Rachel Legmann, PhD ISCT, October 2, 2016 Memphis, TN

Transcription

1 Industrialization of adenoviral vector production in an icellis 500 fixed bed bioreactor for the creation of autologous insulin producing liver cells for the treatment of diabetes: From bench to clinical scale Rachel Legmann, PhD ISCT, October 2, 2016 Memphis, TN This presentation is the work product of Pall Corporation and no portion of this presentation may be copied, published, performed, or redistributed without the express written authority of a Pall corporate officer Pall Corporation.

2 Highlights Technology background: Autologous cell replacement therapy for diabetic patients Liver cell expansion on the Xpansion bioreactor Viral vector production using icellis 500 bioreactor How do these two technologies facilitate the cell therapy approach for treating diabetes by liver trans-differentiation 2

3 Synergistic Relationship Joint Benefits + = Faster Development accelerating time to Diabetic market Xpansion and icellis Bioreactors Disposable bioreactor platforms Full Process Development expertise Liver cells convert to produce Insulin A novel cell therapy for diabetes Reduce the risk to the patients Make the therapy safe and affordable 3

4 The Goal To efficiently generate insulin producing tissue to be used for autologous cell replacement therapy for diabetic patients PDX-1, NeuroD1, Maf-A Propagation Liver biopsy The propagated cells are programmed to produce insulin and secrete it in a glucose regulated manner b-like cells 5-6 weeks QA/QC AIP cells are implanted back to the same patient 4

5 Manufacturing Viral and Cell Products Liver Cells-based Autologous Cell Therapy Schema Industrial scale for Cell proliferation 10 million 1.8 billion 1x10 16 PDX-1 Viral Vector Industrial scale for Virus production 5

6 General Scheme of AIP Cell Manufacturing 200-tray 50-tray Petri Dish T Flasks Cell Factory 2 Cell Factory 10 Cell number 1 x x x x x 10 8 ~2 x 10 9 ~2 x 10 9 Biopsy Expansion Transdifferentiation Passages Weeks Doubling time 2 days*; Passage time 4 days* 6

7 From Bench to Pre-Clinical Autologous Insulin Producer (AIP) cells required 15 million cells per rat, 1 x 10 9 cells for 100 rats (pre-clinical study) Purified adenovirus required for pre-clinical study Adenovirus Dose Per Rat (IFU virus) Pre Clinical Dose (IFU virus, 120 rats) Crude Virus for Pre-Clinical-Site (IFU of viruses for 100 rats) PDX x x x Neuro-D 3.75 x x x Maf-A 7.5 x x x

8 From Bench to Clinical AIP cells required 1.8 billion cells per patient, 1.8 x cells for clinical site phase I Purified adenovirus required for clinical study Adenovirus Dose Per Patient (IFU virus) Clinical Dose (IFU virus, 10 patients) Crude Virus for Clinical-Site (IFU of viruses for 10 patients) PDX-1 1.8x x x10 13 Neuro-D 4.5x x x10 13 Maf-A 9x x x

9 Upstream Industrial Viral Vector Production Industrial Monoclonal Antibody Production Industrial Virus Production Current process CF10 9

10 icellis Process Development Strategy Objectives: 0.63 m m m 2 Tech Transfer of existing process in multitray system to icellis nano ( 2 runs) - Similar cell culture parameters (temp,ph, DO) - Similar seeding density (cells/cm^2 ) - Similar TOT, T, culture duration, Harvest Optimization & scalability in icellis nano ( 4 runs) Robustness (3 runs) Reproduce process to other two Ad5 (NeuroD and MafA) Copy/paste Characterize attachment kinetics Characterize virus absorption Estimate virus productivity Test lower seeding density, simplify seed train Identify optimal infection time Modify media exchange strategy to retain growth factors and maintain glucose > 1g/L Scale-up demonstrate optimized seeding density, infection time, and media exchange strategy Reassess virus absorption Reassess virus productivity through more rigorous harvesting Scale-up to icellis (2 runs) Demonstrate full process in scale down model; obtain optimized viral productivities Reassess all attributes (growth, attachment, viral absorption) m 2 10

11 Optimized and Scale Up Ad5 PDX-1 in icellis Nano Bioreactor Transfer the process to monitor and controlled bioreactor Reduced seed density to simplify seed train at manufacturing scale Optimized process POC in Icellis Robust process Increased product yield by 25 fold (will drive reduce dose cost) control Vessel scale (growth surface area, m 2 ) Robust manufacture process 11

12 Linear Scalability in Disposable Fixed Bed Bioreactor Small Scale icellis Nano Development Predictive Run Large Scale icellis 500 Manufacturing Small footprint reduces facility and labor costs Reduced contamination risks Robust process control enables optimization of cell growth and production conditions 12

13 Metabolite Profile; Small Scale Vs Large Scale BRX Criteria for infection is glucose should be lower than 2.9 g/l 13

14 Growth Reproducibility: Large Scale icellis 500 Infection Harvest 14

15 Specific Productivity :Linear Titer Scalability: From Bench top to Manufacture Scale 25X Linear scalability from small scale BRX to industrial scale Reproducible large scale viral vector production 15

16 Downstream Industrial Purification; mab vs Viral Vector Industrial Monoclonal Antibody Purification Industrial Virus Purification Current process Depth filtration to remove cells and cell debris Harvest Remove DNA, HCP and separate full/empty capsids MustangQ Product concentration and formulation UF/DF Sterilization Sterile Filtration 16

17 Downstream Processing: Purification of Viral Vector SDS-PAGE Analysis of Adenovirus DSP Samples Hexon std ---- Mustang Q fractions ---- TFF samples PDX W2 E1 E pool E3 1M UF,DF permeate MW kda * * * * * * * * PDX Std 4.9 x 10 9 VP All samples reduced, 10% bis-tris gel. Colloidal Coomassie Blue stain 17

18 Biological Activities of Ad5-PDX-1 produced and purified in large scale batch: Death curve analysis No cell death was observed for any of the Concentrations of the three adenovirus examined Trans-differentiation efficiency: Gene expression analysis of ectopic and pancreatic genes Manufacture Scale have generated Functional Viral vector,ad5-pdx-1 18

19 Conclusions Xpansion Bioreactor was successfully used to generate 1.8 billion human liver cells required for one patient Optimization of the culture parameters and harvesting method demonstrate good agreement between small scale icellis Nano bioreactor and large scale icellis 500 bioreactor Linear scalability is proved to produce viral yields of 1.04x10 16 ifu per run at large scale in a 66 m 2 icellis 500 bioreactor icellis Bioreactor was successfully generate functional adenoviruses The icellis disposable fixed-bed bioreactors offer a solution for viral vector manufacturing in large quantities in an adherent environment We have demonstrated how to facilitate the AIP cell therapy approach for treating diabetes 19

20 Acknowledgements Fabien Moncaubeig Andy Reniers Brian Gardell Nicholas Kohlstrom Heather Mallory, PhD Jack Vicalvi Todd Sanderson Lisa Bradbury, PhD Pall Life Science Orgenesis Sarah Ferber, PhD Irit Meivar Levy, PhD Keren Shternhall-Ron, PhD Vered Aviv, PhD Itai Tzchori Shimon Hassin, PhD BIRD Foundation 20

21 Thank You Questions