Papers by Christopher Hewitt
Structures Congress 2008, 2008
Current Opinion in Chemical Engineering, 2013
ABSTRACT Highlights ► Cell based therapies can cure rather than treat medical conditions. ► There... more ABSTRACT Highlights ► Cell based therapies can cure rather than treat medical conditions. ► There is a need to obtain clinically relevant numbers of fully functional hMSCs. ► This need cannot be met by traditional manual culture methods alone. ► Microcarrier technology can be used to generate clinically relevant numbers of hMSCs. ► Concomitant novel downstream processing unit operations need to be developed.
Biotechnology and bioengineering, Jan 28, 2015
Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical developme... more Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal...
Phase inversion temperature (PIT) and self-emulsification (SE) technologies were used to produce ... more Phase inversion temperature (PIT) and self-emulsification (SE) technologies were used to produce novel forms of oil feed for fed-batch fermentations. These novel forms were supplied with the aim of improving both oil utilisation and product titre whilst reducing fermentation broth viscosity. The study involved industrial Streptomyces rimosus fermentations fed with rapeseed oil (RSO) for the production of oxytetracycline. In all cases, oil utilisation increased when compared to the control process. Additionally, a very significant decrease (50%) in the final concentration of residual oil was also achieved when either the PIT or self-emulsifying technologies were used. Further, a significant decrease (75%) in the culture broth viscosity was also measured when using the self-emulsifying method. It was concluded that the measurable increase in the utilization of oil was due to the formation of smaller and more stable oil droplets when using such technologies and/or the lower broth viscosity, which increased the mass transfer from the complex fermentation medium to the cells. In all cases, the final oxytetracycline concentration and time of attenuation was not significantly different for any of the fermentation processes.
Cell Technology for Cell Products, 2007
ABSTRACT
For stem cell therapy to become a routine reality, one of the major challenges to overcome is the... more For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me 2 SO). Me 2 SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me 2 SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me 2 SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.
ABSTRACT Two series of reproducible fed-batch bench scale cultures have been undertaken, one seri... more ABSTRACT Two series of reproducible fed-batch bench scale cultures have been undertaken, one series simulating the impact of spatial variations in pH and nutrients as found at commercial scale on performance, the other, the impact of fluid dynamic stresses associated with agitation. The first was unsuccessful because, somewhat surprisingly, the use of a peristaltic pump to circulate cells and medium through different spatial environments always led to a similar reduction in culture time and resulting product titre compared to uncirculated controls. This fall was sufficient to essentially mask other effects. In the second, even at maximum specific energy dissipation rates up to ∼160 times > with laminar extensional flow and ∼25 times > with turbulent flow compared to typical commercial conditions, no significant effects were observed on cell growth and viability. Most importantly, in all of the cases studied, product quality was unaffected compared to controls. In addition, it is suggested that because of the possibility of cell line specific behaviour and the relationship between damage to entities and the Kolmogorov scale of turbulence, sensitivity to fluid dynamic stresses is best studied in turbulent bench scale bioreactors.
ABSTRACT The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will ... more ABSTRACT The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5 L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7 min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100 mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip((R)) vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/)
ABSTRACT Two series of reproducible fed-batch bench scale cultures have been undertaken, one seri... more ABSTRACT Two series of reproducible fed-batch bench scale cultures have been undertaken, one series simulating the impact of spatial variations in pH and nutrients as found at commercial scale on performance, the other, the impact of fluid dynamic stresses associated with agitation. The first was unsuccessful because, somewhat surprisingly, the use of a peristaltic pump to circulate cells and medium through different spatial environments always led to a similar reduction in culture time and resulting product titre compared to uncirculated controls. This fall was sufficient to essentially mask other effects. In the second, even at maximum specific energy dissipation rates up to ∼160 times > with laminar extensional flow and ∼25 times > with turbulent flow compared to typical commercial conditions, no significant effects were observed on cell growth and viability. Most importantly, in all of the cases studied, product quality was unaffected compared to controls. In addition, it is suggested that because of the possibility of cell line specific behaviour and the relationship between damage to entities and the Kolmogorov scale of turbulence, sensitivity to fluid dynamic stresses is best studied in turbulent bench scale bioreactors.
Regenerative Medicine, 2012
Human pluripotent stem cells will likely be a significant part of the regenerative medicine-drive... more Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential, culture processes must be standardized, scalable and able to produce clinically relevant cell numbers, whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations, referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture, which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture, which may provide a cost-effective platform for low-dose, low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture.
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Papers by Christopher Hewitt