HYPE-293 Transfection Kit Results

Transcription

1 HYPE-293 Transfection Kit Results Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 () T BE +32 () Begonialaan 3a F NL +31 () F BE +32 () TE Huissen E info@bio-connect.nl The Netherlands W

2 HYPE-293 Transfection Kit Results HYPE-293 Transfection Kit is dedicated to achieve High Yield Protein Expression in HEK-293, 293- S, 293-F or any 293-related cells. This Transfection kit has been designed for maximum efficiency in HEK-293 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 & Antibody production yield 2. Suitable for all HEK-293 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-293 Transfection Kit has been designed for large scale up transient transfection and high protein expression such as antibody. The system is optimized for maximum efficiency in all 293 cells. It has been used and validated with cells from different origins (293-F, HEK-293, 293-S etc.) cultured in suspension (flask, spinner and bioreactor) used to produce proteins. Please do not hesitate to contact us at so we can give you all the support you need. HYPE-293 Kit is suitable for any synthetic or regular culture media. It has been tested with various chemically defined media: Medium FreeStyle TM 293 Expi293 Pro293 -CD EX-CELL 293 HEK293 The results shown below have been obtained with 293s and 293-F cells growing in FreeStyle 293 (Life Technologies) culture medium. The maximum efficiency has been reached when HYPE-293 reagent was used in combination with the B293 reagent. 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 are important parameters to obtain maximal protein yield. We advise optimizing experimental conditions (see instruction manual) in order to reach the best efficiency. OZ Biosciences HYPE-293 Transfection Reagent Results Page 1 of 6

3 [IgG] mg/l High production yield for antibody The results presented indicate that HYPE-293 Transfection Kit (HYPE B293) achieves high antibody production level and largely outperforms other commercially available transfection reagents in 293-F cells growing in suspension with chemically defined medium. After 6 days in culture more than 6 mg/l of antibody can be produced under standard bioproduction process (3mL scale) Day 3 Day HYPE-5 HYPE-293 FreestyleMax Fig.1: IgG Rabbit production after transient transfection of 293-F cells with HYPE-293 kit in 3 ml cell culture volume. Transfections were performed using 1.5 µg of plasmid DNA (antibody-expressing positive control vector, ThermoFisher) per ml of cell culture. 2 µl of HYPE-293 reagent per µg of plasmid was used. 293-F cells were cultured in 3 ml of medium in 125 ml shaken flasks. B293 (1X final) was added to the cells just after the addition of the complexes (1µL in 1 ml). Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 3 and day 6 post-transfection, 5µL of supernatant was collected and IgG was measured with Easy-Titer Rabbit IgG Assay Kit (ThermoFisher). OZ Biosciences HYPE-293 Transfection Reagent Results Page 2 of 6

4 µg of SEAP per Liter of cell culture Outperforms Competitors The results presented below indicate that HYPE-293 Transfection Kit (HYPE B293) achieves the highest protein yield in comparison to other commercially available transfection reagents in 293-F cells growing in suspension with chemically defined medium. Fig.2: SEAP Expression with HYPE-293 Kit vs. other reagents. Secreted Alkaline Phosphatase (SEAP) was produced by transient transfection using HYPE-293 Kit or Free Style Max reagent according to manufacturer s instructions. Transfections were performed using.75 µg of plasmid DNA (pvectoz-seap) per ml of cell culture and 2µL of HYPE-293 reagent per µg of plasmid. 293-F cells were cultured in 3 ml of medium in 125 ml shaken flasks. Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 5 post-transfection, 5µL of supernatant was collected and SEAP activity was measured using SEAP assay Kit (#SP5) Day 3 Day HYPE-5 HYPE-293 LP2K FHD 293Fectin ITPro Fig.3: SEAP Expression with HYPE-293 Kit vs. other reagents. SEAP was produced by transient transfection using HYPE-293 Transfection Kit, Free Style Max transfection reagent, LP2K, FHD, 293Fectin or IT-PRO transfection reagents according to manufacturer s instructions. 293-F cells were cultured in 1 ml volume in a 24-well shaken plate Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 3 post-transfection, 2µL of supernatants were collected and SEAP activity was measured using SEAP assay Kit (#SP5). OZ Biosciences HYPE-293 Transfection Reagent Results Page 3 of 6

5 % of SEAP expressed / HYPE-5 as reference % of SEAP expressed / HYPE-5 as reference High production yield for secreted protein Effect of HYPE-293 in small volume of cell culture HYPE-5 HYPE-293 Fig.4: SEAP Expression with HYPE-293 kit in small volume. Transfections were performed using.75 µg of plasmid DNA (pvectoz-seap) per ml of cell culture. 2 µl of HYPE-293 reagent per µg of plasmid was used. 293-F cells were cultured in 1 ml volume in a 24-well shaken plate. B293 (1X final) was added to the cells just after the addition of the complexes (1µL in 1 ml). Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 3 post-transfection, 2µL of supernatant was collected and SEAP activity was measured using SEAP assay Kit (#SP5). Effect of HYPE-293 in large volume of cell culture HYPE-5 HYPE-293 Fig.5: SEAP Expression with HYPE-293 Kit in large volume. Transfections were performed using.75 µg of plasmid DNA (pvectoz-seap) per ml of cell culture and 2 µl of HYPE-293 reagent per µg of plasmid. 293-F cells were cultured in 3 ml of culture medium in 125 ml shaken flasks. B293 (1X final) was added to the cells just after the addition of the complexes (3µL in 3 ml). Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 3 post-transfection, 5µL of supernatant was collected and SEAP activity was measured using SEAP assay Kit (#SP5). OZ Biosciences HYPE-293 Transfection Reagent Results Page 4 of 6

6 Easy and efficient scale-up The results indicate that protein production in 293-F suspension cells can be easily scaled up by using HYPE-293 Transfection Kit. It has been used in volume up to 3 ml of cell culture without any protocol modification and protein yield production is even higher when compared to lower cell culture volume. Fig.6: SEAP Expression with HYPE-293 Kit in increasing volume of culture. Transfections were performed using.75 or 1.5 µg of plasmid DNA per ml of cell culture. Ratio of 2 µl of HYPE- 293 reagent per µg of plasmid was used. Density of cell culture was 1x1 6 cells/ml the day of transfection. High production yield for intracellular expressed protein HYPE-293 Kit is also highly efficient for intracellular protein production on cells adapted in suspension with chemically defined medium and in absence of serum. Fig.7: β-galactosidase Kinetic expression with HYPE-293 Kit vs. FSM reagent. β-galactosidase was produced by transient transfection using HYPE-293 reagent or FSM transfection reagents according to manufacturer s instruction. Transfections were performed using 1.5 µg of plasmid DNA (pvectoz-lacz) per ml of cell culture. Ratio (µl reagent per µg of plasmid) for HYPE-293 and FreeStyle Max reagents were 2 and 1, respectively. 293 cells were cultured in 3 ml in 125 ml shaken flasks. Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 3, 5 and 7, 5µL of supernatants were collected and β-galactosidase expression was measured with ONPG kit (cat # GO11). OZ Biosciences HYPE-293 Transfection Reagent Results Page 5 of 6

7 A B C D Fig.8: GFP Expression with HYPE-293 kit in small volume. GFP was produced by transient transfection using HYPE-293 Kit. Transfections were performed using.75 (A, B) or 1.5µg (C, D) of plasmid DNA (pvectoz-gfp) per ml of cell culture. 2 µl of HYPE-293 reagent per µg of plasmid was used. 293-F cells were cultured in 1 ml volume in a 24-well shaken plate. B293 (1X final) was added to the cells just after the addition of the complexes (1µL in 1 ml). Density of cell culture was 1x1 6 cells/ml the day of transfection. Day 5 post-transfection, photos were captured with a fluorescent microscope. OZ Biosciences HYPE-293 Transfection Reagent Results Page 6 of 6