1 PPCW Workshop NIH 3/28/2004 Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer,...
-
Upload
kelly-hudson -
Category
Documents
-
view
226 -
download
0
Transcript of 1 PPCW Workshop NIH 3/28/2004 Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer,...
1
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?
2
PPCW Workshop NIH3/28/2004
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.
3
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
4
PPCW Workshop NIH3/28/2004
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
5
PPCW Workshop NIH3/28/2004
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
6
PPCW Workshop NIH3/28/2004
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
7
PPCW Workshop NIH3/28/2004
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
8
PPCW Workshop NIH3/28/2004
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)
9
PPCW Workshop NIH3/28/2004
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
10
PPCW Workshop NIH3/28/2004
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
11
PPCW Workshop NIH3/28/2004
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)
12
PPCW Workshop NIH3/28/2004
Large Scale Transient Transfection (2)
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
13
PPCW Workshop NIH3/28/2004
Large Scale Transient Transfection (3)
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
14
PPCW Workshop NIH3/28/2004
Large Scale Transient Transfection (4)
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
15
PPCW Workshop NIH3/28/2004
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
16
PPCW Workshop NIH3/28/2004
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
17
PPCW Workshop NIH3/28/2004
….in Multiparallel Fashion
The “Wave Factory”
18
PPCW Workshop NIH3/28/2004
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
19
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 %.
20
PPCW Workshop NIH3/28/2004
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
21
PPCW Workshop NIH3/28/2004
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
22
PPCW Workshop NIH3/28/2004
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