Transcript HEK-EBNA expression - Rutgers University

PPCW
Workshop NIH
3/28/2004
Transient Transfection into Eukaryotic Cells:
An Alternative to Bacterial and Insect Cell Systems
for the Rapid Generation of Recombinant Proteins?
Bethesda,MD,30.3.2004
Sabine Geisse,
Nicola di Maiuta, Thomas Cremer,
Mario Henke
Novartis Institutes for Biomedical Research,
Basle, Switzerland
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PPCW
Workshop NIH
3/28/2004
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.
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Workshop NIH
3/28/2004
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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Workshop NIH
3/28/2004
A Double-Track Process to Increase
Success Rates
PPC
Transformation/Transfection
ICU
Small Scale Expression
Evaluation
Scale-Up
Evaluate Special
Conditions for Scale-Up
Large-Scale Fermentation
Large-Scale Fermentation
Automated Harvest +
Cell Lysis
Automated Purification
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Standard Harvest + Lysis
Classical Purification
PPCW
Workshop NIH
3/28/2004
Small Scale Expression Evaluation
1-(2) “Entry” clones
7-(14) “Destination” vectors
10-(20) E. coli strains
1-(2) rec. bacmids
1-(2) rec. plasmids
for HEK.EBNA cells
2 clones +
2 fermentation
conditions in
96-well-plates
Transfection
into insect
cells in
24-well-plates
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
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Workshop NIH
3/28/2004
Why HEK.EBNA Cells? The Principle
EBNA-1/ori-P based expression in Human Embryonic Kidney
(293) cells (293 stably transformed with EBNA-1 gene)
EBNA-1 protein
drives episomal
replication of
ori-P containing
plasmids
The cell line is available from ATCC and,
until recently, also from Invitrogen
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integrated Ad5
E1a/E1b fragment
in HEK 293 cells
enhances transcription of CMV
promotor driven
transgene
PPCW
Workshop NIH
3/28/2004
Why HEK.EBNA Cells? Advantages
• In comparison to other eukaryotic expression systems
the HEK.EBNA Expression System is rapid:
from gene to protein in 4-6 weeks
• The cells can be grown adherently and in serum-free
suspension culture
• It can be applied to generate stable cell lines
(pools/ clones) and in transient mode on
small and large scale
• In transient mode not only secreted and membranebound, but also intracellular proteins can successfully
be expressed
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PPCW
Workshop NIH
3/28/2004
HEK.EBNA Expression Vectors
HpaI
EcoRV
ScaI
MluI
Am picillin
OriP
pRS5a
ColE1
6372 bps
BsaM1
DraIII
SV40-EM-Zeocin
BGHpA
CMV
SacI
StuI
XhoI
NheI
• Basic vector (also
Gateway™ adapted)
• Can be decorated with
N- or C-terminal tags,
heterologous leader
sequences
• 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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PPCW
Workshop NIH
3/28/2004
Small Scale Expression in HEK.EBNA Cells
• 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
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• Protein analysis 3 d post
transfection by ELISA
6-well-plate:
4.5 x
105 cells/well
24-well-plate:
Titer: 5-10 mg/l
Titer: 16 mg/l
1.25 x 105 cells/well
48-well-plate:
Titer: 17-24 mg/l
6 x 104 cells/well
96-well-plate:
Titer: > 10 mg/l
6 x 104 cells/well
Note: This protein is extremely well-behaved
in expression, but the same approach works
also for less well expressed proteins
PPCW
Workshop NIH
3/28/2004
Summary of Small Scale Expression
Small Scale Expression Trials using HEK.EBNA Cells
work very efficiently using
• Adherent cultures in DMEM+ 10 % FCS
• Different well-plate formats
• Pre-coating with Poly-D-lysine: facilitates attachment
of cells and minimizes cell losses during transfection
• Lipofectamine 2000™ (Invitrogen): >90 % transfection
efficiency (other reagents also possible – try!)
• Can be also done using serum-free suspension cultures,
but less efficient
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PPCW
Workshop NIH
3/28/2004
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 other
cells (Per.C.6, hybridoma) which can be also
used for cultivation of HEK293 or HEK.EBNA cells
(see Table on Slide 14)
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PPCW
Workshop NIH
3/28/2004
Large Scale Transient Transfection (2)
2. Prerequisite:
a suitable transfection reagent which is cost-effective,
readily available in large quantities and gives rise
to high transfection efficiencies
 Commercially available reagents, such as lipoplexes
are 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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PPCW
Workshop NIH
3/28/2004
Large Scale Transient Transfection (3)
3. Prerequisite:
Generation of sufficient plasmid DNA: an example
Ampicillin
OriP
ColE1
pDEST-R S5a
8013 bp
CMV
SV40-EM-Zeocin
BGHpA
attR1
attR2
ccdB
E. Coli DH5
20-L Fermentation,
CamR
Ampicillin
OriP
ColE1
Ampicillin
OriP
pDEST-R S5a
8013 bp
 300 g wet cell pellet
CMV
ColE1
Ampicillin
SV40-EM-Zeocin
OriP
pDEST-R S5a
8013 bpattR1
BGHpA
attR2
ColE1
ccdB
SV40-EM-Zeocin
CMV
Ampicillin
CamR
OriP
pDEST-R S5a
8013 bpattR1
BGHpA
attR2
ColE1
ccdB
SV40-EM-Zeocin
CMV
CamR
pDEST-R S5a
8013 bpattR1
BGHpA
attR2
CMV
ccdB
SV40-EM-Zeocin
CamR
BGHpA
attR1
attR2
30 g pellet
NucleoBond™,
(Macherey-Nagel)
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10-15 mg
plasmid DNA
ccdB
CamR
PPCW
Workshop NIH
3/28/2004
Large Scale Transient Transfection (4)
Compatibility of Culture Media and Transfection Reagents
Transfectability
HEK.EBNA cells
Medium
14
Price
[CHF/L]
CD 293
140
PRO 293 S
85
293 SFM II
140
FreeStyle
134
Hektor S
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EX-CELL VPRO
30
2055
44
M11
24
M11V3
50
DMEM/F12
45
Growth
characteristics
HEK.EBNA cells
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
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



Calciumphosphate
PEI



n.d.

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







n.d.
n.d.

n.d.


PPCW
Workshop NIH
3/28/2004
Polyethylenimine-mediated Transfection
Step 1
• 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 3
Step 2
• 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
• 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
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PPCW
Workshop NIH
3/28/2004
A Transient Transfection Run…..
25
10
9
Cell density after addition
of 1.4 l transfection mix
20
8
Cell density after addition
of 5 l growth medium
7
15
6
5
10
4
3
5
2
1
0
0
0
20
40
60
80
100
120
time [h]
cell density
16
product titer
140
160
180
product titer [mg/l]
cell density [ x 10 5 cells/ml]
Cell density in 3.6 volume
prior to transfection
PPCW
Workshop NIH
3/28/2004
….in Multiparallel Fashion
The “Wave Factory”
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PPCW
Workshop NIH
3/28/2004
Cell/Supernatant Harvest and Cell Lysis
Secreted
product
in supernatant
or
Cell
concentration
Cell
concentrate
Super
natant
Intracellular
product:
Cell concentrate
+ Lysis buffer
Released product
in cleared lysate
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Cell debris
Clear
Lysate
PPCW
Workshop NIH
3/28/2004
Protein Purification
For Secreted Proteins:
affinity chromatography on antibody or Protein A column
-tag dependentFor 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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PPCW
Workshop NIH
3/28/2004
A Few Examples
Cytokine:
2.5 mg/l
Soluble Rec.:
12 mg/l
Cytokine:
7.5 mg/l
APP family:
3.5 mg/l
Kinase:
8.5 mg/l
Dehydrogenase:
2.6 mg/l
Membrane
Glycoprotein:
0.5 mg/l
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PPCW
Workshop NIH
3/28/2004
To summarize…….
Workflow for Large Scale Transient Transfections
Mol.Biol.:
Cloning +
Plasmid
Generation
Small Scale
Expression
Evaluation
on 6-wellplate
- functionality-testing of
plasmid prior to large
scale plasmid prep
- indication on expression
levels to be expected
Miniaturized
format for multiparallel analyses
available (24/48
well-plate)
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Large-Scale
Plasmid
Prep
Generation of
multiple E.coli
pellets for several
production runs
Multi-parallel
plasmid
preparations
possible
1-2 l-Batch
Production
in Roller
Bottles
10-l-Wave
Production
- Validation of large
scale plasmid prep
- 1st purification trial
- may suffice for entire
production
Shake flasks
cultures or
spinners also
possible
One run sufficient
to meet demands
in most cases
Multi-parallel
production if
several Wave
bioreactors
available
PPCW
Workshop NIH
3/28/2004
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
•
•
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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