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

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

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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

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

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 yield significantly (shaked flasks simulation). Normalised yield improvment (%)

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

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

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

Pictures of ATF (“Darth Vader” sword)

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)

New process scalability from lab to production scale (2009)

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

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

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

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

Conclusion: Yield improvment and quality Process Normalised Productivity (%) Quality parameter 1 Quality parameter 2 D&F 100 ~95 0,03 ATF 250 90-95 0,06 600 ~90 0,07

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

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

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