Process development of human mesenchymal stem cell microcarrier culture using an automated high-throughput microbioreactor

Improvements to process development technology will have a significant impact in reducing the overall costs associated with the manufacture and scale-up of human cell-based therapies. Small-scale models, including microbioreactors, play a critical role in this regard as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system (originally designed for free suspension culture) for adherent hMSC microcarrier culture. We also demonstrated that the ambr15 could be used for bioprocess development of a microcarrier process which was subsequently validated with larger-scale spinner flask studies. The results were achieved by a combination of strategies including adapting the free suspension design of the vessel to improve the suspension and mixing of the microcarriers. A more effective cell attachment method was also developed by using only 50% of the final working volume of medium for the first 24 h combined with an intermittent agitation strategy. These improvements led to a reduction in the initial lag phase which in turn resulted in \u3e 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 ambr 15, similar improvements were obtained in larger scale spinner flask studies. Finally, this improved bioprocess methodology, which was developed for a serum-based medium process, was applied to a serum-free process in the ambr15; this resulted in \u3e 250% increase in yield compared to the ambr15 serum-based process. 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 the serum free medium further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the consistency more than 10-fold compared to the initial manual, serum-based spinner flask work. 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 and automation improves both process yield and consistency. Please click Additional Files below to see the full abstract

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.</p> <p>Results</p> <p>Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of <it>P. pastoris </it>increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD₅₉₅^-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (<it>k_La</it>) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.</p> <p>Conclusions</p> <p>We show that addition of a range of antifoaming agents to shake flask cultures of <it>P. pastoris </it>increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.</p

A potentially scalable method for the harvesting of hMSCs from microcarriers

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

Tariff-based load shifting for domestic cascade heat pump with enhanced system energy efficiency and reduced wind power curtailment

Cascade air-to-water heat pumps may have good potential for retrofitting UK domestic buildings because they can directly replace existing fossil-fuel boilers without the requirement of considerable modifications to heat distribution systems. A widespread uptake of these heat pumps, however, would pose challenges to the grid. Furthermore, wind power generation has increased in the UK to achieve the target of decreasing CO2 emissions by 2050, but there are high levels of wind curtailment due to the mismatch between electricity supply and demand. In this paper, a load shifting study for cascade heat pumps coupled with thermal energy storage addressing these issues is presented. The main objective is to find the best tariff-based schedule load shifting for cascade heat pumps, which can help to avoid peak demand periods while obtaining enhanced system energy efficiency with minimised running costs and reduced wind energy curtailment. How the retrofit performance of the cascade heat pumps with load shifting is further investigated. TRNSYS was used to simulate the system performance validated against experimental results. Northern Ireland (UK) was selected as the evaluated scenario. Simulation results showed that the tank temperature set point of 75 °C and the storage size of 1.2 m3 could wholly shift the cascade heat pumps’ operation to off-peak periods. The best times to start the cascade heat pumps to charge the storage were at 3 am and 2 pm for the morning and afternoon heating demands, respectively. Compared to oil boilers, the cascade heat pumps with load shifting could obtain lower running costs (16–34%) and carbon emissions (20–37%)

Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the kLa by up to 35%. The n-dodecane additions to B. licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher kLa was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. N-dodecane can be used to increase the kLa when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific

Development of a process control strategy for the serum-free microcarrier expansion of human mesenchymal stem cells towards cost-effective and commercially viable manufacturing

Human Mesenchymal Stem Cells (hMSCs) are advancing through clinical development with the first allogeneic adult hMSC therapy receiving approval in Europe. To enable successful large-scale manufacture of hMSC therapies, increased product consistency and yield, and a reduced batch-to-batch variation must be achieved. This paper addresses ways to reduce variation by controlling the processing conditions, in particular the dissolved oxygen concentration (dO2), and the culture medium. Bone marrow derived hMSCs were cultured in DASGIP DASbox bioreactors on Plastic P-102 L microcarriers in FBS-containing and serum free (SFM) media at various dO2 values from 100% to 10%, experiencing the same dO2 value throughout the culture process. The superior control of pH and dO2 in the bioreactor led to improved performances compared to poorly controlled spinner flasks, particularly at reduced dO2 concentrations. At 25% dO2, there was a 300 % increase in the BM-hMSC yield in the bioreactor across the two donor BM-hMSCs in SFM compared to FBS-containing medium. Overall, the process yield increased by an average of around 500% for both donors under controlled conditions in SFM at 25% dO2 in the bioreactor compared to the poorly controlled expansion at atmospheric conditions in FBS-containing medium in spinner flasks. Process control significantly reduced the BM-hMSC variation in yield from 79.1% in FBS-containing medium in spinner flasks to < 15% in controlled SFM bioreactor culture

Agitation and aeration of stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development

The impact of agitation rate and sparged aeration on BM-hMSC expansion in conventional stirred tank bioreactors was assessed. It was found that a decrease in impeller speed to below NJS caused sampling difficulties, clumping and an increase to ~2 NJS decreased the growth rate though an intermediate value of ~1.3 NJS did not. Additionally, over this range of agitation intensities, cell quality remained unchanged post-harvest suggesting that poor growth performance at the highest speed was due to a failure of the cells to attach efficiently to microcarriers rather than damage to the cells due to fluid dynamic stress. Further it was shown that direct aeration of the culture medium both with and without Pluronic F68 via a sparger at NJS was detrimental to BM-hMSC growth. Again, this reduction in growth seems to be associated with poor attachment rather than cell damage, which due to the mechanism of PluronicTM F68 reducing the cell hydrophobicity and thus the affinity of the BM-hMSCs to attach to the microcarrier, leads to a poorer performance in the presence of the surfactant. Certain post-harvest quality characteristics are also detrimentally impacted compared to headspace aeration. This problem is discussed in terms of the need to facilitate future large-scale process development where headspace aeration at NJS may not be sufficient to meet culture needs at higher cell densities