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Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (kLa) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%–66% in biomass and 41%–56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane‐diffusion‐based sensing platform for the integration of low‐cost, noninvasive sensing capabilities for real‐time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide. [ABSTRACT FROM AUTHOR]

Dissolved carbon dioxide (dCO2) is a well‐known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small‐scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small‐scale systems. In this study, we present the implementation of a novel, rate‐based technique for real‐time monitoring of dCO2 in bioprocesses. A silicone sampling probe that allows the diffusion of CO2 through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO2 that had diffused into the probe from the culture broth (sensor was calibrated using air as zero‐point calibration). The gas inside the probe was then allowed to recirculate through gas‐impermeable tubing to a CO2 monitor. We have shown that by measuring the initial diffusion rate of CO2 into the sampling probe we were able to determine the partial pressure of the dCO2 in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO2 in real‐time. Using the proposed sensor, we successfully implemented a dCO2‐based control scheme, which resulted in significant improvement in process performance. [ABSTRACT FROM AUTHOR]

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small‐scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real‐time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results. [ABSTRACT FROM AUTHOR]

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care. [ABSTRACT FROM AUTHOR]