Development and characterisation of affinity devices for cell detection and separation
thesisposted on 24.11.2015, 16:46 by William S.C. Bowen
This thesis seeks to demonstrate technologies capable of improving the purity of pluripotent-derived cells. Such cells offer an enormous opportunity for medical science. However, it is anticipated that a higher proportion of undifferentiated pluripotent cells will remain when manufacturing at scale. As indicated in the literature, the in vivo transplantation of undifferentiated cells is a threat to patient safety and is considered a limiting factor for large-scale manufacture. Pluripotent undifferentiated cells can be identified and removed based on an affinity interaction with the SSEA-4 antigen, which is down-regulated on differentiated cells. Using CD20+ and CD20- (HLA-A2+) lymphocyte cells as a cost-effective alternative, where the CD20+ cells are target impurities and the HLA-A2+ cells are product-designated cells, this thesis presents two technologies to minimise the proportion of undifferentiated cells during manufacture. These technologies rely upon shear stress-induced affinity separation to differentiate between cells with and without target antigens. A small-scale model is used to identify a range of shear stresses (0 25 dynes/cm2) with which these differences can be elucidated. These technologies are: (1) a quartz crystal microbalance (QCM) biosensor to detect pluripotent cell differentiation over a multi-day period, particularly during process development. The limit of quantitation (LoQ) was estimated to be 5,000 cells, which would enable the measurement of target cell purities in excess of 4 %. Findings provide the basis for such a system, but also highlight the technical challenges of development, in particular variability. (2) two affinity membranes (hollow fibre and flat sheet) were used to deplete cells positive for the target antigen, demonstrating a possible downstream-purification tool in instances where clinical purity does not suffice. The shear stress-induced detachment of adsorbed cells incubated for 30 minutes was investigated over 1 25 dynes/cm2 and 1 10 dynes/cm2 for the hollow fibre and flat sheet membranes respectively. Measured output included cell purity, which showed an increase in the relative change in purity (RCP) of 0.2 0.5 for the hollow fibre modules at 5 dynes/cm2 (n = 5) and was as high as 11.8 at 10 dynes/cm2 for the flat sheet membrane cassette (n = 1). A loss of cell membrane integrity, where up to 5 % in hollow fibres and up to 55 % in the flat sheet membrane were observed.
EPSRC, Pall Life Sciences
- Aeronautical, Automotive, Chemical and Materials Engineering
- Chemical Engineering