Scale-up analysis of geometrically dissimilar single-use bioreactors.

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.

Quantifying the hydrodynamic stress for bioprocesses.

Hydrodynamic stress is an influential physical parameter for various bioprocesses, affecting the performance and viability of the living organisms. However, different approaches are in use in various computational and experimental studies to calculate this parameter (including its normal and shear subcomponents) from velocity fields without a consensus on which one is the most representative of its effect on living cells. In this letter, we investigate these different methods with clear definitions and provide our suggested approach which relies on the principal stress values providing a maximal distinction between the shear and normal components. Furthermore, a numerical comparison is presented using the computational fluid dynamics simulation of a stirred and sparged bioreactor. It is demonstrated that for this specific bioreactor, some of these methods exhibit quite similar patterns throughout the bioreactor-therefore can be considered equivalent-whereas some of them differ significantly.

Impact of post-freeze annealing on shrinkage of sucrose and trehalose lyophilisates.

Freeze-drying of pharmaceuticals produces lyophilisates with properties that depend on both the formulation and the process. Characterisation of the lyophilisate in terms of appearance is necessary not only to produce a visually appealing product, but also to gain insight into the freeze-drying process. The present study investigates the impact of post-freeze annealing on the volume of lyophilisates. For this purpose, sucrose and trehalose solutions were freeze-dried with different annealing conditions and the resulting lyophilisates were analysed with a 3D structured light scanner. The external structure of the lyophilisates was found to be dependent on the bulk materials as well as the choice of vials, while the volume was influenced by the annealing time and temperature. Additionally, differential scanning calorimetry was used to determine glass transition temperatures of frozen samples. As a novelty, the volumes of the lyophilisates and their corresponding glass transition temperatures were compared. This resulted in a correlation supporting the theory that the shrinkage of lyophilisates depends on the amount of residual water in the freeze-concentrated amorphous phase before drying. Understanding the volume change of lyophilisates, in combination with material properties such as glass transition temperature, forms the basis for relating physicochemical properties to process parameters in lyophilisation.

Quantitative Analysis of Glassy State Relaxation and Ostwald Ripening during Annealing Using Freeze-Drying Microscopy.

Supercooling during the freezing of pharmaceutical solutions often leads to suboptimal freeze-drying results, such as long primary drying times or a collapse in the cake structure. Thermal treatment of the frozen solution, known as annealing, can improve those issues by influencing properties such as the pore size and collapse temperature of the lyophilisate. In this study we aimed to show that annealing causes a rearrangement of water molecules between ice crystals, as well as between the freeze-concentrated amorphous matrix and the crystalline ice phase in a frozen binary aqueous solution. Ice crystal sizes, as well as volume fractions of the crystalline and amorphous phases of 10% (w/w) sucrose and trehalose solutions, were quantified after annealing using freeze-drying microscopy and image labelling. Depending on the annealing time and temperature, the amorphous phase was shown to decrease its volume due to the crystallisation of vitreous water (i.e., glassy state relaxation) while the crystalline phase was undergoing coarsening (i.e., Ostwald ripening). These results allow, for the first time, a quantitative comparison of the two phenomena. It was demonstrated that glassy state relaxation and Ostwald ripening, although occurring simultaneously, are distinct processes that follow different kinetics.

Numerical and Experimental Investigation of the Hydrodynamics in the Single-Use Bioreactor Mobius® CellReady 3 L.

Two-way Euler-Lagrange simulations are performed to characterize the hydrodynamics in the single-use bioreactor Mobius® CellReady 3 L. The hydrodynamics in stirred tank bioreactors are frequently modeled with the Euler-Euler approach, which cannot capture the trajectories of single bubbles. The present study employs the two-way coupled Euler-Lagrange approach, which accounts for the individual bubble trajectories through Langrangian equations and considers their impact on the Eulerian liquid phase equations. Hydrodynamic process characteristics that are relevant for cell cultivation including the oxygen mass transfer coefficient, the mixing time, and the hydrodynamic stress are evaluated for different working volumes, sparger types, impeller speeds, and sparging rates. A microporous sparger and an open pipe sparger are considered where bubbles of different sizes are generated, which has a pronounced impact on the bubble dispersion and the volumetric oxygen mass transfer coefficient. It is found that only the microporous sparger provides sufficiently high oxygen transfer to support typical suspended mammalian cell lines. The simulated mixing time and the volumetric oxygen mass transfer coefficient are successfully validated with experimental results. Due to the small reactor size, mixing times are below 25 s across all tested conditions. For the highest sparging rate of 100 mL min-1, the mixing time is found to be two seconds shorter than for a sparging rate of 50 mL min-1, which again, is 0.1 s longer than for a sparging rate of 10 mL min-1 at the same impeller speed of 100 rpm and the working volume of 1.7 L. The hydrodynamic stress in this bioreactor is found to be below critical levels for all investigated impeller speeds of up to 150 rpm, where the maximum levels are found in the region where the bubbles pass behind the impeller blades.

CFD-Based and Experimental Hydrodynamic Characterization of the Single-Use Bioreactor XcellerexTM XDR-10.

Understanding the hydrodynamic conditions in bioreactors is of utmost importance for the selection of operating conditions during cell culture process development. In the present study, the two-phase flow in the lab-scale single-use bioreactor XcellerexTM XDR-10 is characterized for working volumes from 4.5 L to 10 L, impeller speeds from 40 rpm to 360 rpm, and sparging with two different microporous spargers at rates from 0.02 L min-1 to 0.5 L min-1. The numerical simulations are performed with the one-way coupled Euler-Lagrange and the Euler-Euler models. The results of the agitated liquid height, the mixing time, and the volumetric oxygen mass transfer coefficient are compared to experiments. For the unbaffled XDR-10, strong surface vortex formation is found for the maximum impeller speed. To support the selection of suitable impeller speeds for cell cultivation, the surface vortex formation, the average turbulence energy dissipation rate, the hydrodynamic stress, and the mixing time are analyzed and discussed. Surface vortex formation is observed for the maximum impeller speed. Mixing times are below 30 s across all conditions, and volumetric oxygen mass transfer coefficients of up to 22.1 h-1 are found. The XDR-10 provides hydrodynamic conditions which are well suited for the cultivation of animal cells, despite the unusual design of a single bottom-mounted impeller and an unbaffled cultivation bioreactor.

Influence of particle size and blender size on blending performance of bi-component granular mixing: A DEM and experimental study.

The effect of particle size enlargement and blender geometry down-scaling on the blend uniformity (BU) was evaluated by Discrete Element Method (DEM) to predict the blending performance of a binary granular mixture. Three 10 kg blending experiments differentiated by the physical properties specifically particle size were performed as reference for DEM simulations. The segregation behavior observed during the diffusion blending was common for all blends, while the sample BU, i.e., standard deviation of active ingredient content % was different among the three blends reflecting segregation due to the particle size differences between the components. Quantitative prediction of the sample BU probability density distribution in reality based on the DEM simulation results was successfully demonstrated. The average root mean square error normalized by the mean of the mean sample BU in the blends was 0.228. Beside the ratio of blender container to particle size, total number of particles in the blender and the number of particles in a sample were confirmed critical for the blending performance. These in-silico experiments through DEM simulations would help in setting a design space with respect to the particle size and in a broader sense with respect to the physical properties in general.

Evaluation and prediction of powder flowability in pharmaceutical tableting.

The focus of this study is to establish a characterization method determining the powder flowability in context of tableting. At first, flowability of different materials is measured using the ring shear tester, and its prediction from particle size is established. Next, the model die-filling system is presented which is a modified version of previous studies. Using this system, flowability of different materials is measured at varying die speeds. A new curve fit to assess die fill ratio vs die speed is suggested improving predictability, and a novel flowability metric, "Die Fill Index" (DFI), is derived. The DFI is appropriate to describe flowability for most of the tested materials, and sensitivity of a material with respect to tableting speed. A correlation is generated predicting DFI from particle size. Additionally, it is shown that model die filling is the preferable method to assess flowability for tableting compared to ring shear tester.