Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools.

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k(L)a) of sensor enabled static and rocking T-flasks is presented and compared with other non-instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k(L)a) in rocking T-flasks. We also carried out a scale-down study at matched k(L) a between a rocking T75-flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale-down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T-flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T-flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k(L)a) and disposability, we argue that rocking T-flasks can be readily integrated with wave bioreactors, making the transition from research-scale to manufacturing-scale a seamless process.

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool.

In this study, we perform mass transfer characterization (k(L) a) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch-based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5-fold resulted in 30% increase in k(L) a (from 2.3 to 3.0 h(-1) ) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control.

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask.

Routine cell culture is done in small-scale disposable vessels (typically 0.1-100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered "black boxes" because they are generally not equipped with sensors. In this study, we show that on-line monitoring of dissolved oxygen (DO) and pH in a T-75 flask-based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on-line monitoring of DO and pH show three distinctive metabolic regions in passages 1-18, 19-28, 29-54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (q(O2)), and the lactate production rate to the oxygen consumption rate yield (Y(Lac/ox)) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre- and seed culture prior to scale-up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques.

Confocal optical system: a novel noninvasive sensor to study mixing.

A novel confocal optical system to study mixing time in small-scale bioreactors is presented. The system is designed to monitor fluorescence upon tracer addition from a localized confocal volume of 0.21 mL within a glass vessel. The key elements of the fluorescence-based confocal system are a pinhole, a lens, an APD (Avalanche photodiode) detector, and light filters. The optical technique was validated by comparison with a pH-based technique. Finally, the optical sensor was tested and a real cultivation media (i.e., spent mammalian cell media) was used to measure mixing time in a 12.5-mL stirred transparent vessel. High accuracy, easy results interpretation, and low costs are the three most attractive characteristics of the sensor. Because of its noninvasive nature and versatility, the results suggest that the confocal system is a promising tool to perform mixing time studies in stirred vessels.

Integrating a 250 mL-spinner flask with other stirred bench-scale cell culture devices: a mass transfer perspective.

The bioprocess development cycle is a complex task that requires a complete understanding of the engineering of the process (e.g., mass transfer, mixing, CO(2) removal, process monitoring, and control) and its affect on cell biology and product quality. Despite their widespread use in bioprocess development, spinner flasks generally lack engineering characterization of critical physical parameters such as k(L)a, P/V, or mixing time. In this study, mass transfer characterization of a 250-mL spinner flask using optical patch-based sensors is presented. The results quantitatively show the effect of the impeller type, liquid filling volume, and agitation speed on the volumetric mass transfer coefficient (k(L)a) in a 250-mL spinner flask, and how they can be manipulated to match mass transfer capability at large culture devices. Thus, process understanding in spinner flasks can be improved, and these devices can be seamlessly integrated in a rational scale-up strategy from cell thawing to bench-scale bioreactors (and beyond) in biomanufacturing.

Optical analysis of liquid mixing in a minibioreactor.

A novel optical sensor was used to study mixing and mean circulation time in a model minibioreactor (12.5 mL stirred vessel, equipped with a paddle impeller). Rotational rates in the range of 10-1,000 rpm corresponding to Reynolds number between 14 and 1,350 were studied. Results suggest that depending on the impeller rotational speed, mixing times up to 214 +/- 87 s can be reproducibly achieved. The minibioreactor was operated in the transitional regime, and it was determined that the non-dimensional form for mixing time, NTheta(M) was linearly dependent on Reynolds number. A linear correlation between mean circulation time and the inverse of rotational speed was also determined. The mean circulation time dependence on rotational speed in the 12.5 mL stirred vessel is similar to those found in large-scale stirred vessels. These results suggest that mixing and circulation times found in large-scale reactors can be replicated in minibioreactors.