nicb-pfizer collaboration: improving production of...

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


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)



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



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”



Growth of Biologic Sales (US)

Source: Aggarwal, S. What's fueling the biotech engine--2008. Nat. Biotechnol. 27, 987-993 (2009).



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



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)




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





1. Cell Culture Facilities

Largest collection of clean rooms (built to Class C / class 10,000 standards) in any research facility in Ireland



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



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





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



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





3. Parallel Proteomics screening project

2-D DIGE(Difference Gel Electrophoresis)

LTQ ion trap

EttanMALDI-ToF

OrbitrapXL

MALDIToF-ToF4800



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



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





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



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



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics






96/384w RT-PCRcDNA OverexpressionsiRNA, shRNA knockdownImpact of altering the expression of gene/protein/miR X on:

Growth, Qp, Viability, etc.Cell CycleApoptosisInvasionDrug Resistance



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



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



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





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?



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




2. Transcriptomics

(microarray)


4. miRNA screening

3. Proteomics

5. Bioinformatics





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



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



NICB Technology Platform Outputs: Publications



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


COUNTRY SERIAL NO.



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


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


COUNTRY SERIAL NO.



PROV AM102912 (L2) WYE-061PR2

United States 61/016,390 Filed: Dec. 21, 2007 CHS#: (2004658-1380)


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



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)

nicb-pfizer collaboration: improving production of...

Documents