RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE...
Transcript of RECENT IMPROVEMENTS TO LONZA‘S GLUTAMINE...
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 3
Meeting the Challenge
Can large quantities simply be obtained by scaling up and up?
Cell linesHighly productiveStable
Cell culture processesRobustScalable
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 4
20,000 Litre Large Scale Reactor
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 5
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 6
The Benefits of Increasing Productivity
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Product Titre (g/L)
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Assumptions:•Product Requirement: 35kg/yr•65% yield across purification
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 7
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 8
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 9
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 10
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 11
Host Cell and Expression System
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 12
Host Cell and Expression System
NH + glutamate glutamine
ATP ADP + Pi
MSX
+
4
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 13
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 14
GS Expression Vectors for Antibodies
Simple construction of a double-gene vector
Both light chain and heavy chain on one vector
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 15
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
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Antibody (mg/L)P
roba
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© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 16
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 17
n=9
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Selection of Media for Transfection I
More transfectants derived using CM25 media
TRANSFECTION MEDIA
DMEM CM25
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 18
TRANSFECTION MEDIA
DMEM CM25
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mg
/L)
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Selection of Media for Transfection II
High productivity of transfectants is maintained
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 19
1973
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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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 20
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
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mg
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© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 21
Optimising Transfection and Selection III
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Stable transfectant
numbersMSX (µM)Electroporation
condition
Evaluation of conditions and MSX concentrations during electroporation
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 22
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 23
Cloning in Constant Region Vectors I
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 24
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 25
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?
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 26
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 27
Improving GS Vectors for Antibodies
Control Reverse Transcription StrongerVector Orientation Blocker Promoter(LC-HC) (HC-LC) (LC-TB-HC) (LC-HC)
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© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 28
Improving GS Vectors for Antibodies
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 29
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 30
Position of MARs
5’
3’
Mid
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 31
Improving GS vectors - MARs
Control 5' Mid 3' 5'and3' Vector MAR MAR MAR MAR
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© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 32
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 33
Finding High Producers II
Shake-flask cultures operated in fed-batch mode
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Candidate Cell Lines
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n (m
g/L)
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 34
Finding High Producers II
Shake-flask cultures operated in fed-batch mode
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© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 35
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 36
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Time (h)
Viab
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ell C
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Process Improvement I
Improving Cell Growth
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 37
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Time Integral of Viable Cell Concentration (109 cell h/L)
Prod
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It. 2 It. 3 It. 4 It. 5 Cl. CY01
Process Improvement II
Improving Product Accumulation
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 38
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
© Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference – Boston October 2004 Slide 39
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