Case study: Challenges and learning in implementing ATF perfusion process

1. Case study:Challenges and learning in implementing ATF perfusion process Dr. Jarno Robin Principal Scientist CMC_Biopharm upstream development Novo Nordisk A/S, Hillerød, Denmark

2. Outline introduction to Novo Nordisk case study: implementation of ATF perfusion process for improving yield prior to fase3 production background & timeline perfusion technology contraints new perfusion process (lab & production scales) process challenges & stabilisation conclusion: yield improvement and quality learnings

3. Novo Nordisk Corporate strategy Novo Nordisk Way

4. Novo Nordisk around the world production R&D, production Headquater, Research Units, R&D, production

5. Diabetes: A global pandemic

6. Part II case study: implementation of perfusion process for improving yield prior to fase3 production background & timeline perfusion technology contraints new perfusion process (lab & production scales) process challenges & stabilisation

7. Background & timeline very stable & robust process (cell growth & product quality) BUT very poor productivity Issues regarding production capacity 2008 • Feb : 3-days-Draw & fill (D&F) process approved for phase3 production. Prod. Conc.

8. Background & timeline Normalised yield improvment (%) 2008 • Feb.: 3-days-Draw & fill (D&F) process approved for phase3 production. • Oct.: process challenge meeting: approval for attempting to change the production process from D&F to a perfusion process based on expectations to increase yield significantly (shaked flasks simulation).

9. Background & timeline 2008 • Feb.: 3-days-Draw & fill (D&F) process approved for phase3 production. • Oct.: process challenge meeting: approval for attempting to change the production process from D&F to a perfusion process based on expectations to increase significantly yield. • Nov-Dec.: Finalising the ATF purchase for lab- & production- scales. • Jan-Jun.: Implementation of ATF-2 in lab scale and ATF-10 in production scale. • Apr-Jul: TDR meetings: • production medium chosen for phase3 production (3 upstream depts. involved). • perfusion process using the ATF chosen for phase3 production (3 upstream + 1 downstream depts. involved, coordination meeting). • Jul-Aug.: Master & production formulas in place for fase3 production planed from Oct. 2009 (2 upstream depts. involved). • Q2/Q3: further process optimisation, MF/PF updated (2 upstream depts. involved). • Q4-2011/Q2-2012: issue regarding impact of complex raw-material quality on process performance (2 upstream depts. involved). • Q2/Q3: Process Justification & transfer to production support department. 2009 2011 2012

10. Why ATF ? : Perfusion devices available Spin filter Applisens BioSep • good in lab (intern filter)  up-scaling: extern at large scale (temp., pH, O2 stress, pump & shear stress) • block at high cell conc. • cells in harvest good in lab • up-scaling, pump • 5-10% cells in harvest ATF (Alternating Tangencial Filtration) Tangential Flow Filtration (TFF) • simple & upscalable (to 5m3) • no temp., pH, O2 stress • no pump & low shear stress • cell free harvest • reasonable cost possible product binding • good in lab • cell free harvest possible product binding • pump, up-scaling

11. ATF perfusion process at Novo Nordisk(Alternative Tangiencial Filtration) Bleed Medium Harvest Harvest Patent no.: WO2012045769

12. Pictures of ATF (“Darth Vader” sword)

13. New ATF-process: constraints • Harvest stability of at least 2 days due to downstream process at large scale. • Short timeline for a complete process change (Q1/Q2-2009,~6 months) • Get a scalable perfusion process to the production facility using the ATF technology. • Get similar product quality as the material produced for tox and fase1 clinical trial using the D&F process. • Improve significantly the expression level (production capacity)

14. New process scalability from lab to production scale (2009)

15. Challenges: Complex raw-material quality impact on process performance at large scale

16. Challenges: Complex raw-material quality impact on process performance. Confirmation at lab scale • Soy hydrolysate has been analysed: • tracer element, • free amino acids • NIR

17. NIR analyses on HySoy batches Work done by Erik Skibsted (ERSK)

18. Process stabilisation: focus on Zn concentration w/o. Zn spiking w/ Zn spiking

19. Conclusion: Yield improvment and quality

20. Conclusion: Learnings targets achieved: new process development & implementation within short timeline (6 months) scalable perfusion process significant increase in yield (up to 600%) identical product quality between D&F and perfusion process process stabilisation after unexpected significant impact of complexe raw-material on process performance increased knowledge: solving challenges increased collaboration across lab & production scales complex raw-material characterisation know-how for next generation process and/or products for early implementation in the project plan: Focus on correlation between cell line/medium/process

21. Aknowledgements: Dept.’s bioreactor team for dedicated technical work Production & Research Unit, up- & down-stream for good collaboration Vendors for support

22. Thank you ! for your attention