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National Institute for Cellular Biotechnology
NICB-Pfizer Collaboration: Improving Production of Biopharmaceuticals using Industrially-relevant CHO
Cell Lines Padraig DoolanPadraig Doolan
National Institute for Cellular Biotechnology (NICB)National Institute for Cellular Biotechnology (NICB)
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
NICB/Dublin City University
National Centre of Expertise in Basic & Applied Molecular & Cell Biotechnology since 1987
State of the art institute, dedicated to cell and tissue culture research
New 3,200m2 building (opened 2006)
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
NICB-Pfizer Collaboration
NICB/DCU’s ongoing collaboration with Pfizer (previously Wyeth Biopharma) Initially funded (€3.9M) in 2004 and successfully re-funded (€2.5M) for four additional years in 2008Bio-Manufacturing Sciences Group, Pfizer Inc., (Grange Castle, Clondalkin, Dublin) and Bioprocess R&D, Pfizer Inc.(Andover, MA, USA)
“Molecular Analysis and engineering of CHO cells for more efficient production of biopharmaceuticals”
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Growth of Biologic Sales (US)
Source: Aggarwal, S. What's fueling the biotech engine--2008. Nat. Biotechnol. 27, 987-993 (2009).
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
NICB-Pfizer/Dublin City University Collaboration: Potential Project Outcomes
Project Scope: Gain a deeper understanding of CHO cell biology
Integrated use of Microarray & Proteomics tools to interrogate selected CHO cell samplesIdentification of gene & protein targets influencing phenotypes of interest
Project Outcome: Move the CHO manufacturing platform to a new level (1-3g MAbs /L >10g /L)
Potential targets for rational cell engineering strategiesIdentify Biomarkers for cell line screeningImproved utilization of existing manufacturing capacityMedia or Process improvements
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
0 2 4 6 8 10 12 14Culture Day
Process 1
Process 2
Prod
uctiv
ityLimit Capital Expenditure by Innovation
Pfizer Bio-
Manufacturing Sciences Group (Grange Castle, Dublin)
PfizerBioprocess R&D(Andover, MA, USA)
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
1. Cell Culture Facilities
Largest collection of clean rooms (built to Class C / class 10,000 standards) in any research facility in Ireland
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Sample Matrix
Focus on Fed Batch Culture8 categories, 4 pairs sub-categories (test vs. control)Multiple time-points
>500 biological samples examined>350 scansLargest collection of CHO transcriptomic data
Test Control Test Control Test Control Test Control Test Control Test Control Test Control
MAb 1 MAb 2 MAb 3 MAb 3Protein
1Protein
1 MAb 3 MAb 2 MAb 3
Fc-Fusion
2Protein
2Protein
1 MAb 3
Fc-Fusion
1
MAb 3
Fc-Fusion
1
Fc-Fusion
2 MAb 5
Fc-Fusion
1
Fc-Fusion
1 MAb 8 MAb 8Protein
1Protein
1 MAb 7 MAb 7 MAb 1 MAb 3
MAb 2 MAb 2 MAb 6 MAb 6 MAb 7 MAb 7 MAb 7 MAb 5
Fc-Fusion
3
Fc-Fusion
3
Fc-Fusion
3
Fc-Fusion
3DUKX
1DUKX
2
MAb 4 MAb 4
Fc-Fusion
2 MAb 5 MAb 8 MAb 8 MAb 9 MAb 9 MAb 9 MAb 9 MAb 9 MAb 5 K1 MAb 3
Low Ammonia Producer
Low Lactate Producer
High Cell Growth Rate
High Cell Density
Sustained High Cell Viability High Max Qp
Sustained High Qp
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
High Cell Growth Rate (HCGR) Comparison
Four CHO MAb-secreting cell lines
Two cell lines with slow growth rates (“Slow”) each paired with a separate cell line with a faster growth rate (“Fast”)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
A BHigh Cell Growth Rate Comparison Pairs
Avg
. D3
Cel
l Den
sity
(x10
e6 c
ells
/ml)
SlowFast
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
2. Transcriptomics
(Microarray)
Affymetrix Genechip system
First Generation CHO Chip (WyeHamster2a)2835 library-derived CHO sequences 732 public hamster sequences125 array quality control sequences22 product/process specific sequencesTotal: 3,714 sequences~10-15% of CHO genome
Oligo-Based microarray (Affymetrix)55 probesets per transcript 18 μm feature size
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR Transcriptomics
118 DE Genes118 DE Genes
3 5 7 3 5 7Control Test
Day1
10
100
1000
1e4
3 5 7 3 5 7Control Test
Day1
10
100
1000
1e4
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
3. Parallel Proteomics screening project
2-D DIGE(Difference Gel Electrophoresis)
LTQ ion trap
EttanMALDI-ToF
OrbitrapXL
MALDIToF-ToF4800
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
3. Parallel Proteomics screening project
Test Control Test Control Test Control Test Control Test Control Test Control Test Control
MAb 1 MAb 2 MAb 3 MAb 3Protein
1Protein
1 MAb 3 MAb 2 MAb 3
Fc-Fusion
2Protein
2Protein
1 MAb 3
Fc-Fusion
1
MAb 3
Fc-Fusion
1
Fc-Fusion
2 MAb 5
Fc-Fusion
1
Fc-Fusion
1 MAb 8 MAb 8Protein
1Protein
1 MAb 7 MAb 7 MAb 1 MAb 3
MAb 2 MAb 2 MAb 6 MAb 6 MAb 7 MAb 7 MAb 7 MAb 5
Fc-Fusion
3
Fc-Fusion
3
Fc-Fusion
3
Fc-Fusion
3DUKX
1DUKX
2
MAb 4 MAb 4
Fc-Fusion
2 MAb 5 MAb 8 MAb 8 MAb 9 MAb 9 MAb 9 MAb 9 MAb 9 MAb 5 K1 MAb 3
Low Ammonia Producer
Low Lactate Producer
High Cell Growth Rate
High Cell Density
Sustained High Cell Viability High Max Qp
Sustained High Qp
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR Proteomics
1
10 11
12
13 14 15 16
17 18
19
2
20 21 22
23 24 25 26 27
28 29
3
30
31 32
33
34
4 5 6
7 8 9
1
10
11 12 13 14
15
16 17 18
19
2
20
21 22
23 24
3 4
5 6 7 8 9
(ii)(i)
58 DE Proteins58 DE Proteins
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
5. Bioinformatics@NICB
Developed initially as essential component of microarray facility, but since extended into all research strands at NICB3 full time bioinformaticians in dedicated laboratoryAnalysis of microarray/proteomic/miRNA data using combination ofopen source and commercial software
R/Bioconductor; Genespring; dChipMascot; Sequest, Progenesis LCMS, etc.
Literature mining (PathwayStudio)Also provide medical statistics support Work with NICB wet-lab scientists from experimental design to publication
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
5. Bioinformatics@NICB
Differential gene expression Significance analysis of microarrays Principal component analysis Hierarchical clustering analysisArtificial neural networks Support vector machines Partial least squaresGenetic algorithms GSEAMeta analysis Coexpression network analysis
Access to the Stokes ICHEC HPC for intensive computations
-15
-10
-5
0
5
10
-6-4
-20
24
68
-8
-6
-4
-2
0
2
4
6
Latent Variable 2
Latent Variable 1
Late
nt V
aria
ble
3
0.81
6.32
11.83
17.34
22.85
28.36
33.87
39.38
44.89
50.40
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR Experiment design+21 priority list
A(MAb-expressing clone V1-5 vs.MAb-expressing clone 9B10)
B(PA DUKX 153.8 vs.
PA DUKX 378)
Proteomics Microarray Proteomics Microarray
Combined Overlap
21 High-priorityTargets
118 DE genes 58 DE Proteins
Combined
A(MAb-expressing clone V1-5 vs.MAb-expressing clone 9B10)
B(PA DUKX 153.8 vs.
PA DUKX 378)
Proteomics Microarray Proteomics Microarray
Combined Overlap
21 High-priorityTargets
118 DE genes 58 DE Proteins
Combined
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
7. Functional Genomics
96/384w RT-PCRcDNA OverexpressionsiRNA, shRNA knockdownImpact of altering the expression of gene/protein/miR X on:
Growth, Qp, Viability, etc.Cell CycleApoptosisInvasionDrug Resistance
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR VCP-siRNA: Cell Density/Viability
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.14 2B6 5C10
Cel
l Den
sity
/Con
trol
Control D3 D7
0
10
20
30
40
50
60
70
80
90
100
1.14 2B6 5C10
% V
iabi
lity
Control D3 Control D7 VCP D3 VCP D7
1.14 2B6
HRS 18 48 65 18 48 65
VCP
GAPDH
18 48 65 18 48 65
VCP siRNA Control siRNA VCP siRNA Control siRNA
Cell DensityCell Density % Viability% Viability
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR VCP-OE: Cell Density/Viability
0.00.20.40.60.81.01.21.41.61.82.02.22.42.62.8
1.14 2B6 3B12
Cel
l Den
sity
/Con
trol
Control D3 D5
0102030405060708090
100
1.14 2B6 3B12
% V
iabi
lity
Control D3 Control D5 VCP D3 VCP D5
1.14 2B6
VCP
GAPDH
DAYS 3 6 3 6 3 6 3 6
Empty vector VCP VCP
Empty vector
Cell DensityCell Density % Viability% Viability
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
HCGR Results of Functional Validation on VCP
INCREASED at the protein level in association with high growth rateDECREASED at the gene level in association with high growth rate
siRNA: Decreases growth & destroys viabilityOE: Increases growth, slight improvement in viability in one cell line
FV results tally with proteomics result
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
4. miRNA Screening
TLDAs~650 miRNAsMultiplex RT2 x 384w plates with target-spec. PCR primers
Anti-mirs and Pre-mirsfor knockdown/knockupTransient transfectionCellular phenotype modified?miRNA target genes?
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
miRs
as engineering targets?
First CHO miRNA
U| CCU GU A A A U AA GUACCA UGUCGG AGCUUAUC GACUG UGUUG CUGU G \ UAUGGU ACAGUC UCGGGUAG CUGAC ACAAC GGUA C U C^ UUU UG - G - - UC
A
B
U| CCU GU A A A U AA GUACCA UGUCGG AGCUUAUC GACUG UGUUG CUGU G \ UAUGGU ACAGUC UCGGGUAG CUGAC ACAAC GGUA C U C^ UUU UG - G - - UC
A
B
0
5
10
15
20
25
0 24 48 72 96 120 144
Time (Hrs)
Via
ble
Cel
ls/m
L (x
10^5
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Technology Platforms available at NICB
2. Transcriptomics
(microarray)
1. Cell Culture Suites
4. miRNA screening
3. Proteomics
5. Bioinformatics
6. Cell Characterisation Suite
7. Functional Genomics
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
6. Cell Characterisation
Core Facility
A key element in studying the behavior of mammalian cells in different biological systems
Interlinking with the Microarray and Proteomic core facilities to further validate and study results obtained
Typical studies have included:Drug localisation within cellsLocation of receptors and target proteinsLocation of fusion proteins within cells
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
6. Cell Characterisation
Core Facility
P.A.L.M. Laser Capture Microdisection microscopeTime-lapse Fluorescent Microscope with CO2 and N2 environmental controlLeica SP2 AOBS Confocal; capable of spectral scanning and unmixingThe facility also has cytometrycapability
Guava EasyCyte Plus platform BD FACS Aria cell sorter
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
NICB Technology Platform Outputs: Publications
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
NICB Technology Platform Outputs: PatentsAM 102427 & AM 102587 - DIFFERENTIAL EXPRESSION PROFILING ANALYSIS OF CELL CULTURE PHENOTYPES AND USES THEREOF
TITLE APPLN TYPE DOCKET NO.
COUNTRY SERIAL NO.
FILING DATE (CHS NO.)
INVENTORS SUBJECT MATTER/CLAIM SUMMARY STATUS
PROV AM102427 WYE-060PR
United States 60/794,299 Filed: Apr. 21, 2006 CHS#: (2004658-1441)
Charlesbois (Wyeth) Clynes (DCU) Doolan (DCU) Gammel (DCU) Leonard (Wyeth) Meleady (DCU) Melville (Wyeth) Sinacore (Wyeth)
Converted - Expired
PROV2 AM102587 WYE-060PR2
United States 60/897,412 Filed: Jan. 25, 2007 CHS#: (2004658-1379)
See above Converted - Expired
US - Non-Prov. AM102427 WYE-060
United States 11/788,872 Filed: Apr. 21, 2007 CHS#: (2004658-1321) U.S. Pub. No.: 2008-0070268 Publication Date: March 20, 2008
Anderson (Wyeth) Barron (DCU) Charlesbois (Wyeth) Clynes (DCU) Di Nino (Wyeth) Doolan (DCU) Gammel (DCU) Kopycinski (Wyeth) Leonard (Wyeth) McCarthy (Wyeth) Meleady (DCU) Melville (Wyeth) Sinacore (Wyeth)
The specification describes differential expression profiling analysis of industrially relevant cell line phenotypes through the use of nucleic acid microarray and proteomics analysis methods. The specification discloses various differentially expressed genes and proteins regulating or indicative of cell growth rate, cellular productivity (such as a maximum cellular productivity or a sustained high cellular productivity), peak cell density, sustained cell viability, rate of ammonia production or consumption, or rate of lactate production. The specification further includes methods for manipulating the identified genes and proteins to engineer improved cell lines.
Response to Restriction Requirement filed 5.15.2009 w/ IDS Claiming priority to U.S.S.N. 60/794,299 (WYE-060PR) and 60/794,299 (WYE-060PR2).
Differential Expression Profiling Analysis of Cell Culture Phenotypes and Uses Thereof
International AM102427 WYE-060PC
PCT PCT/US2007/010002 Filed: Apr. 21, 2007 CHS#: (2004658-1440) Int’l Publication No.: WO/2008054514
See above Nationalized - Expired AU 2007314524 BR PI0710637-8 CA 2649859 CN 200780022890.4 EP 07835734.0 IN 28823/DELNP/2008
AM 102921L1 - DIFFERENTIAL EXPRESSION PROFILING ANALYSIS OF CELL CULTURE PHENOTYPES AND USES THEREOF
TITLE APPLN TYPE DOCKET NO.
COUNTRY SERIAL NO.
FILING DATE (CHS NO.)
INVENTORS SUBJECT MATTER/CLAIM SUMMARY STATUS
PROV AM102912L1 WYE-061PR
United States 60/934,980 Filed: June 15, 2007 CHS#: (2004658-1474)
Anderson (Wyeth) Barron (DCU) Clynes (DCU) Di Nino (Wyeth) Doolan (DCU) Gammel (DCU) Kopycinski (Wyeth) McCarthy (Wyeth) Meleady (DCU) Melville (Wyeth) Ng (Wyeth) Nolan (Wyeth)
Converted - Expired
Non-Prov. AM102912L1 WYE-061
United States 12/139,294 Filed: June 13, 2008 CHS#: (2004658-1790) U.S. Pub. No.: 2009-0017460 Publication Date: Jan. 15, 2009
Anderson (Wyeth) Barron (DCU) Clynes (DCU) Di Nino (Wyeth) Doolan (DCU) Gammel (DCU) Kopycinski (Wyeth) McCarthy (Wyeth) Meleady (DCU) Melville (Wyeth) Ng (Wyeth) Nolan (Wyeth)
The specification focuses on the time course analysis and the genes and proteins identified using the time course analysis.
Awaiting First Office Action Claiming priority to U.S.S.N. 60/934,980 (WYE-061PR) and 61/016,390 (WYE-061PR2).
Differential Expression Profiling Analysis of Cell Culture Phenotypes and Uses Thereof [Time Course Analysis]
INTL AM102912L1 WYE-061PC
PCT PCT/US2008/066845 Filed: June 15, 2008 CHS#: (2004658-1791) Int’l Publication No.: WO/2008157299 Int’l Publication Date: Dec. 24, 2008
See above
Nationalization: December 15, 2009
AM 102921 – PATHWAY ANALYSIS OF CELL CULTURE PHENOTYPES AND USES THEREOF
TITLE APPLN TYPE DOCKET NO.
COUNTRY SERIAL NO.
FILING DATE (CHS NO.)
INVENTORS SUBJECT MATTER/CLAIM SUMMARY STATUS
PROV AM102912 (L2) WYE-061PR2
United States 61/016,390 Filed: Dec. 21, 2007 CHS#: (2004658-1380)
Anderson (Wyeth) Barron (DCU) Clynes (DCU) Di Nino (Wyeth) Doolan (DCU) Gammel (DCU) Kopycinski (Wyeth) McCarthy (Wyeth) Meleady (DCU) Melville (Wyeth) Ng (Wyeth) Nolan (Wyeth)
Converted - Expired
Non-Prov. AM102912
United States 12/340,629 Filed: Dec. 19, 2008 CHS#: (2004658-2219)
Barron (DCU) Doolan (DCU) Gammel (DCU) Meleady (DCU) Clynes (DCU) Melville (Wyeth)
The specification describes pathway analysis of differentially expressed genes and/or proteins identified using various differential profiling methods.
Awaiting First Office Action Claiming priority to U.S.S.N. 61/016,390 (WYE-061PR2).
Pathway Analysis of Cell Culture Phenotypes and Uses Thereof
INTL AM102912
PCT PCT/US2008/087856 Filed: Dec. 19, 2008 CHS#: (2004658-2220)
See above. Nationalization: June 21, 2010
The Pfizer/DCU familyS t a t u s R e p o r t2010
13:15-14:45 03-06-2010 SFI/MMI Technology Platform Workshop
National Institute for Cellular Biotechnology
Questions?/Comments?
The intercourse on terms of equality between the representatives of these different estates of the nation [government and industry] is like a sensitive nervous mechanism endowing the community which possessit with capacities and potentialities realisable in no other way
Sir Cyril Hinshelwood, President, Royal Society, 19th
July 1960(http://www.jstor.org/stable/2413882?cookieSet=1)