Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.

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 5L 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 7min) 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 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® 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

This article was published in the Biochemical Engineering Journal [© Elsevier B.V.] and the definitive version is available at: http://dx.doi.org/10.1016/j.bej.2013.04.011There is a growing body of evidence that the ambrTM workstation from TAP Biosystems performs well\ud in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the\ud physical characteristics of this microscale bioreactor system and found that these are quite different from\ud those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambrTM vessel is\ud transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low\ud whilst the specific power input is much higher (∼400 W/m3) when compared to that used at larger scales\ud (typically ∼20 W/m3). This specific power input is necessary in order to achieve kLa values sufficiently\ud high to satisfy the oxygen demand of the cells and control of dO2. In line with other studies, we find\ud that the culture of CHO cells in a 15 mL ambrTM bioreactor gave similar cell growth and productivity to\ud that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different.\ud Given the differences in physical characteristics between the ambrTM and larger stirred bioreactors, we\ud suggest that this similarity in biological performance is due to their similar control capabilities and the\ud ‘equivalence of the stress parameters’ across the scales when compared with shake flasks

This is an open access article published by Elsevier under the CC BY license\ud (http://creativecommons.org/licenses/by/4.0/)Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells\ud and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the\ud full value of this emerging field can be realized. The characterization of input material for cell-based therapy\ud bioprocesses from multiple donors is necessary to identify and understand the potential implications\ud of input variation on process development. In this work, we have characterized bone marrow derived\ud human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of\ud the measurable input variation on the development of autologous and allogeneic cell-based therapy\ud manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines\ud over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the\ud culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has\ud been demonstrated that this variation results in a range in the process time between these donor hMSC\ud lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing\ud products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT\ud criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the\ud different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of\ud 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent\ud of donor variability throughout the expansion process. Measuring informative product attributes during\ud process development will facilitate progress towards consistent manufacturing processes, a critical step\ud in the translation cell-based therapies