Qasim A. Rafiq

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

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 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Abstract 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.

A quantitative approach for understanding small-scale human mesenchymal stem cell culture - implications for large-scale bioprocess development.

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2 ), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h(-1) ) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing.

Serum-free process development: improving the yield and consistency of human mesenchymal stromal cell production

BACKGROUND AIMS The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. METHODS Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. RESULTS Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R(2) = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. CONCLUSIONS We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate.

BACKGROUND AIMS The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. METHODS In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. RESULTS The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 10(5) and 1.02 ± 0.005 × 10(5) cells/mL compared with 0.79 ± 0.05 × 10(5) and 0.36 ± 0.04 × 10(5) cells/mL in FBS-containing medium. CONCLUSIONS This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Developing an automated robotic factory for novel stem cell therapy production

Advanced therapeutics, specifically cell and gene therapies, provide an opportunity to target previously unmet clinical conditions and offer a potential solution to the social and economic burden associated with an aging population [1,2]. Human mesenchymal stem/stromal cells (hMSCs) are a promising cell therapy candidate for the treatment of numerous clinical indications [3]; however, a transformation is required in the way we isolate, manufacture, characterize and deliver these therapies to ensure they are both efficacious and affordable [2,4]. Manufacture of hMSCs requires in vitro expansion to increase the available number of cells to meet clinical demand; however, progress is impeded by the lack of advanced methods for the isolation and expansion of cells that are scalable, amenable for automation and closed. Many allogeneic processes still require manual intervention which has significant quality and cost implications [5], and developing reproducible, consistent bioprocesses is still a major challenge. Even when a scale-out approach is to be employed (e.g., for autologous therapies), there is a significant practical challenge of manipulating, processing and segregating multiple production batches in an aseptic, closed manner. Such processes demand small units for manufacture in which line segregation is a priority in order to avoid product–patient mismatch and disease transmission. As such, there is an industrial trend toward automated systems as it is increasingly recognized that such systems facilitate consistent manufacture and will play a pivotal role in the translation of cell therapies by improving quality control, process economics, scalability, process capability and provide a platform for understanding process variation and optimization [6]. Through AUTOSTEM (European Commission Horizon 2020 funded research and innovation actions), academic and industrial groups from across the EU are working in collaboration to take a holistic approach to enable large-scale hMSC production, at clinical-grade quality, by implementing a robotic automated pipeline for cell isolation and culture [7]. AUTOSTEM builds on the ground-breaking work of the StemCellFactory project [8] which built and demonstrated an automated robotic pipeline for the production of human induced pluripotent cell lines (Figure 1). AUTOSTEM develops this technology further to enable automated, closed and good manufacturing practice (GMP)ready hMSCs (a ‘StromalCellFactory’) and will focus on the development of:

Bioreactor engineering fundamentals for stem cell manufacturing

Cell-based therapies have the potential to address unmet healthcare needs and improve quality of patient care; effective manufacture is therefore essential. There are, however, many challenges to overcome before this can become a reality and a better understanding of the manufacturing requirements for cell-based products is required. A range of stem cell types are considered as useful cell therapy candidates for various reasons including human embryonic stem cells, induced pluripotent stem cells, and human mesenchymal stem cells (hMSCs). While all have potential as cell-replacement therapies, hMSCs are of particular interest because of their additional immunomodulatory and immune-evasive properties. Until recently, these cells were cultured as a monolayer in tissue culture-treated flasks. However, to produce the number of cells required to meet economically potential demand, it is important to provide greater surface area per unit volume of medium on which the cells can grow. This need has led to the use of microcarriers, particles of some 200. ce:hsp sp=0.25/μm in diameter on which the cells can grow in suspension-based bioreactor systems. After growth, with medium exchange and passaging, the cells have to be harvested. Given the flexibility of stirred tank reactors and the extensive knowledge available from studies of particle suspension within them and their use in free suspension culture, they have become the configuration of choice for culturing adherent cells at increasing scale. This chapter discusses, in detail, the underlying basic principles of stirred tank bioreactors including particle suspension, mass transfer, and the fluid dynamic stresses, which potentially might damage cells. These basic concepts are then related to the cultivation of hMSCs on microcarriers, which has been demonstrated up to the 1000. L scale. It is also shown how concepts from crystallization in stirred tank reactors involving microcarrier-microcarrier and microcarrier-impeller impacts have enabled a successful, insitu harvesting strategy to be developed. The overall concept of growing hMSCs at the minimum agitator speed required for suspension and harvesting them by a short burst of intense agitation in the same bioreactor is introduced. Finally, data are presented showing the success of that approach in 22 combinations involving bioreactors from 15. mL to 5. L with different cell donors, microcarriers (with and without coatings), and detachment enzymes.

Automating decentralized manufacturing of cell and gene therapy products.

Whilst you don’t have to be a botanist to appreciate the first half of this statement, it is only the purest of physicists that will disagree with the latter. The world of drug development, of which cell and gene therapies currently represent the forefront of scientific, clinical and business activity, is an expensive undertaking (where money trees are few and far between). Moreover, global healthcare providers have never been under greater pressure to reduce expenditure in order to meet ever decreasing budgets and an ever increasing patient population. So, whilst addressing unmet clinical needs or improving clinical efficacy does and should remain the primary driver of novel cell and gene therapy development, the expense of implementing such therapies from a manufacturing, logistical and clinical perspective is coming under greater scrutiny and mounting importance – the era of cost-based development is upon us and is central to the approach of the Cell and Gene Therapy Catapult.

Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266.

The early career researcher's toolkit:translating tissue engineering, regenerative medicine and cell therapy products

Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization.