HYPE-5™ Transfection Kit Results

HYPE-5™ Transfection Kit is dedicated to achieve High Yield Protein Expression in mammalian cells. This Kit
has been designed for maximum efficiency in HEK293 and CHO cells growing in suspension. The transient
transfection expression system represents an ideal and faster alternative to the stable expression system
(costly and time-consuming). Scale-up to larger volumes for production of milligrams of protein per liter of cell
culture is straightforward and easy with simple and cost efficient handling steps.

Main features are:

1. High protein production yield.
2. Suitable for both HEK293 and CHO cells growing in suspension.
3. Compatible with any synthetic or regular media used for protein production.
4. Ideal for bioreactor, spinner or flasks.
5. Rapid, simple to scale up and ready-to-use.
6. Free from animal sources.

Applications

HYPE-5™ Transfection Kit has been designed for large scale up transient transfection and high protein
expression. The system is optimized for maximum efficiency in HEK293 and CHO cells. It has been used and
validated with cells from different origins (FreeStyleTM CHO-S/293-F cells from Invitrogen, CHO protein free
from ECACC, etc.) cultured in suspension (flask, spinner and bioreactor).

It is suitable for any kind of mammalian cell models used to produce proteins. Please do not hesitate to
contact us at tech@ozbiosciences.com so we can give you all the support you need.

HYPE-5™ Kit is suitable for any synthetic or regular culture media. It has been tested with various chemically
defined media:

Medium HEK293 CHO

FreeStyleTM CHO-S NA
FreeStyleTM 293 NA
ProCHO™ 4-5 NA
Pro293™S-CDM NA
®
EX-CELL ACF CHO NA
®
EX-CELL 293 NA
BD Select™ CD 1000 NA
NA: Not Applicable

The results shown below have been obtained with FreeStyle™ 293/CHO-S systems (Invitrogen) and the
maximum efficiency has been reached when HYPE-5 reagent was used alone for HEK293 cells or combined
with HYPE-Blast for CHO cells.

Efficient protein production is highly dependent on the cell model used. For instance, adaptation of cells to
growth in suspension, culture medium and cell density parameters are of importance to obtain the maximum
efficiency. We advise optimizing experimental conditions (see instruction manual, section 4) in order to achieve
the best efficiency.

OZ Biosciences HYPE-5 Transfection Reagent - Results Page 1 of 6

High production yield for secreted protein

The results presented below indicate that HYPE-5™ Transfection Kit achieves the highest protein yield in
comparison to other commercially available transfection reagents in HEK293 and CHO cells adapted in
suspension with chemically defined medium and in absence of serum.

CHO cells
1400
µg of SEAP per Liter of cell culture

1200

1000

800

600

400

200

0
HYPE-5 + HYPE-Blast LP FHD

day 3 day 5 day 7

HEK293 cells
12000
µg of SEAP per Liter of cell culture

10000

8000

6000

4000

2000

0
HYPE-5 LP FHD

day 3 day 5 day 7

Fig.1: SEAP Expression with HYPE-5 Kit vs. other reagents.

Secreted Alkaline Phosphatase (SEAP) was produced by transient transfection using HYPE-5 Transfection Kit,
LP or FHD transfection reagents according to manufacturer’s instructions. Transfections were performed
using 1.5 µg of plasmid DNA per mL of cell culture. Ratio (µL reagent per µg of plasmid) for HYPE-5 reagent,
LP and FHD was 2, 2.5, and 3.5, respectively. CHO and HEK293 cells were cultured in 0.5 mL in a 24-well
shaken plate. Density of cell culture was 1 x 106 cells/mL the day of transfection. 4 hours after transfection, for
CHO cells only, HYPE-Blast was added to cell suspension. Day 3, 5 and 7, 20µL of supernatants were
collected and SEAP activity was measured.

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Outperforms competitor

The results presented below indicate that HYPE-5™ Kit outperforms other commercially available reagents
specific for biomanufacturing in HEK293 and CHO cells adapted in suspension with chemically defined
medium and in absence of serum.

CHO cells HEK293 cells

1200,0 14000

µg of protein per Liter of cell culture

µg of SEAP per Liter of cell culture

1000,0 12000

10000
800,0
8000
600,0
6000
400,0
4000

200,0 2000

0,0 0
HYPE-5 + HYPE-Blast FSM HYPE-5 FSM

SEAP Expression SEAP Expression β-Gal Expression

Fig.2: SEAP/β-Gal Expression with HYPE-5 Kit vs. FSM reagent.

Secreted Alkaline Phosphatase (SEAP) or β-Galactosidase (β-Gal) were produced by transient transfection
using HYPE-5 Kit or FSM transfection reagents according to manufacturer’s instructions. Transfections were
performed using 1.5 µg of plasmid DNA per mL of cell culture. Ratio (µL reagent per µg of plasmid) for HYPE-
5 reagent and FSM reagents was 2 and 1, respectively. CHO and HEK293 cells were cultured in 30 mL in 125
mL shaken flasks. Density of cell culture was 1 x 106 cells/mL the day of transfection. 4 hours after
transfection, for CHO cells only, HYPE-Blast was added to cell suspension. Day 5 (CHO cells) and 7 (HEK293
cells) 500µL of supernatants were collected and SEAP or β-Gal activity was measured.

CHO cells
1200,0
µg of SEAP per Liter of cell culture

1000,0

800,0

600,0

400,0

200,0

0,0
HYPE-5 + HYPE-Blast FSM

day 3 day 5 day 7

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 µg of SEAP per Liter of cell culture HEK293 cells
12000

10000

8000

6000

4000

2000

0
HYPE-5 FSM
day 3 day 5 day 7
Fig.3: SEAP Kinetic expression on cells transfected with HYPE-5 Kit vs. FSM reagent.
Secreted Alkaline Phosphate (SEAP) was produced by transient transfection using HYPE-5 Kit or FSM
transfection reagents according to manufacturer’s instructions. Transfections were performed using 1.5 µg of
plasmid DNA per mL of cell culture. Ratio (µL reagent per µg of plasmid) for HYPE-5 reagent and FSM
reagents was 2 and 1, respectively. CHO and HEK293 cells were cultured in 30 mL in 125 mL shaken flasks.
Density of cell culture was 1 x 106 cells/mL the day of transfection. 4 hours after transfection, for CHO cells
only, HYPE-Blast was added to cell suspension. Day 3, 5 and 7, 500µL of supernatants were collected and
SEAP activity was measured.

Easy and efficient scale-up

The results presented below indicate that protein production in CHO or HEK293 suspension cells can be easily
scaled up by using HYPE-5™ Transfection Kit. HYPE-5™ Kit has been used in volume up to 300 mL of cell
culture without any protocol modification and protein yield production is even higher when compared to lower
cell culture volume.
CHO cells HEK293 cells
1400 10000,0
µg of SEAP per Liter of cell culture

9000,0
µg of SEAP per Liter of cell culture

1200
8000,0
1000 7000,0

800 6000,0

5000,0
600
4000,0

400 3000,0

2000,0
200
1000,0

0 0,0
300 mL 30 mL 300 mL 30 mL

Volume of cell culture Volume of cell culture

Fig.4: SEAP Expression with HYPE-5 Kit in increasing volume of culture.

Secreted Alkaline Phosphatase (SEAP) was produced by transient transfection using HYPE-5 Transfection Kit
according to the instruction manual. Transfections were performed using 1.5 µg of plasmid DNA per mL of cell
culture. Ratio of 2 µL of HYPE-5 reagent per µg of plasmid was used. CHO and HEK293 cells were cultured in
30 mL or 300mL of medium in 125 mL or 1000 mL shaken flasks, respectively. Density of cell culture was 1 x
106 cells/mL the day of transfection. 4 hours after transfection, for CHO cells only, HYPE-Blast was added to
cell suspension. Day 5 (CHO cells) and 7 (HEK293 cells) 500µL of supernatants were collected and SEAP
activity was measured.
OZ Biosciences HYPE-5 Transfection Reagent - Results Page 4 of 6
High production yield for intracellular expressed protein

HYPE-5™ Kit is also highly efficient for intracellular protein production on cells adapted in suspension with
chemically defined medium and in absence of serum.

HEK293 cells
14000
µg of β-Gal per Liter of cell culture

12000

10000

8000

6000

4000

2000

0
HYPE-5 FSM
day 3 day 5 day 7
Fig.5: β-Galactosidase Kinetic expression with HYPE-5 Kit vs. FSM reagent.
β-Galactosidase was produced by transient transfection using HYPE-5 reagent or FSM transfection reagents
according to manufacturer’s instruction. Transfections were performed using 1.5 µg of plasmid DNA per mL of
cell culture. Ratio (µL reagent per µg of plasmid) for HYPE-5 reagent and FSM reagents was 2 and 1,
respectively. HEK293 cells were cultured in 30 mL in 125 mL shaken flasks. Density of cell culture was 1 x 106
cells/mL the day of transfection. Day 3, 5 and 7, 500µL of supernatants were collected and β-galactosidase
expression was measured with ONPG kit (cat # GO10001).

Parameters influencing the protein production yield

Efficient protein production is extremely dependent on the cell model used. For instance, adaptations of cells
to growth in suspension, culture medium and cell density (before and during transfection) are of importance
to obtain the maximum efficiency. HYPE-5™ Transfection Kit has been used and validated with different cell
origins and cultivated in different chemically defined medium. Other critical parameters are also important for
efficient protein production. The results presented below show the influence of cell density and DNA quantity
on protein production efficiency.

Fig.6-8: SEAP was produced by transient transfection using HYPE-5 Transfection Kit according to the
instruction manual. CHO/HEK293 cells were cultured in 0.5 mL of medium in a 24-well shaken plate. 4 hours
after transfection, for CHO cells only, HYPE-Blast was added to cell suspension. Day 5 (CHO) or 7 (HEK293),
20µL of supernatants were collected and SEAP activity was measured.

OZ Biosciences HYPE-5 Transfection Reagent - Results Page 5 of 6

 CHO cells HEK293 cells
µg of SEAP per Liter of cell culture

µg of SEAP per Liter of cell culture

1200 9000
8000
1000
7000
800 6000
5000
600
4000
400 3000
2000
200
1000
0 0
1 µg 2 µg 4 µg 1 µg 2 µg 4 µg

DNA quantity per mL of cell culture DNA quantity per mL of cell culture

Fig.6: Influence of DNA concentration on SEAP Expression with HYPE-5 Kit

Transfections were performed using a range of DNA from 1 to 4 µg per milliliter of cell culture. Ratio for
HYPE-5 reagent was fixed at 2µL per µg of DNA. Density of cell culture was fixed at 1 x 106 cells/mL.

CHO cells HEK293 cells

µg of SEAP per Liter of cell culture
µg of SEAP per Liter of cell culture

1400 12000

1200 10000
1000
8000
800
6000
600
4000
400

200 2000

0 0
0,5 1 1,5 0,5 1 1,5

x 10^6 cells per mL of cell culture x 10^6 cells per mL of cell culture

Fig.7: Cell density influence on SEAP Expression with HYPE-5 Kit

Transfections were performed using 1.5 µg per mL of cell culture. Ratio for HYPE-5 reagent was fixed at 2µL
per µg of DNA. Density of cell culture was used in a range from 0.5 to 1.5 x 106 cells/mL the day of
transfection.

CHO cells
1400
µg of SEAP per liter of cell culture

1200

1000

800

600

400

200

1µL/µg 2µL/µg 3µL/µg 4µL/µg

µL of HYPE-5 reagent per µg of DNA

Fig.8: Influence of HYPE-5 / DNA ratio on SEAP Expression.

Transfections were performed using 1.5 µg per mL of cell culture. Ratio of HYPE-5 reagent was used in a
range from 1 to 4µL per µg of DNA. Density of cell culture was fixed at 1 x 106 cells/mL the day of
transfection.

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