Work package 3: Automated expansion of transduced cells
Leader: Dr. Alexandra Stolzing, Loughborough University
Work Package 3 (WP3) addresses the expansion of BOEC cells that have been genetically corrected to include clotting factor VIII (FVIII). The main partners involved are Partner 3 UNILO, Partner 2 IMS and Partner 1 UKW.
The cell expansion will be adapted for automation under good manufacturing practice (GMP) conditions, in order to ensure a standardised production process. The use of single-use bioreactors versus multi-use bioreactors will be evaluated for this purpose.
A series of characterisation assays, ensuring the maximum safety of the therapeutically relevant cell population, will be identified. In addition, the effect of ageing on the expanded cells, during cell propagation, will be assessed as well.
Description of the tasks
Task 1: Expand BOECs from different donors – control donor variability.
Cryopreserved genetically corrected BOEC cells will be sent to UNILO by Partner 3 UPO. Cells’ expansion potential will be tested. Cells which will not pass the expansion test will be produced again by Partner 3, UPO.
Task 2: Expand under different oxygen conditions – control function.
Oxygen levels impact on the ability of cells to expand. Low oxygen levels result slower in vitro aging of the cells during expansion. Cells expanded under hypoxia are more resistant to apoptosis or inflammation and can improve engraftment. Other factors that could also have an effect are serum content, glucose content, cell matrix and growth factors. To be able to maximally expand the genetically corrected BOEC cells, UNILO will optimize the culture media for these cells, aiming for a xeno-free and fully defined culture media, in order to minimise in vitro aging of the cells. Partner 4, UNILO, has extensive experience in cell culture optimization (Stolzing et al., 2012; Efimenko et al., 2011; Stolzing et al., 2006; Stolzing & Scutt, 2006).
The quality control test for the cells at this stage will be their ability to produce factor VIII.
Task 3: Cryopreservation protocol development for BOECs and transfer of optimal conditions to UKW.
Partner 4, UNILO, has extensive experience in designing cryopreservation protocols for different cell products. UNILO’s main objective for this task is to develop a serum and DMSO free cryopreservation method, which would allow reducing centrifugation and washing steps in the cryopreservation process of the expanded BOEC cells. Achieving this means improving the final cell yield and minimising side-effects in the patient (Naaldijk et al., 2012a; Naaldijk et al., 2012b; Stolzing et al., 2011). Production of factor VIII will be the main quality control test for the cells at this stage, in determining the success of the cryopreservation protocol applied.
Once the protocol will have proved to be efficient, the cells will then be sent to partner 3, UPO, to be tested in vivo.
Partner 2, IMS, will then set-up the protocol for the transportation of the cryopreserved cells. Finally the whole process from isolation to cryopreservation of BOECs will be transferred to UKW.
Task 4: Create cost calculation model for the cell production based on market data.
UNILO will analyse the cost of the whole process with a focus on minimising the cost for the cell expansion process, as this is considered to be the main driver for cost in any cell therapy product development. However, other cost drivers will be quantified and included in the whole cost calculation. The goal of the expansion process is to produce cell number in the region of 109 cells per dose, capable of producing factor VIII. This cell dose should be enough for the life-time treatment of the patient.