The scale-up of microbial batch and fed-batch fermentation processes

Micro-organisms are important for both human health and to industry so the fed-batch cultivation of microbial strains, often over expressing recombinant or natural proteins, to high cell density has become an increasingly important technique throughout the field of biotechnology, from basic research programmes to large-scale pharmaceutical production processes (Hewitt et al., 1999). The scale-up of such a process is usually the final step in any research and development programme leading to the large-scale industrial manufacture of such products by fermentation (Einsele, 1978). It is important to understand that the process of scaling-up a fermentation system is frequently governed by a number of important engineering considerations and not simply a matter of increasing culture and vessel volume. Therefore, it is perhaps surprising when the large-scale does not perform as well as the small-scale laboratory process. It is often observed that the biomass yield and any growth associated products are often decreased on the scale-up of an aerobic process (Enfors et al., 2001). For Saccharomyces cerevisiae, the biomass yield on molasses increased by 7% when the process was scaled-down from 120 m3 to 10 l even when a seemingly identical strain, medium and process were employed (George et al., 1993). In an E. coli fed-batch recombinant protein process, the maximum cell density reached was found to be 20% lower when scaling-up from 3l to 9 m3 and the pattern of acetic acid formation had changed. (Bylund et al., 1998). During another study (Enfors et al., 2001), the performance of a recombinant strain of E. coli during fed-batch culture was found to vary on scale-up from the lab-scale to 10-30 m3 industrial bioreactors. This included lower biomass yields, recombinant protein accumulation and surprisingly perhaps a higher cell viability. These findings are typical of those found when scaling up most fermentation processes yet only a few mechanisms have been presented that can satisfactorily explain these phenomena. In this Chapter, we will briefly discuss the main engineering considerations involved in fermentation scale-up and then critically review those mechanisms thought to be responsible for any detrimental change in bioprocessing at the largerscale. Though it addresses mainly E. coli fed-batch fermentations, much of the discussion also applies to batch and other single celled aerobic microbial fermentations too

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

Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO2 evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO2 evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing

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

Expansion of human mesenchymal stem/stromal cells on temporary liquid microcarriers

BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION: The prospect of a temporary microcarrier that can be used to expand cells and then ‘disappear’ for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality

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

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation

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

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

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