Bethesda,MD,30.3.2004

1. Transient Transfection into Eukaryotic Cells: An Alternative to Bacterial and Insect Cell Systems for the Rapid Generation of Recombinant Proteins? Bethesda,MD,30.3.2004 Sabine Geisse, Nicola di Maiuta, Thomas Cremer, Mario Henke Novartis Institutes for Biomedical Research, Basle, Switzerland

2. Background and Rationale • Recombinant proteins are essential research tools for  assay development and screening  structural biology  antibody generation, selectivity assays...... • In the post-genomic era translation of thousands of ORFs into proteins are a major challenge • Technologies and processes increasing the throughput and the success rate in protein production are needed • Novartis Research is currently building a Protein Production Center in which streamlined processes will be applied in a factory-like fashion. In parallel, an Intensive Care Approach is applied to generate proteins by non-generic means.

3. Double-Track Strategy • Target output: 400 tool proteins/year • Quantity 1 - 100 mg • Purity >80 % (application-dependent) • “Protein Factory”: > 300 proteins/year • 3 standardized expression systems • Streamlined, generic, partly automated processes • “Intensive Care”: < 100 proteins/year • Difficult-to-express proteins • Recombinant cell lines, membrane proteins • Monoclonal and recombinant antibodies

4. PPC ICU A Double-Track Process to Increase Success Rates Transformation/Transfection Small Scale Expression Evaluation Scale-Up Evaluate Special Conditions for Scale-Up Large-Scale Fermentation Large-Scale Fermentation Automated Harvest + Cell Lysis Standard Harvest + Lysis Classical Purification Automated Purification

5. 1-(2) “Entry” clones 7-(14) “Destination” vectors 1-(2) rec. plasmids for HEK.EBNA cells 10-(20) E. coli strains 1-(2) rec. bacmids 2 clones + 2 fermentation conditions in 96-well-plates Transfection into insect cells in24-well-plates Transient TF into HEK.EBNA cells in6-well-plates Cleared Lysates/Supernatants for In-Process Analytics Decision on Best Construct/Expression System for Scale-Up Small Scale Expression Evaluation

6. EBNA-1 protein drives episomal replication ofori-P containing plasmids integrated Ad5 E1a/E1b fragment in HEK 293 cells enhances trans- cription of CMV promotor driven transgene Why HEK.EBNA Cells? The Principle EBNA-1/ori-P based expression in Human Embryonic Kidney (293) cells (293 stably transformed with EBNA-1 gene) The cell line is available from ATCC and, until recently, also from Invitrogen

7. Why HEK.EBNA Cells? Advantages • In comparison to other eukaryotic expression systemsthe HEK.EBNA Expression System is rapid:from gene to protein in 4-6 weeks • The cells can be grown adherently and in serum-free suspension culture • It can be applied to generate stable cell lines (pools/ clones) and in transient mode on small and large scale • In transient mode not only secreted and membrane-bound, but also intracellular proteins can successfullybe expressed

8. HEK.EBNA Expression Vectors • Basic vector (alsoGateway™ adapted) • Can be decorated withN- or C-terminal tags, heterologous leadersequences • Co-expression of e.g. GFP via IRES element • Selectable marker for generation of stable cell line Commercially available HEK.EBNA vectors: pREP4 and pCEP4 (Invitrogen)

9. Small Scale Expression in HEK.EBNA Cells • Candidate gene: cytokine (tagged) • HEK.EBNA cells grown in DMEM + 10 % FCS • Cell seeding 24 h prior to transfection • TF reagent: Lipofectamine 2000 (Invitrogen),plasmid/ reagent ratio optimised • Polyfect (Qiagen) and JetPEI (Polyplus) work also effectively • Protein analysis 3 d post transfection by ELISA 6-well-plate: 4.5 x 105 cells/well Titer: 5-10 mg/l 24-well-plate: 1.25 x 105 cells/well Titer: 16 mg/l 48-well-plate: 6 x 104 cells/well Titer: 17-24 mg/l 96-well-plate: 6 x 104 cells/well Titer: > 10 mg/l Note: This protein is extremely well-behavedin expression, but the same approach works also for less well expressed proteins

10. Summary of Small Scale Expression Small Scale Expression Trials using HEK.EBNA Cells work very efficiently using • Adherent cultures in DMEM+ 10 % FCS • Different well-plate formats • Pre-coating with Poly-D-lysine: facilitates attachment of cells and minimizes cell losses during transfection • Lipofectamine 2000™ (Invitrogen): >90 % transfectionefficiency (other reagents also possible – try!) • Can be also done using serum-free suspension cultures, but less efficient

11. Large Scale Transient Transfection (1) What is “large scale”? 1 – 10 liter • Prerequisite: Adaptation of cell culture to serum-free suspension • Several vendors offer cell culture media either specifically developed for HEK293 cells or for other cells (Per.C.6, hybridoma) which can be also used for cultivation of HEK293 or HEK.EBNA cells (see Table on Slide 14)

12. Large Scale Transient Transfection (2) 2. Prerequisite: a suitable transfection reagent which is cost-effective, readily available in large quantities and gives riseto high transfection efficiencies • Commercially available reagents, such as lipoplexesare by far too expensive at this scale • Transfection using CaPO4 precipitation or cationic polymers such as Polyethylenimine, however, meet the above mentioned criteria and have been successfully used at multi-liter scale

13. E. Coli DH5 20-L Fermentation,  300 g wet cell pellet 30 g pellet 10-15 mg plasmid DNA NucleoBond™,(Macherey-Nagel) Large Scale Transient Transfection (3) 3. Prerequisite: Generation of sufficient plasmid DNA: an example

14. Transfectability HEK.EBNA cells Growth Price Medium characteristics Calciumphosphate PEI [CHF/L] HEK.EBNA cells ü ü ü CD 293 140 ü ü û PRO 293 S 85 ü ü û 293 SFM II 140 üü ü FreeStyle 134 n.d. û û û Hektor S 23 üüü û EX-CELL VPRO 30 n.d. ü û ü 2055 44 üü ü ü M11 24 üü üüü M11V3 50 n.d. ü üüü DMEM/F12 45 n.d. Large Scale Transient Transfection (4) Compatibility of Culture Media and Transfection Reagents

15. Step 1 • Back-up cultures kept in roller bottles or Wave reactor • No medium change, no FCS supplementation • Transfer of 3.6 liters at 1.4 x 106 cells/ml into 10-L Wave reactor – Step 2 • Preparation of DNA:PEI complex (10 mg DNA : 20 mg PEI)in 1.4 liters medium M11V3 • Incubation for 15 minutes • Transfer of DNA:PEI complex into 10-L Wave bioreactor Step 3 • 5.0 liters transfection mix is incubated for 4 hours • After 4 hours 5 liters Ex-Cell VPRO medium are added to the culture for the production phase • Starting conditions: 5 x 105 cells/ml in 10 liters Polyethylenimine-mediated Transfection

16. 25 10 Cell density in 3.6 volume prior to transfection 9 Cell density after addition of 1.4 l transfection mix 20 8 Cell density after addition cells/ml] 7 of 5 l growth medium 15 6 5 product titer [mg/l] 5 cell density [ x 10 10 4 3 5 2 1 0 0 0 20 40 60 80 100 120 140 160 180 time [h] cell density product titer A Transient Transfection Run…..

17. ….in Multiparallel Fashion The “Wave Factory”

18. Secreted productin supernatant orCell concentration Wave bag Cell concentrate Cell debris Intracellular product: Cell concentrate+ Lysis buffer Released productin cleared lysate Supernatant Clear Lysate Wave bag Cell/Supernatant Harvest and Cell Lysis

19. Protein Purification For Secreted Proteins: affinity chromatography on antibody or Protein A column -tag dependent- For Intracellularly Expressed Proteins: his and/or his-Strep tag  Ni-chelate and/or Streptactin column To date, > 30 proteins were generated successfully using this approach.The overall success rate in expression trials was >80 %.

20. A Few Examples Cytokine: 2.5 mg/l Soluble Rec.: 12 mg/l APP family: 3.5 mg/l Cytokine: 7.5 mg/l Kinase:8.5 mg/l Dehydrogenase:2.6 mg/l Membrane Glycoprotein:0.5 mg/l

21. Workflow for Large Scale Transient Transfections Small Scale Expression Evaluation on 6-well-plate 1-2 l-Batch Production in Roller Bottles Mol.Biol.: Cloning + Plasmid Generation Large-Scale Plasmid Prep 10-l-Wave Production One run sufficient to meet demands in most cases - functionality-testing of plasmid prior to large scale plasmid prep- indication on expression levels to be expected - Validation of large scale plasmid prep- 1st purification trial- may suffice for entire production Generation of multiple E.coli pellets for several production runs Multi-parallel production if several Wave bioreactors available Shake flasks cultures or spinners also possible Miniaturized format for multi-parallel analyses available (24/48 well-plate) Multi-parallel plasmid preparations possible To summarize…….

22. Acknowledgements: The BTP team • HP Kocher (UH, Biomolecule Production) • S Geisse (Baculo, Mammalian) • M. Buchs • A Patoux • B Rudin • S Hartmann (IPC, In process & funct. analytics) • D Rinaldi • T Kuiper • M Henke (Fermentation) • S Dalcher • R Uhrhahn • F Kolbinger (MolBio, Mammalian) PTH BTP Program • A Schildhauer • M Mahnke (E.coli) PTH EXA Program • S Deutsch • E Eglin • Y Pouliquen • JM Schlaeppi (Protein Pur.) • A Berner • R Schmitz (MolBio) • N Charara • K Leon • T Soellick (Bioinf, ProTrack) • M Zurini (Protein Pur.) • J Causevic • R Enderlin • D Plattner