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FreeStyle® MAX (1.25 µg/mL)

PEI (1 µg/mL)

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GFP

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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)

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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

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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®

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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