Cell and Gene Therapy Catapult 12 th Floor, Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT +44 (0) 203 728 9500 | [email protected] | ct.catapult.org.uk Cell and Gene Therapy Catapult is a trading name of Cell Therapy Catapult Limited, registered in England and Wales under company number 07964711. DEVELOPMENT OF A SCALABLE PLATFORM FOR AAV MANUFACTURING Juline Guenat* , Adrien Soula* , Florian Leseigneur, Quentin Bazot, Nishanti Weeratunge, Nishant Pawa, Lily Li, Tony Bou Kheir, Gregory Berger, Tristan Thwaites, Mike Delahaye, Julie Kerby, Hadi Mirmalek-Sani and Jonathan Appleby *equally contributed Presenting author contact: [email protected] , [email protected] Conclusion: Here is presented a scalable AAV manufacturing platform using STRs bioreactors for production. Production process was defined and optimised at small scale and is now being scaled up at production scale. Purification and analytics are already enabling efficient end to end process but will be further optimised. Introduction: AAV vectors are an appealing tool for both ex-vivo and in-vivo gene therapy. The number of AAV gene products entering early and late phase clinical trials is significantly on the increase. High cost of goods, low process yield, and poor product characterisation are all metrics that require substantial development and improvement within this emerging field. Currently most AAV vectors are manufactured using an adherent process and the demand currently outstrips capacity. Whilst switching from a classical 2D approach to a suspension process using single use bioreactors might be appealing to meet the requirement in term of doses and patient number for late stage of development (e.g Phase III study), this can come at the expense of laborious and costly comparability study. Hence, a reliable, low risk manufacturing platform delivering at the desired scale should be identified early on during development. Therefore, it becomes clear that there is a need to develop the next generation of upstream platform processes using a suspension cell line in STRs. Following Quality by design principles, we sought to develop this platform for AAV manufacturing. Using a scale down model, we investigated the impact of a broad range of process parameters using a design of experiment approach on AAV productivity. Scalability of the newly designed process as well as the impact of our USP on full capsid enrichment during our purification process has been investigated. Overall, our latest efforts in developing an end to end scalable suspension platform for AAV manufacturing will be presented. Manufacturing challenges USP Optimisation Statistical design of experiments (DoE) was applied to determine the relationship between factors affecting the process and the output of that process. Transfection efficiency and AAV titre were selected as outputs of the process. Titre 1 (vg/mL) 5E+010 1E+011 1.5E+011 3 3 4 Titre 2 (vg/cell) 5000 5000 10000 15000 3 3 4 Transfection efficiency (%) 20 40 60 80 100 3 DoE #2 3D surface response of modelled outputs. Y axis represent input (1) and X axis input (2). Red colour indicates higher response and Blue lower. The stars show predicted best combination. 2 3 4 5 6 2 3 4 5 6 Transfection efficiency (%) 75 80 85 2 2 2 2 5 2 3 4 5 6 2 3 4 5 6 Titre 1 (vg/mL) 2.7E+011 2.7E+011 2.8E+011 2.8E+011 2.9E+011 3E+011 3.1E+011 2 2 2 2 5 2 3 4 5 6 2 3 4 5 6 Titre 2 (vg/cell) 44000 44000 46000 46000 48000 50000 2 2 2 2 5 DoE #5 3D surface response of modelled outputs. Y axis represent input (1) and X axis input (2). rpm #1 rpm #2 rpm #1 rpm #2 0 20 40 60 80 100 Transfection efficiency GFP positive cells (%) Baseline process Optimised process rpm #1 rpm #2 rpm #1 rpm #2 b*0 b*1 b*2 b*3 b*4 b*5 0 25 50 75 100 Viable Cell Density at harvest Viable Cell Density (vc/ mL) Baseline process Optimised process Viability (%) Viability pH #1 Viability pH #2 rpm #1 rpm #2 rpm #1 rpm #2 0*10^c 5*10^c 10*10^c 15*10^c 20*10^c 25*10^c 30*10^c 35*10^c Titer at harvest Titer (vg/mL) Baseline process Optimised process Optimised process led to a eight times titer increase while decreasing the cost. pH #1 pH #2 Despite all efforts… ’Viral vector manufacturing capacity is a barrier that is limiting the development of therapies and places the industry at risk. We’ve seen an improvement in capacity but demand has increased’ Our Approach for process optimisation USP DSP Media 1 Media 2 Media 3 Media 1 Media 2 Media 3 0*10^c 5*10^c 10*10^c 15*10^c 20*10^c 25*10^c 30*10^c 35*10^c Titer at harvest Titer (vg/mL) HEK293-A HEK293-B Media 1 Media 2 Media 3 Media 1 Media 2 Media 3 0 20 40 60 80 100 Transfection efficiency %GFP positive cells HEK293-A HEK293-B a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 b*0 b*1 b*2 b*3 b*4 b*5 b*6 Growth Viable Cell Density (vc/ mL) hrs post seeding Media 1 Media 1 Media 2 Media 2 Media 3 Media 3 HEK293-A HEK293-B Two cell lines were cultivated in three medias using historical culture parameters, on the Ambr® 15 system, transfection in triplicates. Cell line and media selection a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 a*1 a*2 a*3 a*4 0 20 40 60 80 100 Aggregation Aggregates (%) hrs post seeding Media 1 Media 1 Media 2 Media 2 Media 3 Media 3 HEK293-A HEK293-B Screen Optimisation Scale up Proving Run Optimisation Scale up Scale-up from Ambr® 15 to Ambr® 250 • Maintenance of geometric similarity • Equal specific energy dissipation rates (volumetric power input P/V) USP Scale-up a*1 a*2 a*3 a*4 b*0 b*2 b*4 b*6 b*8 Growth Viable Cell Density (vc/ mL) hrs post seeding Ambr 15 Ambr 250 VVM #1 Ambr 250 VVM #2 0*10^c 5*10^c 10*10^c 15*10^c 20*10^c 25*10^c 30*10^c 35*10^c Titer at harvest Titer (vg/mL) Ambr 15 Ambr 250 VVM #1 Ambr 250 VVM #2 Successful scale-up to 250mL using the optimised process developed at Ambr® 15 scale. Next steps: further optimisation at Ambr® 250 scale, and scale up to 2.0L UniVessels®. DSP optimisation Multi-angle dynamic light scattering (MADLS®) from Zetasizer®. X axis represent size (d.nm) and Y axis represent intensity of the signal. Two-steps purification with overall yield >75% With 60% full capsids. Load Post-capture Post-polishing Capture Polishing Post capture (left) and post polishing (right) material purified on AKTA®.