improving transient cho and hek-293 expression systems ... · production in both cho and hek cells...
TRANSCRIPT
0
10
20
30
40
50
0 1 2 3 4 5 6
Co
nce
ntr
atio
n (
mg
/L)
Days post-transfection
1 L
100 mL
30 mL
0
10
20
30
0 1 2 3
Co
nce
ntr
atio
n (
mg
/L)
Days post-transfection
1 L
100 mL
30 mL
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5
Co
nce
ntr
atio
n (
mg
/L)
Days post-transfection
FectoPRO®(0.8 µg/mL)
FreeStyle® MAX (1.25 µg/mL)
PEI (1 µg/mL)
0
20
40
60
80
100
0.8 µg DNA/mL 1.25 µg DNA/mL 1 µg DNA/mL
FectoPRO® FreeStyle™ MAX TransIT-PRO®C
on
cen
trat
ion
(m
g/L
)
0
20
40
60
80
100
0.8 µg DNA/mL 1.25 µg DNA/mL
FectoPRO® FreeStyle™ MAX
Perc
enta
ge (
%)
GFP
-exp
ress
ing
cells
CD CHO CD FortiCHO™
HyClone™ HyCell™ TransFX™-C FreeStyle™ CHO
FreeStyle™ F17 CHO-S-SFM II
Improving transient CHO and HEK-293 Expression systems with a powerful transfection solution for high protein production yields: FectoPRO®
Mathieu Porte, Jonathan Havard, Valérie Moreau, Jelena Vjetrovic, Fabrice Stock, Patrick ErbacherPolyplus-transfection, Bioparc, 850 Boulevard S. Brant, 67400 Illkirch, France
Development process for biotherapeutic protein production usually begins with generating a high-performing stable cell line which can be used for manufacturing. As this step takes a lot of time, transienttransfection offers a great alternative to quickly produce milligram to gram quantities of recombinantproteins and antibodies. A various number of culture media are available to perform transient proteinproduction in both CHO and HEK cells but the limiting factor often remains the transfection reagent.Therefore, Polyplus-transfection® has developed a novel technologically advanced transfection solutionnamed FectoPRO®. Here we show that FectoPRO® outperforms currently available PEI-based and lipid-based transfection reagents in all the transient expression systems tested, offering great transfectionefficiency and amazing protein yields.
Protocol
Conclusion
Remarkably efficient in HEK-293 cells
M: Marker
1: untransfected cells
2: FectoPRO® (0.8 µg/mL)
3: FreeStyle™ MAX Reagent (1.25 µg/mL)
4: TransIT-PRO® (1 µg/mL)
CHO-S HEK-293F
Significantly better yield of full mouse IgG is obtained with lower DNA amount when using FectoPRO® in comparison withFreeStyle™ Max and PEI. Mouse IgG production in CHO-S cells was achieved by co-transfection of plasmids coding for the Heavychain & Light chain. Quantification was performed using protein G Biosensors (fortéBIO®). Qualitative analysis was done on 8%non reducing PAGE and 12% reducing PAGE 5 days post-transfection. Data kindly provided by ProteoGenix SA.
High-yield production of full mouse IgG in CHO-S cells
Great protein production in HEK-293F cells
Significantly higher protein production yields are reached when using FectoPRO® in HEK-293F cells in comparison withcompetitors. FreeStyle™ 293F cells were seeded at 1 x 106 cells/mL in 30 mL of FreeStyle™ 293 Expression Medium andtransfected with FectoPRO® and its competitors following the recommended protocols. Quantitation of IgG3-Fc fragment wasperformed by using protein G affinity column (HPLC) and qualitative analysis was done by Western Blot 72 hours post-transfection .
1- The day before transfection, prepare cell suspensionat 1 x 106 cells/mL.
2- On the day of transfection, prepare the transfection mixin the serum free medium.
3- Add the FectoPRO®-DNA transfection mix to the cells,homogenize the culture.
4- If FectoPRO® Booster is to be added, add it directly tothe cell culture 0 to 4 hours post-transfection,homogenize.
5- Harvest protein or antibody when required.
A perfect scalability for protein production is observed with FectoPRO® in both CHO and HEK-293 cells. FreeStyle™ CHO-Sand HEK-293F cells were seeded at 1 x 106 cells/mL in either 30 mL, 100 mL or 1 L of their recommended FreeStyle™ mediaand transfected with an IgG3-Fc expressing plasmid using FectoPRO®+ FectoPRO® Booster (0.5 µg DNA/mL). Quantification wasperformed every day using protein G affinity column (HPLC).
(1)
(2)
(3)
Advantages of FectoPRO®
• Amazing antibody yields in CHO & HEK-293 suspension cells, including high cell density systems• Cost-effective Transient Gene Expression using low DNA amount (<1 µg/mL of cell culture)• Sustained protein and antibody production over several days• Easily scalable from a few mL to several liters of cell culture• Compatible with various mammalian expression media and cell systems
FectoPRO®’s high transfection efficiency isindependent of the cell culture medium.FreeStyle™ CHO-S were seeded at 1 x 106
cells/mL in 30 mL of the mentioned mediaand transfected with FectoPRO® (0.8µg/mL) or FreeStyle™ MAX following thestandard protocols. GFP expression wasassayed using fluorescence cytometry 24hours post-transfection.
FectoPRO® allows a sustained proteinproduction with yields similar toExpiFectamine™ 293 while using 20% lessDNA. Expi293F™ cells were seeded followingthe recommended protocol in Expi293™Expression Medium, and transfected withFectoPRO® + FectoPRO® Booster (0.8 µgDNA/mL), ExpiFectamine™ 293 + Enhancers(1 µg DNA/mL), or FreeStyle™ MAX (1.25µg/mL) following the standard protocols.IgG3-Fc production was assayed at differentdays by protein G affinity quantification(HPLC).
Sustained protein production with low DNA amount in the Expi293™ system
0
25
50
75
100
0 1 2 3
Co
nce
ntr
atio
n (
mg
/L)
Days post-transfection
FectoPRO® + FectoPRO® Booster (0.8 µg/mL)
ExpiFectamine™ 293 + Enhancers (1 µg/mL)
FreeStyle™ MAX (1.25 µg/mL)
Higher yields obtained faster in the ExpiCHO™ system
FectoPRO® protocol, to triple protein productionyields in the ExpiCHO™ system compared toExpiFectamine™-mediated transfection, as earlyas 3 days after transfection.ExpiCHO-S™ cells were seeded following therecommended protocol in ExpiCHO™ ExpressionMedium, and transfected with FectoPRO® (0.8 µgDNA/mL) or ExpiFectamine™ CHO + Feed +Enhancer (0.8 µg DNA/mL) following the standardprotocols. IgG3-Fc production was assayed atdifferent days using protein G Biosensors(fortéBIO® BLItz).Detailed protocol available upon request.
www.polyplus-transfection.com
FreeStyle™, Expi293™, ExpiCHO™, ExpiFectamine™, FortiCHO™ are trademarks of Life Technologies™ Corporation.TransIT-PRO® is a trademark of Mirus® Bio LLC. HyClone™, HyCell™ and TransFx™ are trademarks of General Electric Company.fortéBIO® is a trademark of Pall Corporation.FectoPRO® is a trademark of Polyplus-transfection®.
FectoPRO™ gives high transfection efficiency in suspension HEK-293F cells. HEK-293F cells were seeded at 1 x 106 cells/mL in 30mL of FreeStyle™ 293 Expression Medium and transfected using a GFP expressing plasmid with FectoPRO™ (0.8 µg DNA/mL), PEI(1 µg/mL), FreeStyle™ MAX Reagent (1.25 µg DNA/mL) or TransIT-PRO® (1 µg DNA/mL). GFP expression was observed 24 hoursafter transfection using fluorescence microscopy.
Specifically developed for CHO cells
Non transfected cells
0%
FectoPRO®
64%
PEI
3%
FreeStyle™ Max
15%
Superior transfection efficiency in CHO-K1
Easily implementable in a production process
FectoPRO® shows a remarkable transfection efficiency in CHO-K1 cells in comparison to PEI and FreeStyle™ MAX. Suspension-adapted CHO-K1 cells were seeded following the recommended protocol, and transfected with FectoPRO® (0.8 µg DNA/mL), PEI(1 µg DNA/mL), and FreeStyle™ MAX (1.25 µg/mL) following the standard protocols. Transfection efficiency was determined bymeasuring the percentage of GFP-expressing cells by capillar cytometry 24 hours post-transfection.
High transfection efficiency in HEK-293F cells
Compatible with various synthetic media
Abstract
Great scalability for antibody production
FectoPRO® PEIFreeStyle™ MAX TransIT-PRO®
0
20
40
60
80
0 2 4 6 8 10
Co
nce
ntr
atio
n (
mg
/L)
Days post-transfection
FectoPRO optimized protocol
FectoPRO standard protocol
Expifectamine standard protocol
Culture amplification
for 4 days(seeding at
0,3 x 106/ml)
ExpiFectamine™standard protocol
FectoPRO® optimized protocol
D1 D2D0
Dilution 6 x 106/ml
Transfection
ExpiFeed
HARVEST
D3 – D10
Dilution 4 x 106/ml
Transfection
Dilution 6 x 106/ml
Add FectoPRO® into an emptytube
Pour transectionmix into the cells
Vortex immediately and incubate 10 min at RT
Add FectoPRO® Booster if needed
Pour diluted DNA intoFectoPRO®
Dilute DNA into serum-free medium