![P 3 —a Protein Production Platform for mAb Generation : a Series of Case Studies Part I Carlo Ciatto, PhD [email_address]](https://image.slidesharecdn.com/parti-13073642910923-phpapp02-110606075159-phpapp02/85/PCUBE-Protein-Production-Platform-for-mAb-generation-Part-I-1-320.jpg)
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PCUBE--Protein Production Platform for mAb generation. Part I
- 1. P 3 —a Protein Production Platform for mAb Generation : a Series of Case Studies Part I Carlo Ciatto, PhD [email_address]
- 2. Cases 1 and 2
- 3. 1: Transient Gene Expression in 293E in suspension culture: fast and reliable for medium-scale production
- 5. Adherent cells were split: 1:6 on Day 1 1:4 on Day 4 1:2 on Day 7 1:12 on Day 8 1443-hFC cloned into pFUSE (non-episomal) and pCEP4 (episomal) vectors Episomal vector is advantageous 2-fold serial dilutions pFUSE pCEP4 (-) D1 D4 D5 D6 D1 D4 D5 D6 pFUSE pCEP4 (-) D6 D7 D8 D12 D6 D7 D8 D12 2-fold serial dilutions
- 6. Transfection with PEI is effective Suspension-adapted cells were transfected with EGFP (in PCEP4) Flow cytometry was performed on Day 2 Untransfected PEI Lipofectamine 2000 MFI 6 MFI 50 MFI 75
- 7. Protein expression is sustained over time EGFP was used a co-transfection marker 3.8 x 10 5 viable cells/mL 70 % viability 1.1 x 10 6 viable cells/mL 87 % viability 2.2 x 10 6 viable cells/mL 90 % viability Total cells Green fluorescent Overlay Day 3 Day 6 Day 8
- 8. WB analysis with anti-Strep-tag and target-specific antibody Single-step affinity purification of culture supernatant on 5 mL StrepTrap column Protein of interest (ligand) contains a Strep-tag Purification is straightforward D0 D3 D6 D8 D0 D3 D6 D8 Anti-Strep-tag antibody Target -specific antibody 170 kDa - 130 kDa - 95 kDa - 72 kDa - 55 kDa - 43 kDa - 34 kDa - 26 kDa - 17 kDa - 11 kDa - 170 kDa - 130 kDa - 95 kDa - 72 kDa - 55 kDa - 43 kDa - 34 kDa - 26 kDa - 17 kDa - 11 kDa -
- 9. Double-decker sandwich ELISA Purified ligand recognizes receptor, but not (-)controls Recombinant protein is active Goat anti-mouse IgG/M Goat anti-hFC Rec-hFC Mouse anti-Streptag Lig- Streptag Receptor-hFc
- 10. 2: When suspension culture is not good enough: protein production in adherent cells in a disposable packed-bed bioreactor 2-fold serial dilutions suspension adherent (-) D3 D4 D5 D6 D7 D9 PEI L2K (both on D2) mIgG
- 11.  How to pack 12000 cm 2 of growth surface in an easy-to-handle volume 500 mL Would you rather deal with this... or this (FibraStage bottle)?
- 12. Planning for protein production 262-mFc, pCEP4 episomal vector, adherent 293E cells, FibraStage bottle, repeat-batch/fed-batch culture
- 13. Protein is still being expressed one month post transfection Dot blot analysis of culture supernatants 2-fold serial dilutions mouse IgG, 5 ng/ L Batch 1 - D4 Batch 2 - D7 Batch 3 - D9 Batch 4 - D12 Batch 6 - D18 Batch 5 - D15 Batch 7 - D22 Batch 8 - D25 Batch 9 - D28 Batch 10 - D32 Medium
- 14. Seed-transfect produces higher titer than transfect-seed An alternative transfection protocol Transfect-seed: Bioreactor was seeded with 2 x 10 8 pre-transfected cells Seed-transfect: Bioreactor was seeded with 2 x 10 8 cells; transfection was performed in the bioreactor after expansion mouse IgG, 5 ng/ L Seed-transfect B1 – D3 Medium Seed-transfect B2 - D7 Seed-transfect B3 - D12 Transfect-seed B1 - D5
- 15. 262-mFc purified on protein A column (5 mL ) One-step purification 170 kDa - 130 kDa - 95 kDa - 72 kDa - 55 kDa - 43 kDa - 34 kDa - 26 kDa - 17 kDa - 11 kDa - The expected molecular weight of non-glycolsylated 262-mFC is 75 kDa Mass spec analysis confirms identity of 262-mFc
- 16. Sera from immunized mice work by ELISA on recombinant protein Sera screen by direct ELISA
- 17. Mouse #1 Mouse #3 Pre-bleed 1:200 Final-bleed 1:200 Sera recognize native protein on cell surface Flow cytometry analysis on cell line expressing native protein
Notes de l'éditeur
- P cube working in a core facility presents its own challenges I want to show how I tried to overcome them, and how I tried to solve problems. Also I would like to show how I approach things
- The first couple of case studies fall into the category: reagent generation and validation
- Comparable to what is used in industry
- First question I wanted to answer: does the use of episomal vector gives an advantage in practice? Just let me explain what dot blots are since I am going to show them in several slides It ’s a fast way to detect the presence of proteins in culture supernatants, and to compare expression level You make serial dilutions in a multiwell plate, and transfer with a multichannel pipette on a membrane; then you analyse with an antibody. Supernatants from adherent cells In pfuse expression peaks after 4 days, and then starts to peter out after 5 days This confirms that expression with pcep4 vector provides an advantage
- How does PEI transfection compare to L2K? Suspension 293 transfected with EGFP in episomal vector
- And finally I putting the two things together, I wanted to verify that suspension cells transfected with PEI provide sustained protein expression Cotransfection with a small percentage of plasmid carrying the gene for the green fluorescent protein. This provides a fast way of verifying transfection efficiency and expression level by using a fluorescence microscope just by looking at cells This slides in turn proves that transient gene expression in suspension cells with an episomal vector is actually sustained over time
- But what about the protein of interest? Is it being expressed? Is it easy to purify? The answer to both questions is yes
- The final goal is to produce a functionally active protein In each column the second reagent is rec-hFC or an irrelevnt protein
- cell to cell contact, the presence of serum
- Describe how the system works: cell transfection in dishes, seeding of disks. Then monitor culture, and feed or change medium
- After some preliminary experiments, we came to the conclusion that we could plan and extend the culture for quite a long time. Here I am showing a gantt chart detailing a typical run ....give description
- This a dot blot analysis of supernatants from the corresponding run Protein expression peaks after 18 days, during batch number 6, and after 32 days, in batch 10, there is still some protein being produced
- Things can always be improved..
- Is the protein easy to purify? Is it active?
- But the real proof that a recombinant protein used for immunization is of high quality lies in its ability to induce antibodies that recognize the native protein