycosylated (~0.5%)
and other variants.
•Low percentage of acidic
and basic variants
•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
• Predominant glycan
species did not change
between host cell lines
• Man5 varied from 2 to
15% between cell lines
•Some minor differences with DG44-2B but
this had different medium / feed system
Clone-27 derived cell line
• Adapting all cell lines to this system
removed the minor differences
•No considerable aggregation detected
CONFIDENTIAL

35. 35
Comparison of top producers in the ambr15TM
screen
point size: Qp
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
IVC (x 106 cells.h/mL)
Clone-27 Sort-5
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Harvest Titre (g/L)
► The highest titre was achieved by a Clone-27 derived cell line
• 3 g/L
► Cell lines from Sort-5 generally had higher Qp but lower IVC

36. 3.0
2.5
2.0
1 .5
1 .0
Product Concentration (g/L)
ambr15TM
Boxplot of IVC data from Dolly and Phoenix in fed shake-flasks and Ambr
36
Decision on lead host cell line
Box plot summary of key data for cell lines derived from Clone-27 and Sort-5
Fed-batch shake-flask ambr15TM
Clone-27
Sort-5 Sort-5
Dolly FOG IVC Phoenix FOG IVC Dolly Ambr IVC Phoenix Ambr IVC
200
1 50
1 00
50
0
IVC (x1 E6 cells.day/mL)
Boxplot of Qp data from Dolly and Phoenix in fed shake-flasks and Ambr
Qp (pcd)
Dolly FOG Qp Phoenix FOG Qp Dolly Ambr Qp Phoenix Ambr Qp
45
40
35
30
25
20
1 5
1 0
Qp (pcd)
Clone-27
► 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
Fed-batch shake flask
Fed-batch shake flask ambr15TM
IVC (x 106 cells.d/mL)
Clone-27 Sort-5 Clone-27 Sort-5
Clone-27 Sort-5 Clone-27 Sort-5

37. 37
New
hosts/vector
system
Original
host/vector
system
Higher Titres
Better growth
Suitable PQ
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. Summary
► 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
• Performance of both superior to original system, which had been included
as a comparator
► A final new host cell line was selected
• Product concentrations up to 3 g/L observed in ambr15™ using a non-optimised
process
• Improved growth characteristics
• Acceptable product characteristics
38

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
39




40. Acknowledgements
►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
40

41. 41