RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE …bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_Powerpoint... · synthetase (gs) gene expression system ... (msx) - a specific

Dr Robert Gay

© Lonza Biologics plc, 2004

RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE SYNTHETASE (GS) GENE EXPRESSION SYSTEM

© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 2

The Challenge of the MAb Market

Global market for Monoclonal Antibody Therapeutics reached a total of $7.2 billion in 2003Compound average annual growth rate of 53% over previous 5 years

Marketresearch.com 2004

More than 370 recombinant antibody product are currently in the pipeline

Research and Markets 2004

Currently fifteen rMAbs on the market Several are ‘blockbuster’ therapueticsHigh dose requirement – 10s to 100s Kg per annum requiredChallenge: produce large quantities with cost and time efficiency


Meeting the Challenge

Can large quantities simply be obtained by scaling up and up?

Cell linesHighly productiveStable

Cell culture processesRobustScalable


20,000 Litre Large Scale Reactor


Meeting the Challenge

Can large quantities simply be obtained by scaling up and up?

Cell linesHighly productiveStable

Focus onDevelopment of GS-CHO platformExpression of antibodies


The Benefits of Increasing Productivity

0

40

80

120

160

200

0.1 0.3 0.6 1.9 2.8

Product Titre (g/L)

Num

ber o

f Bat

ches

Req

uire

d

0%

20%

40%

60%

80%

100%

Rel

ativ

e Co

st %

2000L scale5000L ScaleCost

Assumptions:•Product Requirement: 35kg/yr•65% yield across purification


Highly Productive Cell Lines I

Host cellExpression systemTransfection and selection protocolRapid creation

Goal to create stable, high producing cell linesGrow in suspension cultureGrow in a chemically defined, animal component-free media

Optimise culture High cell numbers which can be maintained for extended time


Highly Productive Cell Lines II

Host cell CHOK1SVExpression system GSElectroporation and selection protocol MSXRapid creation 20 weeks

Early phase clinical supply (Uncloned)cDNA to cGMP in a generic process in

<12 months

Late phase clinical supply (Clonal)Marry cell line with optimised process


Host Cell and Expression System

Developed CHOK1SV (suspension variant)Grows as single cell suspensionPre-adapted to growth in chemically defined, animal component-free mediaExhibits good growth characteristics

Reach high maximum viable cell concentrationAble to maintain cultures at high cell viability

The GS Gene Expression System


0 10 20 30 40 50 60

Time (weeks)

Serum-Containing Process

Vector construction

Selection and ExpansionTransfection

Adaptation to SuspensionGrowth

CDACF Process

20 weeks

Vector construction

Selection and ExpansionTransfection

Adaptation to SuspensionGrowth

Timeline Reduction with CHOK1SV

Use of suspension variant of CHO-K1 pre-adapted to growth in chemically defined, animal component-free (CDACF) media substantially reduces time taken to generate cell lines



Developed CHOK1SV (suspension variant)

The GS Gene Expression SystemGlutamine synthetase (GS) used as a selectable markerGlutamine omitted from culture media as selective pressureFurther selection pressure applied with methionine sulphoxamine (MSX) - a specific inhibitor of GS

Focus on recent developments creating GS-CHO cell lines



NH + glutamate glutamine

ATP ADP + Pi

MSX

+

4


GS Expression Vectors

Gene of interest driven by strong promoterGS gene driven by weak promoterBiases for selection of rare integration into transcriptionally active sites in genomeRange of unique restriction enzyme sites present in polylinkerAccessory vector available for cloning of antibodies


GS Expression Vectors for Antibodies

Simple construction of a double-gene vector

Both light chain and heavy chain on one vector


Finding High Producers

High producers are infrequent

Probability distribution of antibody productivities for primary GS-CHO transfectants ( 24 well plates )90% transfectants produce less than 90 mg/L1.5% transfectants produce more than 150 mg/L

0.000

0.004

0.008

0.012

0.016

0.020

0 50 100 150 200 250 300

Antibody (mg/L)P

roba

bilit

y


Cell Line Screening

Highly productive transfectants are rare even with a good selection systemHow can the hit rate for finding highly productive cell lines be increased?

Various approaches to improve screening processIncrease number of colonies createdImprove stringency of selectable marker to eliminate low producersHigh throughput methods (FACS + cell surface product capture)Early screening will not necessarily indicate growth characteristics in manufacturing process


n=9

Nu

mb

er

of

tran

sfect

an

ts p

er

20

pla

tes

0

100

200

300

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500

Selection of Media for Transfection I

More transfectants derived using CM25 media

TRANSFECTION MEDIA

DMEM CM25


TRANSFECTION MEDIA

DMEM CM25

An

tib

od

y (

mg

/L)

0

50

100

150

200

n=30

Selection of Media for Transfection II

High productivity of transfectants is maintained


1973

1242

681

Numbers of stable

transfectants

Electroporationcondition

2.5 x 106 cells electroporated

Optimising Transfection and Selection I

Influence of electroporation conditions for GS-CHO cell lines with cB72.3 antibody


Cell lines have not been amplified

Influence of selection conditions for GS-CHO cell lines with cB72.3 antibody

Optimising Transfection and Selection II

Selection conditions - MSX concentration

25 µM 50 µM

An

tib

od

y (

mg

/L)

0

50

100

150

200

250

300

350


Optimising Transfection and Selection III

7050

197253

5750

124252

3250

68251

Stable transfectant

numbersMSX (µM)Electroporation

condition

Evaluation of conditions and MSX concentrations during electroporation


GS Expression Vectors for Antibodies

Constant region vectors

Time consuming to clone constant regions when drug discovery is focused on generating variable regions

Preformatted to receive antibody variable regions

Class switching of antibodies possible


Cloning in Constant Region Vectors I


Cloning in Constant Region Vectors II

Wide range of constant regions precloned into GS vectors

Allows easy subcloning of variable regionssingle ligation step

Fast construction of chimaeric, humanised or fully human mAbs


Improving GS Vectors for Antibodies

Free light chain often seen in culturesDoes first gene (LC) interfere with expression of second gene (HC)?Can levels of LC and HC be balanced?

Transcription blockerMust the LC gene be first?

Reverse the order and put HC first

Can stronger promoters be used?


Evaluating Improvements to GS Vectors

Transfect host cells with vector

100 data points productivity assessment(quantitative)

STATIC CULTURE100 transfectants

3 - 4 weeks

2 weeks

Identify single colonies per well

100 transfectants

Transfer to 24 well plate

Compare against control



Control Reverse Transcription StrongerVector Orientation Blocker Promoter(LC-HC) (HC-LC) (LC-TB-HC) (LC-HC)

Ant

ibod

y (m

g/L)

0

20

40

60

80

100

120

140

160

180

200

n=100



Order of LC and HC is important even in CHO cells

LC preferred upstream

Presence of a transcription blocker provides no benefit

Promoter strength appears to be finely balanced


Improving GS vectors - MARs

Matrix Attachment Regions (MARs)DNA sequencesCapable of specific binding to nuclear proteinsTypically localised at boarders of gene domainsInvolved in formation of higher order chromatin

Thought to stimulate transgene expressionReduce incidence of gene silencingMay increase stability of gene expression


Position of MARs

5’

3’

Mid


Improving GS vectors - MARs

Control 5' Mid 3' 5'and3' Vector MAR MAR MAR MAR

Ant

ibod

y (m

g/L)

0

20

40

60

80

100

120

140

160

180

200

n=100


Finding High Producers I

Transfect 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

3 - 6 week

4 weeks

8 weeks

3 weeks

5-10 transfectantsFed-batch assessment of

growth and productivity3 weeks

Single colonies per well

60-100 transfectants

productivity assessment(semi-quantitative)1 week


Finding High Producers II

Shake-flask cultures operated in fed-batch mode

0

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1200

1400

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1 2 3 4 5 6 7 8 9 10

Candidate Cell Lines

Har

vest

Pro

duct

Con

cent

ratio

n (m

g/L)


Finding High Producers II

Shake-flask cultures operated in fed-batch mode

0

200

400

600

800

1000

1200

1400

1600

1800

2000

1 2 3 4 5 6 7 8 9 10

Candidate Cell Lines

Har

vest

Pro

duct

Con

cent

ratio

n (m

g/L)


Bioreactor

Maximum Viable Cell

Concentration (106 cells/mL)

Product Concentration

(g/L)

Specific Production

Rate (pg/cell/h)

Harvest day

Laboratory-scale (10 L) 9.42 1.6 0.78 15

Pilot-scale (130 L) 10.78 1.9 0.76 17

Manufacturing (200 L) 9.66 1.4 0.78 15

Finding High Producers III


1

10

100

1000

0 100 200 300 400 500

Time (h)

Viab

le C

ell C

once

ntra

tion

(105 /m

L)

It. 2 It. 3 It. 4 It. 5 Cl. CY01

Process Improvement I

Improving Cell Growth


0

1000

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5000

6000

0 500 1000 1500 2000 2500 3000 3500

Time Integral of Viable Cell Concentration (109 cell h/L)

Prod

uct C

once

ntra

tion

(mg/

L)

It. 2 It. 3 It. 4 It. 5 Cl. CY01

Process Improvement II

Improving Product Accumulation


Summary

Creation of highly productive cell lines is the sum of many parts

Host cell and expression systemTransfection and selectionStrategies to identify the highest expressers

Constant region vectors facilitate antibody cloning and expression

Vector architecture can influence expression levels


Acknowledgements

Development – Lonza Biologics Slough, UK

Cell Culture Process DevelopmentAssay DevelopmentProcess Scale Up and Support

Rinat Neuroscience Corp.

Dr Robert Gay – Lonza Biologics224 Bath Road,Slough, UK

Email – [email protected]

© Lonza Biologics plc, 2004

RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE SYNTHETASE (GS) GENE EXPRESSION SYSTEM

Documents

RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE …bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_Powerpoint... · synthetase (gs) gene expression system ... (msx) - a specific