Large-Scale Manufacture of Therapeutic Human Stem Cells - Pharmaceutical Technology

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Large-Scale Manufacture of Therapeutic Human Stem Cells
Large-scale manufacturing of human stem cells for therapeutic use is a leap in technology and science for the current biotechnology industry.

Pharmaceutical Technology
Volume 33, Issue 7, pp. 74-79

For many systems, there are question marks over true scalability and potential for CGMP compatibility because of their complexity. Furthermore, the level of control and monitoring for successful scaled production is not established, and these systems will be relatively expensive if simpler systems prove adequate. There may ultimately be an economic trade off between suboptimal production conditions and simpler or more generic production facilities. Finally, there is a surge in interest in applying conventional bioprocess stirred-tank reactors to the scale up of stem-cell production. These systems offer the advantages of decades of development in process control and knowledge of scale-up, much of which would carry over to stem-cell applications. Unfortunately, they are most suitable for nonadherent cell types and most stem cells are adherent cells. Attempts to 'adapt' cells to nonadherent culture using cytokines or cell adhesion molecule ligands, or to use microcarriers to achieve adherent growth in tanks, have had mixed success and are at an early stage.

There may be room for all of these approaches. Stirred tanks would be very good for large batch production of allogeneic therapies where T-flasks might be more appropriate for autologous production of patient-specific therapies. Complex bioreactors may find a role for niche stem-cell types that require highly specific environments, or as closed minimal intervention systems for stem cell manipulation within a clinical environment.

The underpinning manufacturing science:

Automation and process improvement

Although the stem-cell manufacturing requirements are not fully defined, and final solutions are still to be resolved, some spot solutions for the large-scale manufacturing of stem-cell based therapies have begun to emerge. It is clear that, from an economic and regulatory process-control perspective, manual processing of cellular therapies cannot be a long-term solution to cell-therapy production. To address this, commercial automated scalable systems have been marketed, including the Wave Bioreactor Systems technology from GE Healthcare (Piscataway, NJ) and the robotic T-flask handling platforms from The Automation Partnership (Hertfordshire, UK). These systems aim to create an automated robotic parallel to many of the current manual processes. They offer the opportunity to increase process reproducibility by removing operator variation, although they do not move the processes forward in terms of on-line monitoring and advanced environments. Instead, these systems were developed based on the reality that many products are moving toward the clinic using "simple" culture systems, and that these processes will be difficult to change significantly after clinical trials, given the problems of product measurement and importance of process validation. Such "simple" automated solutions will be essential to facilitate the movement of the industry to the next phase of mass market products. However, despite their similarity to manual handling, they face challenges with regard to the sensitivity of the cells to those process changes imposed through automation. They have also faced the skepticism of scientists steeped in the ethos that continual experience- based intervention, as opposed to rigorous process control, is the route to a consistent quality product.

Figure 1: The CompacT SelecT automated cell-culture platform can simultaneously manipulate 2 x T175 flasks and house 90 x T175 culture flasks in a robot-accessible incubatabor. All culture processes are carried out within a sterile class II environment and require no manual intervention. The inset shows an enlarged image of the manipulation chamber and pipette head. Major processing components are labeled: A, Robot arm; B, Flask incubator; C, Plate incubator; D, Flask decappers; E, Flask holders; F, Media pumps; G, Pipette head; H, Cedex automated cell counter.
In the Loughborough University Healthcare Engineering group, we have worked with academic and commercial stem-cell therapy researchers to demonstrate the successful and reproducible use of one of these automated cell-culture platforms, The Automation Partnership's CompacT SelecT (see Figure 1), to expand a range of clinically important human stem cell types at large scale. The CompacT SelecT automated cell culture platform can simultaneously manipulate 2 x T175 flasks and house 90 x T175 culture flasks in a robot-accessible incubator. All culture processes are carried out within a sterile class II environment and require no manual intervention. The CompacT SelecT closely mimics the normal manual cell-culture operation with the key exception of a centrifugation step.

Figure 2: Sample data from the successfully adapted automated bioprocessing of human embryonic stem cells (hES) and neural stem cells:A) the growth rate and normal morphology of automation-processed neural stem cells, B) Nestin expression of the neural stem cells C) growth rate and morphology of automation-processed hES cells, and D) normal expression of pluripotency markers by the hES cells.
Our objective was to automate commercial near-clinic and publicly available platform stem-cell types to have maximum broad impact on potential regenerative medicine applications. In the initial stage, human Mesenchymal stem cell (hMSC) expansion was automated (7). hMSCs are an adult stem cell, usually derived from bone marrow or umbilical cord blood, that are of clinical interest for mesenchymal tissue repair, including bone, cartilage, and muscle as well as a supporting cell in other tissue-repair scenarios such as post stroke. These target conditions represent a large clinical market. Bone-marrow-derived primary hMSCs that underwent automated expansion replicated at the equivalent rate to cells processed manually and maintained hMSC typical surface marker expression. More advanced automated stem-cell scale-up successes include hES cells and human neural stem cells. hES cells are important because they are pluripotent and therefore have the potential to be a complete platform cell type for any regenerative tissue therapy. They are notoriously difficult to culture. They grow in colonies and show both phenotypic and genotypic sensitivity to culture conditions. In collaboration with colleagues at Nottingham University we demonstrated that it is possible to maintain key cell attributes, including genetic stability, pluripotency markers, and differentiation potential over ten automated passages for two separate cell lines using a novel monolayer culture technique (8). The human neural stem-cell culture was conducted in collaboration with ReNeuron (Surrey, UK). This stem-cell line is entering clinical trials this year for the treatment of stroke, so the established culture conditions are designed to be compatible with CGMP and other regulatory requirements. Neural stem cells expanded in the automated system passed the cell-bank quality control tests including expansion rate, gene expression equivalence to master cell bank, and Nestin expression (9). Data from this work are shown in Figure 2.

Figure 3: The concepts of systematic process improvement and quality tools need to be applied to regenerative medicine manufacturing. A typical systematic process improvement cycle as applied to automated stem-cell processes is shown alongside an interaction chart from a designed experiment. The chart shows that a change in cell density significantly affects the impact of serum concentration on the rate of large-scale automated expansion of human mesenchymal stem cells. This type of complex interaction requires the application of sophisticated process optimization techniques. FCS is fetal calf serum.
Automated cell expansion is not only a route to large-scale stem cell production. It provides a platform for the application of manufacturing and process-improvement tools essential to a controlled manufacturing process. We have employed design of experiments (DOE) within a systematic process improvement methodology to improve the efficiency and quality of stem cells from the automated platform (see Figure 3). This work has identified the sensitivity of cells to cell culture factor interactions and reinforced the importance of a systematic approach to process characterization (10).


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