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 CA1

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)

Scope

►Aims and Objectives

►Host cell line development (CLD)

• Directed evolution approaches

• Results

►CLD run-through

• Experimental design

• Results

►Summary

3

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

Host cell line development

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

6

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

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

ChemostatSubculture

regimeCloningFACS

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

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

50% CD-DG44

50% OPTI-CHO

75% CD-DG44

25% OPTI-CHO

C3

100% CD-DG44

9

0.00

0.25

0.50

0.75

1.00

0 50 100 150 200

Gro

wth

rate

(d

ay

-1)

Days in culture

The long and winding road to CD media

adaptation: The C2 story

120 rpm110 rpm 140 rpmStatic

100% MEMα

+10%FBS

75% OptiCHO

80% OptiCHO

85% OptiCHO

90% OptiCHO 100% CD DG44

Clumping issues

3-day sub3/4-day sub

0.2 x 106 seeding0.3 x 106 seeding0.2 x 106 seeding

1:100 anti-clump 1:700 anti-clump No anti-clump

10

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

► 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

11

0

5

10

15

C1 C2 C3 2B-1 2B-2

IVC

(x

10

6cell∙day/m

L)

0

10

20

30

C1 C2 C3 2B-12B-2

Meta

bo

lite

C

on

cen

trati

on

(m

M)

Amm

Lac

0

20

40

60

80

C1 C2 C3 2B-1 2B-2

Tra

ns

fec

tio

n

eff

icie

nc

y (

%)

► 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

Higher transfection efficiency and transient

titre obtained with cell line C2

12

0

1

2

3

4

C1 C2 C3 2B-1 2B-2

Tra

nsie

nt

mA

b

exp

ressio

n (

mg

/L)DG44-2B in ACF

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 mAb2

13

0

5

10

15

20

25

30D

G44

-2B

C2

Sort

-1

Sort

-2

Sort

-3

Sort

-4

Sort

-5

Sort

-6

Clo

ne

-7

Clo

ne

-11

Clo

ne

-26

Clo

ne

-27

Clo

ne

-36

Clo

ne

-46

Clo

ne

-56

Clo

ne

-59

Sta

t-1

Sta

t-2

Sta

t-3

Sta

t-4

Sta

t-5

Sta

t-6

8 m

M

6 m

M

4 m

M

2 m

M

1 m

M

Do

ub

lin

g tim

e (

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

0

2

4

6

8

10

12

14

DG

44

-2B

C2

Sort

-1

Sort

-2

Sort

-3

Sort

-4

Sort

-5

Sort

-6

Clo

ne

-7

Clo

ne

-11

Clo

ne

-26

Clo

ne

-27

Clo

ne

-36

Clo

ne

-46

Clo

ne

-56

Clo

ne

-59

Sta

t-1

Sta

t-2

Sta

t-3

Sta

t-4

Sta

t-5

Sta

t-6

8 m

M

6 m

M

4 m

M

2 m

M

1 m

M

Batc

h s

hake-f

lask s

cre

en

harv

est

IVC

(x

10

6cell∙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)

0

1

2

3

DG

44

-2B

C2

Sort

-1

Sort

-2

Sort

-3

Sort

-4

Sort

-5

Sort

-6

Clo

ne

-7

Clo

ne

-11

Clo

ne

-26

Clo

ne

-27

Clo

ne

-36

Clo

ne

-46

Clo

ne

-56

Clo

ne

-59

Sta

t-1

Sta

t-2

Sta

t-3

Sta

t-4

Sta

t-5

Sta

t-6

8 m

M

6 m

M

4 m

M

2 m

M

1 m

M

mA

b 2

tra

ns

ien

t e

xp

res

sio

n (

mg

/L)

0

2

4

6

8

10

12D

G44

-2B

C2

Sort

-1S

ort

-2S

ort

-3S

ort

-4S

ort

-5S

ort

-6C

lone

-7C

lone

-11

Clo

ne

-26

Clo

ne

-27

Clo

ne

-36

Clo

ne

-46

Clo

ne

-56

Clo

ne

-59

Sta

t-1

Sta

t-2

Sta

t-3

Sta

t-4

Sta

t-5

Sta

t-6

8 m

M6 m

M4 m

M2 m

M1 m

M

mA

b 1

tra

ns

ien

t ex

pre

ssio

n (

mg

/L)

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

0

20

40

60

80

100

120D

G44

-2B

C2

Sort

-4

Sort

-5

Sort

-6

Clo

ne

-7

Clo

ne

-11

Clo

ne

-26

Clo

ne

-27

Clo

ne

-36

Clo

ne

-46

Clo

ne

-56

Clo

ne

-59

Sta

t-1

Sta

t-2

Sta

t-3

Sta

t-4

Sta

t-5

Sta

t-6

8 m

M

6 m

M

4 m

M

2 m

M

1 m

M

% C

olo

ny s

urv

ival

in s

em

i-so

lid

med

iaGrowth 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

0

2

4

6

8

10

12

14

16

18

Clo

ne

11

Sort

5

Sort

4

1 m

M

Clo

ne

27

2 m

M

Sta

t 6

Clo

ne

56

Sort

6

6 m

M

Clo

ne

26

Sta

t 2

Sta

t 5

Sta

t 1

Clo

ne

59

DG

44

-2B

4 m

M

Clo

ne

7

8 m

M C2

Sta

t 4

Sta

t 3

Clo

ne

46

Clo

ne

36

Avera

ge R

an

kin

gAverage ranking used to select host cell lines

to progress

Top 5 cell

lines

progressed

18

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

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

20

Experiment biased towards

mAb 1 as mAb 2 and mAb 3

have previously proven to be

“difficult-to-express”

0

5

10

15

20

25

30

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

mA

b 1

mA

b 2

mA

b 3

DG44-2B Clone 11 Clone 27 Sort 4 Sort 5 1 mM (in1mM GLN)

1 mM (in8mM GLN)

24w

p ti

tre (

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

24-well plate screen titre

21

Cell Line Development

Run-Through

22

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

CLD run-through: Transfection to recovery

Pools from

Clone-27 hostrecovery

Pools from

Sort-5 host

Pools from

DG44-2B host

recovery

recovery

TransfectionHost 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

CLD run-through: ClonePix™2 screening

25

FIT

C1

00

0 E

xte

rior

Me

an I

nte

nsity

DG44-2BAlternative Host Cell LineApollo

40000

30000

20000

1 0000

0

FIT

C1

00

0 E

xte

rio

r M

ean

In

ten

sity

Clone-27 Sort-5 DG44-2B

Exem

pla

r

co

lon

y im

ag

e

► The fluorescence intensity and colony size were lower for

the original host/vector system (DG44-2B)

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

Clo

ne-2

7S

ort

-5D

G4

4-2

B

0

50

100

150

200

250

0 100 200 300 400 500 600 700 800

Tit

re (

mg

/L)

0

50

100

150

200

250

0 100 200 300 400 500 600 700 800

Tit

re (

mg

/L)

0

50

100

150

200

250

0 100 200 300 400 500 600 700 800

Tit

re (

mg

/L)

Colony Number

0

50

100

150

200

0 50 100 150 200 250

Tit

re (

mg

/L)

CLD run-through: 24-well plate screen

27

Tit

re (

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

Clo

ne

-27

So

rt-5

DG

44

-2B

0

50

100

150

200

0 50 100 150 200 250

Tit

re (

mg

/L)

0

50

100

150

200

0 50 100 150 200 250

Colony number

28

CLD run-through: Suspension culture

Clone-27 Sort-5 DG44-2B

4

3

2

1

0

VC

D (

x1

E6

)

100

90

80

70

60

50

Via

bilit

y (

%)

VC

D (

x 1

06

ce

lls

/mL

)

Via

bilit

y (

%)

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):

► 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

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

Clo

ne-2

7S

ort

-5D

G4

4-2

B

0

100

200

300

400

500

600

Tit

re (

mg

/L)

0

100

200

300

400

500

600

Tit

re (

mg

/L)

0

100

200

300

400

500

600

Tit

re (

mg

/L)

* * ** *

* cell growth was not

acceptable for progression

CLD run-through: Growth profiles from the

fed-batch shake-flask screen

30

► 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

DG44-2BClone-27 Sort-5

Note: different

medium/feed system

required for DG44-2B

0

5

10

15

20

25

0 2 4 6 8 10 12 14

VC

D (x

10

6c

ells

/mL

)

Culture time (days)

0

5

10

15

20

25

0 2 4 6 8 10 12 14

Culture time (days)

0

5

10

15

20

25

0 2 4 6 8 10 12 14

Culture time (days)

►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

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

Clo

ne-2

7S

ort

-5D

G4

4-2

B

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Tit

re (

g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Tit

re (

g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Tit

re (

g/L

)

33

CLD Run-through: Specific productivity

from the fed-batch shake-flask screen D

G4

4-2

BC

lon

e-2

7S

ort

-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

0

10

20

30

40

50

Qp

(p

g/c

ell.d

ay

)

0

10

20

30

40

50

Qp

(p

g/c

ell.d

ay

)

0

10

20

30

40

50

Qp

(p

g/c

ell.d

ay

)

No substantial difference in product characteristics

between cell lines from different hosts

34

Size exclusion

chromatography

Capillary

electrophoresis-SDS

(reduced)

N-linked glycansCation-exchange

chromatography

Exemplar Overlay

graph

•The majority of the

product from all

recombinant cell lines in

monomeric form (≥98%) Exemplar

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

• Adapting all cell lines to this system

removed the minor differences

Clone-27 derived cell line

•No considerable aggregation detected

CONFIDENTIAL

35

Comparison of top producers in the ambr15TM

screen

point size: Qp

► 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

Sort-5Clone-27

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Ha

rve

st

Tit

re(g

/L)

IVC (x 106 cells.h/mL)

3.0

2.5

2.0

1 .5

1 .0

Pro

du

ct C

on

cen

trat

ion

(g

/L)

36

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 Phoenix Ambr IVCDolly Ambr IVCPhoenix FOG IVCDolly FOG IVC

200

1 50

1 00

50

0

IVC

(x1

E6 c

ell

s.d

ay/m

L)

Boxplot of IVC data from Dolly and Phoenix in fed shake-flasks and Ambr

Qp

(pc

d)

Phoenix Ambr QpDolly Ambr QpPhoenix FOG QpDolly FOG Qp

45

40

35

30

25

20

1 5

1 0

Qp

(p

cd)

Boxplot of Qp data from Dolly and Phoenix in fed shake-flasks and Ambr

► 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

ambr15TMFed-batch shake flask

ambr15TM

IVC

(x

10

6c

ell

s.d

/mL

)

Clone-27 Sort-5 Sort-5 Clone-27

Clone-27 Sort-5 Sort-5 Clone-27

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 stageVector

Host

+

Suitable PQ

CLD

run-t

hro

ugh

► 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

Summary

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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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►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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