MASS TRANSFER ASPECTS OF CELL CULTURE IN A BIOREACTOR

Abstract

The widespread use of stirred tank bioreactors (STBRs) with agitation system as their core elements can be explained by their long tradition. STBRs being multiphase reactors are most widely used in industrial applications including chemical, biochemical, pharmaceutical and biological processes owing to their excellent operational flexibility and mixing capability. They play a vital role in the biopharmaceutical industry, particularly in aerobic bioprocesses and can find applications in fermentation and cell culture systems. STBRs have attracted much greater attention in the bioprocesses owing to their potential for integrating the development of high value-added products and thus replacing the need for conventional chemical based processes. STBRs have huge industrial importance as nearly 50% of the chemical reactants and products have passed through stirred tank reactors at one stage or the other and thus translating into over USD 1200 billion turnover per annum worldwide. To enhance the heat and mass transfer in such systems, baffles and impeller and other internals for specific applications are used. The oxygen transfer in such systems is an important parameter for determining their efficiencies and successful scale-up and is generally characterized by volumetric mass transfer coefficient, kLa being recognized as the most important parameter characterizing gas-liquid mass transfer in STBRs. It also serves as an important transport characteristic used in the scale-up, design and performance optimization of STBRs. The oxygen transfer is often considered as a rate limiting factor for the bioprocesses due to its low solubility in the liquid medium and therefore controlling dissolved oxygen in the liquid medium, i.e. broth is essential for cell growth. It is generally affected by agitation or (stirring) rate, aeration or (air flow rate) rate, media properties, different impeller types and their configurations, etc. Power consumption is also very important parameter in STBRs. It is an indispensable and the mostly used parameter to describe hydrodynamics, mixing and mass transfer and is also important scaling up parameter in stirred tank reactors. In this work, experiments have been carried out in stirred tank bioreactors of different volumes, i.e. 7.5 L, 5 L and 1 L. Dissolved oxygen concentration for the prediction of volumetric mass transfer coefficient, kLa has been measured by using the most widely used physical method, i.e. dynamic gassing-out-gassing-in method. It was observed that with an increase in scale of the reactor, irrespective of the impeller configuration, the kLa decreases when employing the same iii agitation speed (50-800 rpm) and aeration rate (0.5-3.5 L/min.). The effect of other parameters such as impeller diameter, liquid volume inside the reactor, liquid medium viscosity on kLa has also been studied. The power input per unit volume is also studied for single and dual Rushton turbine systems. It is observed that the power consumption in aerated system is lower than the unaerated system, because the transfer of power from impeller to the fluid is greatly influenced by aeration. It may also be attributed to the formation of cavities behind the impeller blades and the fluid having different density under gassed and ungassed conditions. The difference between gassed and ungassed power inputs is more pronounced at higher agitation rates (400-800 rpm). A new correlation has been proposed for kLa and P/VL based on a mathematical and statistical approach using response surface methodology (RSM) with Box-Behnken design (BBD) of experiments. This correlation includes the effect of various parameters such as agitation rate (50-800 rpm), air flow rate (0.5-3.5 L/min.) and temperature (10-40 °C) for different impeller configurations. Among the operating parameters, the most significant variable affecting kLa was found to be agitation rate, followed by aeration rate and temperature. The effect of temperature in most cases was insignificant. This may be most likely due to the range of temperature examined in this study was relatively narrow, typically used in commercial bioreactors. Among the investigated impeller configurations, dual Rushton turbine demonstrated the highest value of kLa. However, taking into account both kLa and shear force generated by agitation, the pitched blade turbine appears to be most effective for aerobic fermentation and cell culture applications. Models developed using RSM successfully interpreted experimental kLa and have been further validated under other operating conditions. More importantly, it is also observed that, compared with conventional power-law models, the RSM approach enables a more efficient correlation procedure and formulates simplified models with comparably high accuracy. Further to study the effect of impeller spacing on kLa and power input per unit volume (P/VL), RSM-BBD study has been carried out considering three factors, i.e. agitation rate (100-600 rpm) and aeration rate (1-12 L/min.) and impeller spacing (4-8 cm) for dual Rushton and mixed impeller (Rushton-marine propeller) configurations. It is found that kLa and P/VL were mainly affected by agitation rate, however the interaction between agitation rate and aeration rate is significant for both configurations. It is also observed that the effect of impeller spacing on kLa and P/VL was insignificant. Correlations developed using RSM for P/VL have been found to better predict as compared with the available correlations in the literature. It is also established that P/VL is lower iv for mixed impeller, thus suggesting its wide applicability in cell culture applications. A new power-law correlation is proposed for the mixed impeller configuration. Higher level of accuracy for both the original and simplified RSM models is observed as compared with conventional power-law models. Finally, the proposed simplified models successfully validated with the experimental data. A power-law correlation proposed for dual Rushton turbine has been found to well predict kLa and comparison has also been made with the available correlations in the literature and the RSM models, both original and simplified. Further, mass transfer and rheological behavior are characterized during the growth of E. coli BL21 in a stirred tank bioreactor. During the culture of E. coli BL21 in a 1 L stirred tank bioreactor, effects of various key operating variables such as agitation rate (50-600 rpm), aeration rate (0.5-2 L/min.), impeller diameter (4-5 cm), bioreactor working volume (0.25-0.75 L) for different impeller configurations on kLa has been investigated. It is observed that kLa increases with all the examined operating variables except the bioreactor working volume. Among the impeller configurations investigated, pitched blade turbine showed the highest kLa value (2.72 min-1), suggesting that it is promising for its successful cell culture as it also generates relatively less shear force owing to its low power number. A comparison evaluating mass transfer with and without cells has also been investigated in this study. A new impeller type, i.e. dislocated Rushton turbine has been investigated for its mass transfer performance having same dimensions as that of the standard Rushton turbine and is found to display superior mass transfer performance for E. coli BL21 culture, thus showing its potential for its application in bioprocess industry. Further, kLa for different impeller configurations is also correlated using dimensionless groups such as Reynolds, Froude and Flow numbers, suggesting this approach can be used for predicting kLa in different scale of stirred tank bioreactors. To understand rheological properties of the culture medium, in the present work, samples of the liquid medium with a dual Rushton turbine have been collected at specific time intervals, and their viscosity is evaluated. The rheological analysis showed that the viscosity of the liquid medium used in this study is independent on the shear rate, indicating that it behaves as a Newtonian liquid. Further, it is also observed that the shear stress linearly increases with the shear rate, which indicates that the liquid medium can be classified as a Newtonian liquid.