1 PPCW Workshop NIH 3/28/2004 Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer,...

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PPCW Workshop NIH3/28/2004

Bethesda,MD,30.3.2004

Sabine Geisse, Nicola di Maiuta, Thomas Cremer,

Mario Henke

Novartis Institutes for Biomedical Research, Basle, Switzerland

Transient Transfection into Eukaryotic Cells: An Alternative to Bacterial and Insect Cell Systems for the Rapid Generation of Recombinant Proteins?

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Background and Rationale• Recombinant proteins are essential research tools for

assay development and screening structural biology antibody generation, selectivity assays......

• In the post-genomic era translation of thousands of ORFs into proteins are a major challenge

• Technologies and processes increasing the throughput and the success rate in protein production are needed

• Novartis Research is currently building a Protein Production Center in which streamlined processes will be applied in a factory-like fashion. In parallel, an Intensive Care Approach is applied to generate proteins by non-generic means.

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PPCW Workshop NIH3/28/2004 Double-Track Strategy

• Target output: 400 tool proteins/year – Quantity 1 - 100 mg– Purity >80 % (application-dependent)

• “Protein Factory”: > 300 proteins/year – 3 standardized expression systems– Streamlined, generic, partly automated processes

• “Intensive Care”: < 100 proteins/year – Difficult-to-express proteins– Recombinant cell lines, membrane proteins– Monoclonal and recombinant antibodies

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Transformation/Transfection

Small Scale Expression Evaluation

Scale-Up

Large-Scale Fermentation

Automated Harvest + Cell Lysis

Automated Purification

Evaluate Special Conditions for Scale-

Up

Large-Scale Fermentation

Standard Harvest + Lysis

Classical Purification

PPC ICU

A Double-Track Process to Increase Success Rates

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Small Scale Expression Evaluation

1-(2) rec. plasmids for HEK.EBNA cells

Transient TF into

HEK.EBNA cells in

6-well-plates

Cleared Lysates/Supernatants for In-Process Analytics

Decision on Best Construct/Expression System for Scale-Up

1-(2) “Entry” clones

7-(14) “Destination” vectors

10-(20) E. coli strains

2 clones + 2 fermentation conditions in 96-well-plates

1-(2) rec. bacmids

Transfection into insect

cells in24-well-plates

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Why HEK.EBNA Cells? The Principle

integrated Ad5E1a/E1b fragment in HEK 293 cells enhances trans-cription of CMVpromotor driventransgene

EBNA-1 protein drives episomal replication ofori-P containing plasmids

EBNA-1/ori-P based expression in Human Embryonic Kidney (293) cells (293 stably transformed with EBNA-1 gene)

The cell line is available from ATCC and, until recently, also from Invitrogen

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Why HEK.EBNA Cells? Advantages

• In comparison to other eukaryotic expression systemsthe HEK.EBNA Expression System is rapid:from gene to protein in 4-6 weeks

• It can be applied to generate stable cell lines (pools/ clones) and in transient mode on small and large scale

• The cells can be grown adherently and in serum-free suspension culture

• In transient mode not only secreted and membrane-bound, but also intracellular proteins can successfullybe expressed

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HEK.EBNA Expression Vectors

pRS5a

6372 bps

HpaIEcoRV

MluI

SacI

NheIXhoI

StuI

DraIII

BsaM1

ScaI

OriP

CMV

BGHpASV40-EM-Zeocin

ColE1

Ampicillin

• Basic vector (alsoGateway™ adapted)

• Can be decorated withN- or C-terminal tags, heterologous leadersequences

• Co-expression of e.g. GFP via IRES element

• Selectable marker for generation of stable cell line

Commercially available HEK.EBNA vectors: pREP4 and pCEP4 (Invitrogen)

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Small Scale Expression in HEK.EBNA Cells

6-well-plate:

4.5 x 105 cells/well

24-well-plate:

1.25 x 105 cells/well

48-well-plate:

6 x 104 cells/well

96-well-plate:

6 x 104 cells/well

• Candidate gene: cytokine (tagged)

• HEK.EBNA cells grown in DMEM+ 10 % FCS

• Cell seeding 24 h prior to transfection

• TF reagent: Lipofectamine 2000

(Invitrogen),plasmid/reagent ratio optimised

• Polyfect (Qiagen) and JetPEI (Polyplus) work also effectively

• Protein analysis 3 d post transfection by ELISA

Titer: 5-10 mg/l

Titer: 16 mg/l

Titer: 17-24 mg/l

Titer: > 10 mg/l

Note: This protein is extremely well-behavedin expression, but the same approach works also for less well expressed proteins

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Summary of Small Scale Expression

Small Scale Expression Trials using HEK.EBNA Cellswork very efficiently using

• Adherent cultures in DMEM+ 10 % FCS

• Different well-plate formats

• Lipofectamine 2000™ (Invitrogen): >90 % transfectionefficiency (other reagents also possible – try!)

• Pre-coating with Poly-D-lysine: facilitates attachment of cells and minimizes cell losses during transfection

• Can be also done using serum-free suspension cultures, but less efficient

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Large Scale Transient Transfection (1)

What is “large scale”? 1 – 10 liter

1. Prerequisite:Adaptation of cell culture to serum-free suspension

Several vendors offer cell culture media either specifically developed for HEK293 cells or for othercells (Per.C.6, hybridoma) which can be alsoused for cultivation of HEK293 or HEK.EBNA cells

(see Table on Slide 14)

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2. Prerequisite:a suitable transfection reagent which is cost-effective, readily available in large quantities and gives riseto high transfection efficiencies

Commercially available reagents, such as lipoplexesare by far too expensive at this scale

Transfection using CaPO4 precipitation or cationic polymers such as Polyethylenimine, however, meet the above mentioned criteria and have been successfully used at multi-liter scale

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3. Prerequisite:Generation of sufficient plasmid DNA: an example

20-L Fermentation,

300 g wet cell pellet

pDEST-RS5a8013 bp

CMV

CamRccdB

SV40-EM-Zeocin

OriP

attR1

attR2

BGHpA

ColE1

Ampicillin

E. Coli DH5

NucleoBond™,(Macherey-

Nagel)

30 g pellet

10-15 mg plasmid DNA

pDEST-RS5a8013 bp

CMV

CamRccdB

SV40-EM-Zeocin

OriP

attR1

attR2

BGHpA

ColE1

Ampicillin

pDEST-RS5a8013 bp

CMV

CamRccdB

SV40-EM-Zeocin

OriP

attR1

attR2

BGHpA

ColE1

Ampicillin

pDEST-RS5a8013 bp

CMV

CamRccdB

SV40-EM-Zeocin

OriP

attR1

attR2

BGHpA

ColE1

Ampicillin

pDEST-RS5a8013 bp

CMV

CamRccdB

SV40-EM-Zeocin

OriP

attR1

attR2

BGHpA

ColE1

Ampicillin

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Compatibility of Culture Media and Transfection Reagents

MediumPrice

[CHF/L]

Growth characteristics

HEK.EBNA cells Calciumphosphate PEI

CD 293 140 PRO 293 S 85 293 SFM II 140 FreeStyle 134 n.d. Hektor S 23 EX-CELL VPRO 30 n.d. 2055 44 M11 24 M11V3 50 n.d. DMEM/F12 45 n.d.

Transfectability HEK.EBNA cells

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Polyethylenimine-mediated Transfection

• Back-up cultures kept in roller bottles or Wave reactor

• No medium change, no FCS supplementation

• Transfer of 3.6 liters at 1.4 x 106 cells/ml into 10-L Wave reactor –

Step 1

• Preparation of DNA:PEI complex (10 mg DNA : 20 mg PEI)in 1.4 liters medium M11V3

• Incubation for 15 minutes• Transfer of DNA:PEI complex

into 10-L Wave bioreactor

Step 2

• 5.0 liters transfection mix is incubated for 4 hours

• After 4 hours 5 liters Ex-Cell VPRO medium are added to the culture for the production phase

• Starting conditions: 5 x 105 cells/ml in 10 liters

Step 3

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A Transient Transfection Run…..

0

5

10

15

20

25

0 20 40 60 80 100 120 140 160 180

time [h]

cell

den

sity

[ x

10

5 c

ells

/ml]

0

1

2

3

4

5

6

7

8

9

10

pro

du

ct t

iter

[m

g/l]

cell density product titer

Cell density in 3.6 volume

prior to transfection

Cell density after additionof 1.4 l transfection mix

Cell density after addition

of 5 l growth medium

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….in Multiparallel Fashion

The “Wave Factory”

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Cell/Supernatant Harvest and Cell Lysis

Cell concentrat

e

Supernatant

Wave bag

Secreted productin supernatant

orCell concentration

Cell debris

Clear Lysate

Intracellular product:

Cell concentrate+ Lysis buffer

Released productin cleared lysate

Wave bag

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PPCW Workshop NIH3/28/2004 Protein Purification

For Secreted Proteins: affinity chromatography on antibody or Protein A column

-tag dependent-

For Intracellularly Expressed Proteins: his and/or his-Strep tag

Ni-chelate and/or Streptactin column

To date, > 30 proteins were generated successfully using this approach.

The overall success rate in expression trials was >80 %.

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A Few Examples

Cytokine: 2.5 mg/l

Soluble Rec.: 12 mg/l

APP family: 3.5 mg/l

Cytokine: 7.5 mg/l

Dehydrogenase:2.6 mg/l

Kinase:8.5 mg/l

Membrane Glycoprotein:

0.5 mg/l

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To summarize…….

Mol.Biol.: Cloning + Plasmid Generation

Small Scale Expression Evaluation on 6-well-plate

Large-Scale Plasmid Prep

1-2 l-Batch Production in Roller Bottles

10-l-Wave Production

- functionality-testing of plasmid prior to large

scale plasmid prep- indication on expression levels to be expected

Generation of multiple E.coli pellets for several production runs

- Validation of large scale plasmid prep- 1st purification trial- may suffice for entire production

One run sufficient to meet demands in most cases

Miniaturized format for multi-parallel analyses available (24/48 well-plate)

Multi-parallel plasmid preparations possible

Shake flasks cultures or spinners also possible

Multi-parallel production if several Wave bioreactors available

Workflow for Large Scale Transient Transfections

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Acknowledgements: The BTP team

• HP Kocher (UH, Biomolecule Production)

• S Geisse (Baculo, Mammalian)• M. Buchs• A Patoux• B Rudin

• S Hartmann (IPC, In process & funct. analytics)

• D Rinaldi• T Kuiper

• M Henke (Fermentation)• S Dalcher• R Uhrhahn

• F Kolbinger (MolBio, Mammalian) PTH BTP Program

• A Schildhauer

• M Mahnke (E.coli) PTH EXA Program• S Deutsch• E Eglin• Y Pouliquen

• JM Schlaeppi (Protein Pur.) • A Berner

• R Schmitz (MolBio) • N Charara• K Leon

• T Soellick (Bioinf, ProTrack)

• M Zurini (Protein Pur.)• J Causevic• R Enderlin• D Plattner

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1 PPCW Workshop NIH 3/28/2004 Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer,...

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