Development of Cell Lines for Controlled Proliferation and Apoptosis

Mohamed Al-Rubeai

University College Dublin

Cell Line Development and Engineering, 23 May, Zurich

Criteria for Cell Line Selection

• Stability

• Product biological activity

• Product expression: level, duration and inducibility

• Growth and productivity in large scale culture–ease of selection of high producers

–Adaptation to protein free suspension culture

–apoptosis; proliferation rate; max cell number

• Safety issues

Mammalian Cell Lines Used For Protein Production

PropertyCell Line

CHO NS0 PER.C6 BHK

Cell number ( cells/ml) 107 107 107 <107

Productivity (pg/cell/day) 15-65 15-65 10-20 10Product quality ++ + ++ +Impurities ++ + ++ ++Ease of manufacture +++ +++ ++ ++Economics ++ ++ ++ ++Time to clinic + + + +Intellectual property + + - +Regulatory issues +++ ++ + +++

Based partially on information from Robert D Kiss, Genetic Engineering News

Improvement of Product Expression

• Expression Engineering - development of genetic tools comprising cloning and expression vectors

• Cell (Metabolic) Engineering - design or redirection of metabolic pathways

Selection of cell lines with high-level, regulated gene expression

• Rational cell engineering– Multicistronic expression: Coordinated, constitutive or

adjustable high expression level of several genes– Suppressing gene expression (siRNA technology)

• Selection of high producers and monitoring of stability– Selection markers and reporter genes

• DHFR and GS• Flow cytometric based methods

– DHFR/fluorescent MTX– Cell encapsulation (Gel Microdrops) and Affinity matrix based

secretion assay (Carroll and Al-Rubeai, 2004, Expert Opin. Biol. Ther. 4, 1821)

Approaches for Cell line Engineering

• Identification of genes/proteins that are specifically up-regulated in bioprocessing conditions (-omics approach)– Engineering of cells and selection of a new cell line

• Engineering of cells to over-express the gene(s) of interest (historical approach)– Examine the effect and select new cell line– Further understanding of genes/pathways directly

regulated by the gene of interest

Key Genes in Proliferation and Apoptosis

1. bcl-2 suppresses cell death

2. p21 arrests the cell proliferation and enhance specific productivity

3. c-myc enhances proliferation rate, reduces serum dependency and induces anchorage independence

4. hTERT reduces apoptosis, enhances proliferation and increases attachment tendency in the absence of serum

Proliferation and Cell Death

Stimulated bycyclins, cdk’s, c-myc,signals from environment

Proliferation Cell Population Cell Death

(numbers increase) (numbers decrease)

Inhibited by cytostasisinducers, e.g. excess thymidine,hydroxyurea, nitrous oxide cdki p21Cip1, cdki p27Kip1

Inhibited by bcl-2,bclXL, p35, hTERTsignal from environment

Stimulated by chemical compounds, NGF, Fas ligand

The Mammalian Cell Cycle

G0

DNA synthesis

preparation for mitosisreversible quiescent phase

Apoptosis

bcl-2hTERT

cyclin

p21

P cdk

Growth of antibody-producing GS-CHO with and without anti-apoptosis gene-

laboratory results

0

2

4

6

8

10

12

14

16

18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (days)

Via

ble

cel

l co

un

t (c

ells

/ml)

E5

Parental CHO

CHO Bcl2

Growth of antibody-producing GS-NS0: with and without anti-apoptosis gene-

Industrially optimised condition

0

2

4

6

8

10

12

0 100 200 300 400

Time (hr)

X v (

106 /m

L)

parent

bcl-2 transfectant

Bcl-2 over-expression: The Advantages

• Increases cell viability

• Prolongs culture duration

• Reduces serum dependency

• Improves nutrient metabolism

• Protects cells in stressful conditions

• Enhances adaptation in serum free media

Bcl-2 over-expression: Productivity?

• Culture dependant– Increased productivity

• laboratory scale• stressed conditions• serum supplemented culture

– Decreased productivity• optimised culture conditions• Fed batch industrial scale

• Cell line dependant

The effect of ectopic p21CIP1 and Bcl-2 expression on IgG production in batch and

perfusion culture

02468

10121416182022

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (days)

Cel

l cou

nt (

cell/

ml)

x

arrested p21 bcl2 viable cell count arrested p21 bcl2 total cell count

p21 bcl2 viable cell count p21 bcl2 total cell count

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

DaysCe

ll de

nsity

(10

5 /ml)

0

5

10

15

20

25

30

35

40

45

50

SPR

(pg/

cell/

day)

IPTG

IPTGTotal cell count

Viable cell count

Arrested total & viable cell count

Batch culture Perfusion culture

Time (days)

Effect of p21 expression on cell proliferation and cell productivity

3A1 1B5 2C12 2B7 2H3 3B2

MA

b P

rod

uc

tiv

ity

(pg

/ce

ll/d

ay)

0

50

100

150

200

250

300

Col 15 Col 18

3A1 1B5 2C12 2B7 2H3 3B2

Ce

ll D

en

sit

y (

x 1

05 c

ells

/ml)

0

2

4

6

8

10

12Col 15 Col 18 ControlIPTG

p21- transfected CHO clones

Increased productivity: cell cycle vs cell volume?

P21-dependent productivity: Cell size vs cell cycle

• P21 arrests cells in G1phase– G1 is less productive than S and

G2

• P21 leads to increased:• mitochondrial activity• oxygen uptake rate• cell volume• total cellular protein• dry cell weight• ribosomal biogenesis• intracellular IgG• H:L chain ratio

• Larger cells are more productive than smaller cells

Time

0 20 40 60 80 100 120

Ce

ll vo

lum

e (

um

3)

1000

2000

3000

4000

5000

6000

Controlinduced

cell dissociation over 10 minutes following exposure to cell disociation solution

0

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10

sampling time (mins)

cell

den

sity

x10

5

+ IPTG

- IPTG

The effect of cell cycle arrest on cell adhesion cells grown in static for 48 hours with or without IPTG. Cells dissociated using 50% dilution cell dissociation solution (non enzymatic)

Adaptation of NS0 cells to serum free media using the p21 technology

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

0 10 20 30 40 50

Passages (2 day)

Peak C

ell D

ensity (cells/m

l)

0

2

4

6

8

10

12

[Feo

tal C

alf S

eru

m] v/v

NS0 p21 (4 Day Arrest)

NS0 p21

Feotal Calf Serum (v/v)

IPTG ArrestInitiated

IPTG Arrest Removed

10%

5%

4%

3%

2%

1%

0.5%

Adaptation of CHO cells to serum free media using the p21 technology

Time (days)0 20 40 60 80 100

Via

ble

ce

ll c

ou

nt

(ce

lls/m

l) E

5

3

4

5

6

7

8

9

10

No arrest

3 days arrest6 days9 days

Time (days)

0 10 20 30 40 50 60

Via

ble

ce

ll c

ou

nt

(ce

lls/m

l) E

5

2

4

6

8

10

12

Adaptation of CHO cells to serum free media using the p21-Bcl2

technology

No arrest

3 days6 days9 days

Summary of time taken for adaptation to serum free in CHO cells using P21 and

bcl2 technology

0

10

20

30

40

50

60

70

80

P21 arrestedfor 3 days

P21 BCL2arrested for 3

days

P21 arrestedfor 6 days

P21 BCL2arrested for 6

days

P21 arrestedfor 9 days

P21 BCL2arrested for 9

days

P21 nonarrested

throughout

P21 BCL2 nonarrested

throughout

Tim

e ta

ken

(day

s)

Why arresting cells in G1 makes adaptation to serum-free easier?

DC

G1 S G2 M

DC

DC

Highly variable duration, Less variable durationDependent on soluble growth factors and Growth conditions to proceed to S

R

Cells survive on minimum nutrition

Adaptation of CHO cells to suspension using the p21 technology

Time (Days)

0 20 40 60 80

Via

ble

ce

ll c

ou

nt (c

ells/m

l) E

5

1

2

3

4

5

6

7

8

No arrest3 days

6 days9 days

Adaptation of CHO cells to suspension using the p21-Bcl2 technology

Time (days)

0 10 20 30 40 50

Via

ble

ce

ll c

ou

nt

(ce

lls/m

l) E

5

1

2

3

4

5

6

7

Why arresting cells in G1 makes adaptation to suspension easier?

DN

A s

ynth

esis

agitation rate

agitation progression ofcells from S to G2/M

Mi to

ti c in

de

x (%

)

Before After

Mitotic index of remaining cells after capillary flow test

Disruption of cells by turbulent capillary Flow

stimulates cell proliferation decreases attachment

dependency improves adaptation to

suspension enhances response to

feeding works synergistically with

growth factors to promote proliferation

control IGF-1 Transferrin IGF-1+Transferr

% IN

CR

EA

SE

IN C

EL

L N

UM

BE

R /

3 D

AY

S

0

20

40

60

80

100

neo-cho cmyc-cho

(A) with serum

Control cmyc

The effect of c-myc expression on growth of CHO cells

Time (Days)

0 5 10 15 20 25

Via

ble

cell

num

ber

(cel

ls/ m

l)

5.0e+5

1.0e+6

1.5e+6

2.0e+6

2.5e+6

T1F11 T2G7T1G11 T1A8 B1E7 B3E12B2F6

Over-expression of hTERT in CHO cells

Cell Attachment and Survival in the Absence of Serum

Acc No. Gene Ratio T/B

Function

AF296282 Icam4 2.2 Adhesion molecule that binds to LFA-1 adhesion protein. Binds to integrins (By similarity)

M18933 Col3a1 26.5 Collagen type III occurs in most soft connective tissues along with type I collagen

NM_015734 Col5a1 11.3 Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin

X65582 Col6a2 2.2 Collagen VI acts as a cell-binding protein

NM_019759 Dpt 94.5 Mediate adhesion by cell surface integrin binding. Link between cell surface and its ECM

NM_009242 Sparc 2.4 Regulate cell growth through interactions with the extracellular matrix and cytokines

NM_010577 Itga5 2.6 Integrin alpha-5/beta-1 is a receptor for fibronectin and fibrinogen. Play a role in the survival of adult skeletal muscle

M59912 C-kit ligand 2.2 Mediates cell-cell adhesion

NM_008608 Mmp14 3.0 Endopeptidase that degrades various components of the extracellular matrix, such as collagen

NM_011777 Zyx 2.4 Component of a signal pathway that mediates adhesion-stimulated changes in gene expression

NM_011780 Adam23 2.4 May play a role in cell-cell and cell-matrix interactions

NM_013798 Actg1 3.4 Actins are highly conserved proteins that are involved in various types of cell motility

NM_021293 Cd33 9.0 Adhesion molecule that mediates sialic-acid dependent binding to cells

NM_013681 Syn2 10.4 Phosphoprotein that binds to the cytoskeleton

AB009674 Adam22 2.7 Probable ligand for integrin

Telomerase cells Blank cells

T: telomerase clonesB: blank clones

cDNA Microarray analysis of specific genes involved in cell attachment and formation of extra cellular matrix gene regulation in the CHO K1 cell lines

DNA Microarrays

Over-expression of hTERT enhances chromosomal stability and possibly production stability

Micronucleus

Aneuploidy (loss of several chromosomes)

Aneuploidy

Act

ivity

(m

U/m

l)

0

1

2

3

4

5

The distribution of chromosomal number at different times. A: one day, B: 4 months and C: one year. The x axis represents the number of chromosomes in the cell line and the y axis represents the percentage of cells in population.

CHO is not stable cell line!

Cells were transfected with the SEAP gene, passaged in culture for one year and activity of protein measured in the supernatants of batch cultures

Telomerase

Blank

Conclusions

• Cell proliferation and apoptosis are co-ordinately linked processes.

• Genetic engineering of cellular and metabolic pathways can enhance cell robustness, adaptation and productivity.

• Genetic and chromosomal instability may affect production stability.

Acknowledgments

• Kelly Astley• Paul Clee• Gary Khoo• Darrin Kuystermans• Amelia Petch• Jenny Bi

• Funding: Lonza Biologics (p21 work), BBSRC, EU Framework (Bcl-2 work), SFI (Ireland), Cambrex Biosciences

• John Birch, Lonza• Andy Racher, Lonza