Nucleofector technology and transient protein production

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1 amaxa xxxxxxxxxxxx Nucleofector technology research Nucleofector technology and transient protein production your link to transfection

2 The Nucleofector technology and transient protein production Transient protein expression systems have gained increasing relevance as they enable fast and flexible protein production. Protein amounts up to mg can be produced within a few days in contrast to production periods up to several months using stably transfected cell clones. Transient protein production offers: fast production of small scale protein amounts low costs for cell maintenance Easy and fast nucleofection procedure: harvest of protein Combine cells of interest, expression vector and the appropriate celltype specific Nucleofector Solution and transfer to an amaxa certified cuvette. Insert the cuvette into the Nucleofector and choose the cell-type specific Nucleofector program. Press the start button X. After a few seconds nucleofection is completed. Rinse the cuvette with culture medium and transfer the cells into the culture flask. For 1 x 8 cells, ten cuvettes have to be nucleofected. Culture cells for several days. In the past gene transfer into serum-free cultivated mammalian cells suitable for protein production was hampered by inefficient transfection methods. The Nucleofector technology represents the ideal tool for gene transfer even into hard-to-transfect cell lines like suspension CHO cells. It has now been successfully tested and validated for its use in transient protein production. Benefit from transient protein production using nucleofection: suitable for scho, shek-293, BHK-21, NS and many other cells applicable even under serum-free conditions high transfection efficiencies low DNA amounts page 2 Nucleofector technology

3 Nucleofection is superior to lipofection for transient protein production In a comparative study the Nucleofector technology (amaxa) and the lipofection reagent Lipofectamine 2 (Invitrogen) have been tested in regard to their suitability for transient protein production. Nucleofection achieves -3 times higher expression rates in CHO cells compared to cells transfected with lipofection. A similar ratio has been determined for both the specific and the volumetric productivities. Experimental setup: 2 different suspension CHO cell clones have been transfected with a plasmid encoding the human secreted alkaline phosphatase (SEAP) either using Lipofectamine 2 (Invitrogen) or nucleofection. Transfection conditions: Cell line scho-clone1 (ECACC) scho-clone2 (Invitrogen) Transfection method Lipofectamine 2 nucleofection Lipofectamine 2 nucleofection Transfection conditions 29 µl Cell Line Kit V Program U µl Cell Line Kit V Program U-24 DNA 58 µg 25 µg 116 µg 25 µg Cell number 5 x 7 5 x 7 5 x 7 5 x 7 Post transfection cells were cultivated as batches in spinner flasks containing 5 ml culture medium for 5 days under non-regulated conditions. Suspension HEK-293 cells (DSMZ, adapted from adherent cell clone) have also been tested successfully. 5 x 7 cells have been nucleofected with 25 µg pdrive-hseap using Cell Line Nucleofector Kit V in combination with program X-1. A specific productivity of 2.4 µu/c/d and a volumetric productivity of.5 U/ml could be obtained. These cells were not transfectable using lipofection. Specific productivities (µunits/cell/ day) of hseap referred to a cell density of 6 cells/ml seeded after the respective transfection. specific productivity hseap [µu/c/d] lipofection nucleofection scho-clone1 scho-clone2 page 3 Nucleofector technology

4 Volumetric productivities (Units/ml) of hseap. volumetric productivity hseap [U/ml] scho-clone1, lipofection scho-clone1, nucleofection scho-clone2, lipofection scho-clone2, nucleofection time [d] Efficient antibody production using nucleofection The Nucleofector technology was also tested successfully for its suitability to produce functional proteins. 1.7 mg of a human IgG1 antibody was produced within five days. antibody concentration [µg/ml] time [d] Antibody production after nucleofection of CHO suspension cells 1 x 8 scho cells (clone 2, ^= samples) were nucleofected using Cell Line Nucleofector Kit V in combination with program U-24 or U-24. Cells were cotransfected with plasmids encoding either the DNA sequence of the light chain or the heavy chain of a human IgG1 antibody. Post nucleofection cells were cultivated for 5 days in 5 ml serum-free medium. Cells reached a specific productivity of 5.5 pg/c/d and a volumetric productivity of 39 µg/ml. Within 4-5 days 1.7 mg of the antibody could be produced (determined by ELISA). The integrity of the produced antibody was verified via Western blotting. q For more scientific data go to or contact our Scientific Support Team. page 4 Nucleofector technology

5 High transfection efficiencies in hard-to-transfect cells such as scho and shek-293 CHO cells are the predominant cell line used for protein production. Suspension CHO cells have so far been difficult to transfect by conventional non-viral methods. The same is true for many other suspension cell lines used for protein production like BHK-21 and NS. High transfection efficiencies after nucleofection. Transfection efficiencies for two different suspension CHO cell clones and two different suspension HEK- 293 cell clones after nucleofection of pmaxgfp. maxgfp positive cells were determined 24h post nucleofection by FACS analysis (light bars). Viabilities were assessed after propidium iodide staining by FACS analysis (dark bars). transfection efficiency/viability (%) efficiency viability scho-clone1 scho-clone2 shek-293-clone1 shek-293-clone2 How to find your cell line of interest The Nucleofector technology is the ideal tool for efficient non-viral gene transfer into different suspension CHO and HEK-293 cell clones. Please find the Optimized Protocols for these and many other cell lines on amaxa s homepage. Visit the amaxa online cell line database Enter your cell line of interest or view complete list Your cell line of interest Screen results Cell type listed Cell type not listed Click on cell name Use recommended Nucleofector Kit in combination with the respective Optimized Protocol or use the settings optimized by other users. Use Cell Line Optimization Nucleofector Kit or contact amaxa s Scientific Support Team for further advice. page 5 Nucleofector technology

6 How Nucleofector technology works The Nucleofector Device delivers unique electrical parameters that are different from any commercially available electroporation device. The electrical settings are pre-programmed and each program is optimized for the requirements of a particular cell type and can be used for the delivery of different substrates such as sirna duplexes, DNA vectors or RNA. The Nucleofector Kits contain amaxa specified cuvettes, pipettes, Nucleofector Solution, Supplement and the positive control vector pmaxgfp. For cell lines, three different Nucleofector Solutions, R, T and V, are available. All solutions provide a cellfriendly environment that ensure the highest transfection results and cell viability. Nucleofector Kits are only functional in combination with the Nucleofector Device. Ordering information Nucleofector Kits for cell lines* VCO-1 VCA-1 VCA-2 VCA-3 Cell Line Optimization Nucleofector Kit Cell Line Kit R Cell Line Kit T Cell Line Kit V d For an up-to-date overview or more detailed information about amaxa products, please see * Each Kit contains Nucleofector Solution, Supplement, Cuvettes and Pipettes for 25 reactions and the pmaxgfp plasmid as a positive control. VCO-1 contains Nucleofector Solution, Supplement, Cuvettes and Pipettes for 5 reactions and the pmaxgfp plasmid as a positive control. WKB-2_5.25 amaxa s Nucleofector Process, Nucleofector Device and Nucleofector Solutions are covered by PCT Applications PCT/EP1/7348, PCT/DE2/1489, PCT/DE2/1483, and other pending patents, and domestic or foreign applications corresponding thereto. amaxa, Nucleofector, nucleofection and maxgfp are trademarks of amaxa GmbH. Lipofectamine 2 is a trademark of Invitrogen. amaxa GmbH, Europe/World amaxa Inc., USA Scientific Support Scientific Support +49 () (24) scientific-support@amaxa.com scientific-support.us@amaxa.com