Use of the XCell ATF in the Manufacture of Suspension Lentiviral Vector

Transcription

1 Use of the XCell ATF in the Manufacture of Suspension Lentiviral Vector Jesse Milling 20 June 2017 Nasdaq : BLUE

2 bluebird bio: Why We Do What We Do Ethan Aidan Cameron Our Vision Make Hope a Reality Seeking to transform the lives of patients with severe genetic and rare diseases through the development of innovative gene therapy products. 2

3 How Our Gene Therapy Approach Works Produce Viral Particle With Therapeutic Payload Isolate Target Cells From Patient Transduce Target Cells ex vivo Test & Re-infuse Gene Modified Cells Viral Particle Targeted Cells Gene Modified Cells Gene therapy seeks to correct disease by ultimately delivering functional genes (and the protein it expresses) to the DNA of the target cell. 3

4 bluebird Pipeline Overview 4

5 Adherent LVV Manufacturing Process Cell seeding in CF10 cell stacks Gag /Pol Env Rev GOI Transfect CaPO 4 + DNA Media replacement Harvest Media exchange No serum Acceptable for orphan diseases, but not for diseases with larger patient populations Process scales out but does not scale up Need to develop a larger production scale process 5

6 Comparability Challenge Options for LVV Production and Scalability Stainless Steel Bioreactor Single Use Bioreactor Suspension Producer Cell Line Cytodex Microcarriers Pall icellis Suspension Transient Tfxn HYPERstack 10-Tray Cell STACK Adherent Transient Tfxn Adherent Transient Tfxn Adherent Transient Adherent Transient Tfxn Tfxn Scale of LVV Production Process

7 Suspension Adapted cell line to serum-free suspension Developed in AMBR-15 and Eppendorf 5-L stirred tank reactors Similar to adherent process Transfection step optimized Manual media exchange/cell resuspension Gag /Pol Env Rev GOI Media Exchange Transient Transfection Growth Media Exchange Harvest 7

8 Titers Show Necessity of Media Exchanges No media exchange before or after transfection cuts titer in half Dilutions are also not as effective as complete media exchanges How does this fit into our goal of scale-up?

9 ATF Method Chosen and Purchased 3x volume exchange selected based on 95% theoretical exchange efficiency Developed appropriate flow rates and filtration rates based on volumes ATF Pump Rate Flow per fiber, Flow per fiber length, and shear Permeate Flux LMH ATF Pump to Filtration Ratio Maintain positive ratio state Volumes % Exchanged % Unexchanged % 60.7% % 36.8% % 22.3% % 13.5% % 8.2% % 5.0% % 1.8% % 0.7%

10 Engineering Run Growth Characteristics Reactor 1 = media exchange using ATF system Reactor 2 = media exchange using centrifugation and resuspension Pre-Tfxn CME Post-Tfxn CME

11 Engineering Run Transfection and Titer % GFP+ cells Titer Values Production Kinetics are Similar

12 Scale process up to 50L 50L SUB with an ATF4 for the media exchange 12

13 Scale-Up to 250L Scale The Good Scalable Constant flow per fiber length, flux values, and ATF:Filtration ratios Low shear (< 2500 s-1) Rooms for Improvement A lot of media Low end of recommended ATF:Filration ratio High end of recommended flow per fiber length values for scale-up Higher than recommended flux values in order to fit within a single working day High end of pump capability Confidential - 13

14 Further Development Are 3x reactor volumes necessary for CME? Confidential

15 Reasses ATF Parameters with Reduced Volume Exchange Where do we want to be? Balance between ATF Flow Rate and Filtrate Rate start with flow rate From Repligen (Refine) ATF Engineering Considerations for Scale-up 15

16 Further ATF Parameter Optimization Rooms for Improvement A lot of media reduced by 300% Low end of recommended ATF:Filration ratio able to increase ATF:Filtration Ratio by 100% High end of recommended flow per fiber length values for scale-up closer to recommended range for linear scale-up Higher than recommended flux values in order to fit within a single working day reduced flux values by 100% and within one working day (< 7 hours) High end of pump capability due to reduced flow rates, able to bump down to ATF6 use. Major benefit due to disposable considerations 16

17 Titer Results with ATF Optimization Optimized ATF with 1x CME Control with 3x CME 17

18 Further ATF Scale-Up Considerations Based on discussions with the Repligen team 1) It is important to keep the wv/day equal so that specific perfusion rates are equivalent across all scales (assuming equivalent cell densities) Complete 2) It is important to keep the Flow per Fiber equal so that cell shear is maintained across all scales 3) It is important to keep the Flux equal so that filtration capacity is maintained across all scales. ATF4 scaled on flow per fiber length due to shorter fiber length 4) It is important that the ATF to Filtration RATIO is approximately equal so that backflush efficiency is maintained across all scales 5) It is important that residence time is similar so that cells are exposed to the same level of Oxygen stress. Taken into consideration when designing bioreactor connections Also began to explore single use filter options with launch of the ATF2 and ATF6 single use filters by Repligen 18

19 ATF2 SU Comparability - Growth/Viability 19

20 ATF2 SU Comparabilty Transfection Kinetics R4 = SU R5 = control 20

21 Future Work and Acknowledgements Continue use of ATF2 system for platform process development Continue scale-up of the bbb developed process utilizing the ATF technology with partner sites to GMP scale Utilize the ATF6 single use system for GMP scale manufacturing Kelly Kral Susan Abu-Absi Michael Kuczewski Maya Miatkowski Tatiana Laivins Nolan Sutherland 21