Search

• Low-aspect-ratio partially-filled cylinder examined with multi experimental methods. • Fundamental knowledge for improved mixing in the intermittently rotating bioreactor. • Interaction between unstable boundary layers and inviscid interior. • Scalability of macromixing is demonstrated with strategies to optimise micromixing. This work explores the mixing and fluid dynamics in a novel bioreactor currently used for the automated manufacturing of CAR-T cell therapy, which offers a single-dose cure for several forms of advanced blood cancer. The cylindrical bioreactor has a low aspect ratio and a free surface. Agitation is achieved by intermittent rotation of the entire vessel around its central axis. No engineering characterisation has been conducted to date for this system in a bioprocessing context. The study examines the fluid dynamics problems of spin-up from rest and spin-down to rest. Novel Particle Image Velocimetry and Planar Laser-Induced Fluorescence data is presented alongside reflective flakes results, shedding light on the different transient flow regions inside the intermittently rotating bioreactor, and determining the timescales of macro- and micromixing. The results presented can be used to design a custom rotation pattern of the bioreactor for improved mixing performance during the cell expansion step. [ABSTRACT FROM AUTHOR]

Abstract: Reliable models of enzyme kinetics are required for the effective design of bioconversion processes. Kinetic expressions of the enzyme-catalysed reaction rate however, are frequently complex and establishing accurate values of kinetic parameters normally requires a large number of experiments. These can be both time consuming and expensive when working with the types of non-natural chiral intermediates important in pharmaceutical syntheses. This paper presents an automated microscale approach to the rapid and cost effective generation of reliable kinetic models useful for bioconversion process design. It incorporates a model driven approach to the experimental design that minimises the number of experiments to be performed, while still generating accurate values of kinetic parameters. The approach has been illustrated with the transketolase mediated asymmetric synthesis of L-erythrulose. Experiments were performed using automated microwell studies at the 150 or 800μL scale. The derived kinetic parameters were then verified in a second round of experiments where model predictions showed excellent agreement with experimental data obtained under conditions not included in the original experimental design. In comparison with conventional methodology, the modelling approach enabled a nearly 4-fold decrease in the number of experiments while the microwell experimentation enabled a 45-fold decrease in material requirements and a significant increase in experimental throughput. The approach is generic and could be applied to a wide range of enzyme catalysed bioconversions. [Copyright &y& Elsevier]

Abstract: Pressures on pharmaceutical companies to speed bioprocess development have led to significant interest in small scale, parallel experimentation. A particular focus is cell cultivation and the optimisation of protein synthesis because of the number of biological and engineering variables involved. In this work, we briefly review the current understanding of mixing and mass transfer phenomena in shaken bioreactors with a view to defining criteria for the scale-up of results obtained in shaken microwell systems to conventional laboratory scale. Scale-up approaches are illustrated for two different cell cultures. The first involves an automated microscale process for the aerobic fermentation of E. coli JM107:pQR706 overexpressing transketolase (TK) which is subsequently used for asymmetric carbon–carbon bond formation. The kinetics of both the fermentation and bioconversion stages are first quantified as a function of fermentation medium composition (LB or LB-glycerol) and shaking frequency with oxygen transfer rates being identified as rate limiting in certain cases. Successful scale-up of the microwell process (in terms of maximum cell growth rate, biomass yield and specific TK activity) to a 1.4l scale mechanically stirred bioreactor is then demonstrated based on experiments performed at constant values. The second process investigated involved antibody production in suspension cultures of VPM8 hybridoma cells. Initial results suggest that experiments performed at constant mean energy dissipation rates provide a satisfactory basis for scale translation from shaken microwells to conical flasks (100ml) and are indicative of results obtained in a mechanically stirred bioreactor (3.5l). Overall this work provides an initial insight into the engineering characterisation of shaken bioreactors and how key parameters may be used to define suitable scale-up criteria for different cell cultures. [Copyright &y& Elsevier]

Microcarrier suspension performance and ensemble-averaged flow fields at the just-suspended condition are studied in a scale-down mimic of the Allegro STR single-use bioreactor range (abbreviated A-STR) and compared to an equivalent standard cylindrical configuration (abbreviated S-STR). Four commercial microcarriers were assessed to determine the required impeller agitation rate and corresponding power input per unit volume to achieve suspension – Cytodex™ 1, Cytodex™ 3, Cytopore™ 1, and Hillex® II microcarriers. The A-STR operating in down-pumping mode (DP) achieved homogenous microcarrier suspension at considerably lower power input when compared to the other baffled configurations. For the A-STR (DP), the power input per unit volume demand was 67.8 % lower than the A-STR in up-pumping mode (UP), 77% lower than the S-STR (DP) and 66.8 % lower than the S-STR (UP). Flow structures investigated at the just-suspended condition, in the transitional regime, were in line with those previously presented in the turbulent regime at considerably higher Reynolds number. This indicates that the normalised flow structures presented in this work are representative of those in the commercial Allegro STR range, despite operating at significantly reduced scale and Reynolds number. Removal of baffles resulted in substantially reduced power input required to achieve homogenous suspension in all configurations – other than the A-STR (DP), which exhibited relatively similar performance to the corresponding baffled configuration. Considering adherent cell sensitivity and mixing implications, and based on the results found in this work, the baffled A-STR (DP) is recommended for achieving microcarrier suspension at the lowest specific power input. • Microcarrier suspension is characterised in a scale-down mimic of the Allegro STR. • Homogenous suspension can be achieved at reduced power input in the cubical geometry. • The implications of baffles on suspension efficiency are explored. [ABSTRACT FROM AUTHOR]

• Allegro STR 50–2000 L bioreactor range is successfully scaled down to 1 L. • Agreement of power and mixing number across 1 to 200 L bioreactor scale. • Square cross-section of vessel increases impeller power number. • Mean flow characterisation of unique vessel geometry. • Allegro STR geometry enhances mixing and kinetic energy distribution. The development of new biopharmaceuticals relies on robust scale-up from small-scale screening studies to industrial bioreactors. Novel SUB designs can prove highly beneficial, although they lack the extensive characterisation of traditional STRs. Pall Corporation's Allegro™ STR 50–2000 L bioreactor range has a unique design of square cross-section, with three wedge-shaped baffles. A scale-down 1 L prototype (abbreviated A-STR) was developed for characterisation studies and compared to a standard cylindrical STR (abbreviated S-STR). Agreement of power and mixing number data with that of the Allegro STR 200 L indicated successful scale-down. In down-pumping mode, mixing times in the A-STR were approximately 53 % lower than in the S-STR. However, in up-pumping mode both configurations exhibited similar mixing times. This study utilises a scale-down prototype to provide quantitative data on the commercial Allegro STR bioreactor range, to define operating parameters for enhanced scalability, and for comparison with a standard bioreactor geometry. [ABSTRACT FROM AUTHOR]

Abstract: The commercial success of mammalian cell-derived recombinant proteins has fostered an increase in demand for novel single-use bioreactor (SUB) systems that facilitate greater productivity, increased flexibility and reduced costs (Zhang et al., 2010). These systems exhibit fluid flow regimes unlike those encountered in traditional glass/stainless steel bioreactors because of the way in which they are designed. With such disparate hydrodynamic environments between SUBs currently on the market, traditional scale-up approaches applied to stirred tanks should be revised. One such SUB is the Mobius® 3L CellReady, which consists of an upward-pumping marine scoping impeller. This work represents the first experimental study of the flow within the CellReady using a Particle Image Velocimetry (PIV) approach, combined with a biological study into the impact of these fluid dynamic characteristics on cell culture performance. The PIV study was conducted within the actual vessel, rather than using a purpose-built mimic. PIV measurements conveyed a degree of fluid compartmentalisation resulting from the up-pumping impeller. Both impeller tip speed and fluid working volume had an impact upon the fluid velocities and spatial distribution of turbulence within the vessel. Cell cultures were conducted using the GS-CHO cell-line (Lonza) producing an IgG4 antibody. Disparity in cellular growth and viability throughout the range of operating conditions used (80–350rpm and 1–2.4L working volume) was not substantial, although a significant reduction in recombinant protein productivity was found at 350rpm and 1L working volume (corresponding to the highest Reynolds number tested in this work). The study shows promise in the use of PIV to improve understanding of the hydrodynamic environment within individual SUBs and allows identification of the critical hydrodynamic parameters under the different flow regimes for compatibility and scalability across the range of bioreactor platforms. [Copyright &y& Elsevier]

Abstract: Objective: A survey entitled FEMME was conducted during 2002 in order to evaluate among French women doctors their own actual or future menopause perception and this before the WHI publication. The results of this American trial possibly modified the perception of these French women doctors. Therefore the same experts group 1 conducted a new survey, from May to September 2003. The main aim of this survey was to evaluate the possible changes in the medical management of the actual or future menopause of these women, and secondarily to evaluate the changes in their patients'' behaviour towards hormone replacement therapy (HRT). Population and methods: Postal auto administered questionnaires were sent to the same 10 000 French women doctors (GP or gynaecologist) whatever their menopausal status or their age. 1365 women doctors (respectively 18,5 or 11% of the gynaecologists or GPs contacted) were volunteers to participate in this survey. Among them, 1120 (84,9%) had already participated in the first part of this survey which took place before the WHI publication. Results: 80% of these women doctors have been informed on WHI results principally by professional press or conferences. 70,9% changed their own actual or future menopause perception as follows. No additional selection of non hormonal treatment have been mentioned in comparison with the first part of the survey. On the other hand for HRT, selections of free estrogen plus progestin associations increased whereas those of fixed combinations decreased: this might be linked to the greater variety of estrogen doses, types of progestin and schedules of treatment (mostly with bleeding) offered by this kind of associations. Finally, duration of HRT is included between three and ten years in most cases. Discussion and conclusion: Thus, unlike most of their patients, these women''s physicians always preferred hormonal treatment for their own actual or future menopause. Only the conditions of these treatments have changed. [Copyright &y& Elsevier]