RECENT IMPROVEMENTS TO LONZA S GLUTAMINE SYNTHETASE (GS) GENE EXPRESSION SYSTEM. Dr Robert Gay
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1 RECENT IMPROVEMENTS TO LONZA S GLUTAMINE SYNTHETASE (GS) GENE EXPRESSION SYSTEM Dr Robert Gay Lonza Biologics plc, 2004
2 The Challenge of the MAb Market Global market for Monoclonal Antibody Therapeutics reached a total of $7.2 billion in 2003 Compound 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 therapuetics High dose requirement 10s to 100s Kg per annum required Challenge: produce large quantities with cost and time efficiency Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 2
3 Meeting the Challenge Can large quantities simply be obtained by scaling up and up? Cell lines Highly productive Stable Cell culture processes Robust Scalable Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 3
4 20,000 Litre Large Scale Reactor Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 4
5 Meeting the Challenge Can large quantities simply be obtained by scaling up and up? Cell lines Highly productive Stable Focus on Development of GS-CHO platform Expression of antibodies Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 5
6 The Benefits of Increasing Productivity % Number of Batches Required % 60% 40% 20% Relative Cost % 2000L scale 5000L Scale Cost Assumptions: % Product Requirement: 35kg/yr 65% yield across purification Product Titre (g/l) Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 6
7 Highly Productive Cell Lines I Host cell Expression system Transfection and selection protocol Rapid creation Goal to create stable, high producing cell lines Grow in suspension culture Grow 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 7
8 Highly Productive Cell Lines II Host cell CHOK1SV Expression system GS Electroporation and selection protocol MSX Rapid 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 8
9 Host Cell and Expression System Developed CHOK1SV (suspension variant) Grows as single cell suspension Pre-adapted to growth in chemically defined, animal component-free media Exhibits good growth characteristics Reach high maximum viable cell concentration Able 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 9
10 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 Serum-Containing Process Vector construction Transfection Selection and Expansion Adaptation to Suspension Growth CDACF Process Vector construction Transfection Selection and Expansion Adaptation to Suspension Growth 20 weeks Time (weeks) Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 10
11 Host Cell and Expression System Developed CHOK1SV (suspension variant) The GS Gene Expression System Glutamine synthetase (GS) used as a selectable marker Glutamine omitted from culture media as selective pressure Further 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 11
12 Host Cell and Expression System ATP ADP + Pi + 4 NH + glutamate glutamine MSX Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 12
13 GS Expression Vectors Gene of interest driven by strong promoter GS gene driven by weak promoter Biases for selection of rare integration into transcriptionally active sites in genome Range of unique restriction enzyme sites present in polylinker Accessory vector available for cloning of antibodies Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 13
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 14
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/l 1.5% transfectants produce more than 150 mg/l Probability Antibody (mg/l) Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 15
16 Cell Line Screening Highly productive transfectants are rare even with a good selection system How can the hit rate for finding highly productive cell lines be increased? Various approaches to improve screening process Increase number of colonies created Improve stringency of selectable marker to eliminate low producers High 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 16
17 Selection of Media for Transfection I More transfectants derived using CM25 media Number of transfectants per 20 plates n=9 TRANSFECTION MEDIA DMEM CM25 Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 17
18 Selection of Media for Transfection II High productivity of transfectants is maintained Antibody (mg/l) n=30 TRANSFECTION MEDIA DMEM CM25 Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 18
19 Optimising Transfection and Selection I Influence of electroporation conditions for GS-CHO cell lines with cb72.3 antibody Electroporation condition 1 Numbers of stable transfectants x 10 6 cells electroporated Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 19
20 Optimising Transfection and Selection II Influence of selection conditions for GS-CHO cell lines with cb72.3 antibody 350 Cell lines have not been amplified 300 Antibody (mg/l) µm 50 µm Selection conditions - MSX concentration Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 20
21 Optimising Transfection and Selection III Evaluation of conditions and MSX concentrations during electroporation Electroporation condition MSX (µm) Stable transfectant numbers Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 21
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 22
23 Cloning in Constant Region Vectors I Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 23
24 Cloning in Constant Region Vectors II Wide range of constant regions precloned into GS vectors Allows easy subcloning of variable regions single 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 24
25 Improving GS Vectors for Antibodies Free light chain often seen in cultures Does first gene (LC) interfere with expression of second gene (HC)? Can levels of LC and HC be balanced? Transcription blocker Must 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 25
26 Evaluating Improvements to GS Vectors Transfect host cells with vector STATIC CULTURE 3-4 weeks 100 transfectants 100 transfectants Identify single colonies per well Transfer to 24 well plate 2 weeks 100 data points productivity assessment (quantitative) Compare against control Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 26
27 Improving GS Vectors for Antibodies Antibody (mg/l) n=100 Control Reverse Transcription Stronger Vector Orientation Blocker Promoter (LC-HC) (HC-LC) (LC-TB-HC) (LC-HC) Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 27
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 28
29 Improving GS vectors - MARs Matrix Attachment Regions (MARs) DNA sequences Capable of specific binding to nuclear proteins Typically localised at boarders of gene domains Involved in formation of higher order chromatin Thought to stimulate transgene expression Reduce incidence of gene silencing May increase stability of gene expression Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 29
30 Position of MARs Mid 5 3 Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 30
31 Improving GS vectors - MARs Antibody (mg/l) n=100 Control 5' Mid 3' 5'and3' Vector MAR MAR MAR MAR Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 31
32 Finding High Producers I Transfect host cells with vector STATIC CULTURE 3-6 week 1 week 4 weeks transfectants transfectants Single colonies per well productivity assessment (semi-quantitative) productivity assessment (quantitative) SUSPENSION (shake flask culture) 8 weeks transfectants Adapt to protein-free 3 weeks 3 weeks transfectants 5-10 transfectants Select 3 cell lines for further analysis Preliminary productivity assessment (quantitative) Fed-batch assessment of growth and productivity Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 32
33 Finding High Producers II Shake-flask cultures operated in fed-batch mode 2000 Harvest Product Concentration (mg/l) Candidate Cell Lines Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 33
34 Finding High Producers II Shake-flask cultures operated in fed-batch mode 2000 Harvest Product Concentration (mg/l) Candidate Cell Lines Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 34
35 Finding High Producers III Bioreactor Laboratory-scale (10 L) Pilot-scale (130 L) Manufacturing (200 L) Maximum Viable Cell Concentration (10 6 cells/ml) Product Concentration (g/l) Specific Production Rate (pg/cell/h) Harvest day Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 35
36 Process Improvement I Improving Cell Growth 1000 Viable Cell Concentration (10 5 /ml) Time (h) It. 2 It. 3 It. 4 It. 5 Cl. CY01 Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 36
37 Process Improvement II Improving Product Accumulation 6000 Product Concentration (mg/l) Time Integral of Viable Cell Concentration (10 9 cell h/l) It. 2 It. 3 It. 4 It. 5 Cl. CY01 Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 37
38 Summary Creation of highly productive cell lines is the sum of many parts Host cell and expression system Transfection and selection Strategies 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 38
39 Acknowledgements Development Lonza Biologics Slough, UK Cell Culture Process Development Assay Development Process Scale Up and Support Rinat Neuroscience Corp. Dr Robert Gay - Lonza Biologics 3rd International Cell Culture & Upstream Processing Conference Boston October 2004 Slide 39
40 RECENT IMPROVEMENTS TO LONZA S GLUTAMINE SYNTHETASE (GS) GENE EXPRESSION SYSTEM Dr Robert Gay Lonza Biologics 224 Bath Road,Slough, UK robert.gay@lonza.com Lonza Biologics plc, 2004
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