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1 | © 2013 Life Technologies
ELRIG: Advances in Recombinant Protein Technology Alderley Park UK 19-20 Nov 2013
Stepping Up Transient Expression: The Expi293™Expression System
Henry C Chiou, Ph.D.Gibco Cell Biology SystemsLife Technologies
2 | © 2013 Life Technologies
Protein Expression from Mammalian Cells
5 to 7 days
High EfficiencyTransfection Expression
ProteinProduction
Transient Expression
Months
Transfection
Isolation,Adaptation,Banking & Expansion
ProteinProduction
Expression,Selection &
Sub-culturing
Classic Stable Expression
3 | © 2013 Life Technologies
The Need: Keep fast turnaround, get MORE PROTEIN
5 to 7 days
High EfficiencyTransfection Expression
ProteinProduction
Transient Expression
Months
Transfection
Isolation,Adaptation,Banking & Expansion
ProteinProduction
Expression,Selection &
Sub-culturing
Classic Stable Expression
4 | © 2013 Life Technologies
Published high density transient protocol forincreased yield
600mg/L at Day 7
Protocol:Cell prep:
Expand cells in growth medium
Concentrate cells → 20x106/ml transferred into transfection compatible medium
Transfection:PEI + 37.5% IgG HC, 37.5% IgG LC, 10% p18h, 10% p21h, 5%
aFGF(2.5X more DNA/cell than standard protocol)(1.7X more PEI/cell than standard protocol)
Post Transfection Treatment:
3-4 hours post-transfection → dilute culture with growth medium to 4x106/ml
Add medium supplements
7 days to reach 600mg/L Human IgG, 14 days to 1 gm/L
Backliwal et al., Biotechnol Bioeng 2007
Protocol is requires spin down of cells, multiple plasmids, proportionately higher
DNA and transfection reagent per cell. Is not readily scalable
5 | © 2013 Life Technologies
Strategy to Increase Transient Protein Yields
++
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+
+
++
++ ++
++
+
+
++
+ +++
+
+
+
++
+ +++
+
+
+
3-10X Yield
Expi293 Innovations:• Culture medium that achieves 3-fold higher cell density
• Cells that express more protein per cell
• Transfection reagent that effectively delivers DNA into high cell density cultures
• System plugs directly into users current 5-7 day workflow
More Cells Per mL High EfficiencyTransfection More ProteinHigher Productivity
Per Cell
6 | © 2013 Life Technologies
• Chemically defined, protein-free, AOF, serum free medium• Supports viable cell densities up to 18 x 106 cells/ml• High culture viabilities• Single medium solution for cell growth and transient expression
•Transfection compatible formulation•Supports high expression yields
Expi293 Medium
Medium 2
Medium 3
Medium 4
Expi293™ Expression Medium: A high density growth & transfection medium
Unless otherwise stated, all data shown was generated at Life Technologies.
7 | © 2013 Life Technologies
0
200
400
600
800
1000
1200
hIgG
(ug/
ml)
hIgG titer (passage-to-passage)
d5
d6
d7
Adapted 293F cellshigh yield Expi293F™ cells Bank 2
Day
Bank 1
• high yield cells have higher viability, cell density, and cell size vs. other 293 cells
• Significant increase in protein production
Derived from FreeStyle™ 293F cells
Adapted into Expi293™ Expression Medium
Culture viability
95
95.5
96
96.5
97
97.5
98
98.5
FreeStyle293F
Kentaro-adapted
293F
High Yield 293F
perc
ent v
iabl
e ce
lls
Expi-
Culture Viability
Cell size
16
16.5
17
17.5
18
18.5
FreeStyle293F
Kentaro-adapted293F
High Yield 293F
mic
rons
Expi-
Cell Size
VCD
0
1
2
3
4
5
6
FreeStyle293F
Kentaro-adapted
293F
High Yield 293F
cells
/ml
(x10
^6)
Expi-
VCD
Expi293F™ cells: higher per cell productivity
8 | © 2013 Life Technologies
ExpiFectamine™ 293 Transfection Performance
0
10
20
30
40
50
60
70
80
90
30 mL 1 mL Neg
% G
FP-P
osit
ive
Cells
Transfection Efficiency(day 3 pt)
Transfection Enhancers(added 18-24 hr pt)
(125 mL shake flask) (24-well plate)
• ExpiFectamine™ 293 achieves high efficiency transfection of high density cultures
• Enhancers combine to maximize yields
0
1
2
3
4
Fold
Cha
nge
In E
xpre
ssio
nEnhancer 1 Enhancer 2
−−
+−
−+
++
9 | © 2013 Life Technologies
Human IgG
0
200
400
600
800
1000
1200
1ml 30ml 1000ml
ug/m
l hIg
G
ExpiFectamine™ 293 Transfection: Yield and scalability
EPO
0
1
2
3
4
5
6
7
8
9
FS 1ml
Expi 1ml
FS 30ml
Expi30ml
FS 1L
Expi 1L
EPO
(yie
ld re
lativ
e to
1m
l sca
le F
S st
anda
rd)
1ml 30ml 1000ml
FreeStyle™ 293Expi293™
B2 Adrenergic Receptor VLPs
0
20
40
60
80
100
120
FS 30ml Expi 30ml FS 1L Expi 1L
VLP
yie
ld (u
g/m
l of c
ultu
re)
30ml 1000ml
Cripto
0
200
400
600
800
1000
1200
FS 1ml Expi 1ml FS 30ml Expi 30ml FS 1L Expi 1L
prot
ein
mg/
L
1ml 30ml 1000ml
Human IgG
1 ml = 24 well plate30 ml = 125 mL shake flask1 L = 3 L shake flask
1ml 30ml 1000ml
11 | © 2013 Life Technologies
External Results
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22 Labs, 98 Proteins,average 4.6-fold increase
Fold
Yie
ld I
ncre
ase
• 87% demonstrated increased expression in Expi293™ vs users current system
• 4.6X average increase for all proteins (n=98)
• 4.0X average increase for mAbs (n=54) highest level – 826 mg/L
• 5.3X average increase for non-mAbs (n=44 ) highest level – 790 mg/L
12 | © 2013 Life Technologies
96 well plate (square well)2 mL deep well format700 µl fill volume3 mm-throw shaker1000-1250 rpm
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
3.0E+07
6 well 12well 96well
GFP
TU
/mL
plate format
Lentivirus (from pLenti6.3-EmGFP) produced in microplate format using Expi293™
96 well optimization
0100200300400500600
190 rpm(0.75 inch
throw)
700 rpm 1000 rpm 1250 rpm
IgG
yie
ld (
mg/
L)
96 deep well optimization
(19 mmthrow)
(3 mm throw)
Adaptation of Expi293™ to microplate formatsMost plates only require adjustments in shaker rotational speeds.
EppendorfMixMate
Benchmark Orbi-Shaker MP
13 | © 2013 Life Technologies
Protein Functionality: equivalent to FreeStyle™ 293
0 5 10 15 20 25 300
10
20
30
40
Total BoundSpecific BoundNon-Specific Bound
BmaxKd
Total Bound31.411.143
Specific Bound30.081.069
[3H] DHA (nM)
Rad
ioac
tivity
Bou
nd (p
mol
/mg)
0 5 10 15 20 25 300
10
20
30
40
Total BoundSpecific BoundNon-Specific Bound
BmaxKd
Total Bound30.010.7543
Specific Bound29.141.057
[3H] DHA (nM)
Rad
ioac
tivity
Bou
nd (p
mol
/mg)
Expi293™ FreeStyle™
Beta2 adrenergic receptor VLPs challenged with increasing 3[H] DHA antagonist in a saturation binding experiment +/-100uM cold propanolol.0.5ug VLP protein per reaction
12 25 50 100 200ng60504025
12 25 50 100 200ng
Expi293™ FreeStyle™
IgG functionality: Abs show equivalent reactivity against target
Equivalent IgG glycosylation profiles:
GPCR:Comparable and physiological antagonist binding activity
pmol ligand/mg proteinnM
pmol ligand/mg proteinnM
Expi293™
3xyield
>10xyield
Expi293™
FreeStyle™
[Erythropoeitin] ng/mL
Rela
tive
Lum
ines
cenc
e Un
its (R
LU)
0.0001 0.01 1 100 10000
50000
100000
150000
200000
Expi293
Freestyle293
EPO Activity:Measured by TF-1 cell-based proliferation assay
5xyield
14 | © 2013 Life Technologies
Clone GOI into pEF6 TOPO® Expression
Vector
MembranePro™
Reagent+
Add MembranePro™
Precipitation Mix
Re-suspend MembranePro™ particles (50-500ug protein, enough for up to
1,000 ligand binding assay data points)
Lipofectamine™ 2000 Transfection Reagent
Decant culture medium with MembranePro™ particles (48hrs post-transfection)
Precipitate for 18hrs at 4ºC
and centrifuge
Add transfection
mix to
HEK-293FT Cells
+
Expression of Membrane Proteins onVLPs – MembranePRO
15 | © 2013 Life Technologies
Clone GOI into pEF6 TOPO® Expression
Vector
MembranePro™
Reagent+
Add MembranePro™
Precipitation Mix
Re-suspend MembranePro™ particles (50-500ug protein, enough for up to
1,000 ligand binding assay data points)
ExpiFectamine™ 293 Transfection Reagent
Decant culture medium with MembranePro™ particles (48hrs post-transfection)
Precipitate for 18hrs at 4ºC
and centrifuge
Add transfection
mix to
Expi293F™ Cells
+
Expression of Membrane Proteins onVLPs – MembranePRO
16 | © 2013 Life Technologies
Scalable lentivirus production in Expi293™
0.0E+00
2.0E+07
4.0E+07
6.0E+07
6 well 12well 96well
Tite
r TU
/mL
plate format
Lentivirus from pLenti6.3-EmGFP produced in microplate format
Expi293™ protocol for 30ml suspension culture production of lentivirus:
9ug pLenti construct27ug Virapower™ packaging mixin 4ml Opti-MEM I
180ul Expifectamine™ 293in 4ml Opti-MEM I
20 minutes RT.Add to 22ml Expi293™ cells(3x10^6 vcd/ml).Shake 125rpm, 8% CO2, 37oC.Next day: add 150ul of Enhancer 1
1.5ml of Enhancer 2Harvest 24-48hrs later.
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1.0E+07
1.2E+07
1.4E+07
7.3TOPO 7.3GW
GFP
TU
/ml
AdherentLenti Yield
17 | © 2013 Life Technologies
Acknowledgements
Expi293™ Development team:Carlsbad, CASanjay VasuIsabel CisnerosJeff RogersJingqiu LiDan BezdekStacey WinterToby JordanGabriella JaharisMaggie Abo-HebeishAlda ChangSahil NockRik BasiHenry Chiou
Frederick, MDMeredith JonesShyam KumarJon ZmudaAlice FanJim SanfordDana MeentsBrandon HarrisDianna FoxBryan Poltilove
Grand Island, NYChao Yan LiuRichard Del MaestroChang ChoiBryan GreeneBrian BurginBob KenersonDave Dugan
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