Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

Diffuse fluorescence fiber probe for in vivo detection of circulating cells.

There has been significant recent interest in the development of technologies for enumeration of rare circulating cells directly in the bloodstream in many areas of research, for example, in small animal models of circulating tumor cell dissemination during cancer metastasis. We describe a fiber-based optical probe that allows fluorescence detection of labeled circulating cells in vivo in a diffuse reflectance configuration. We validated this probe in a tissue-mimicking flow phantom model in vitro and in nude mice injected with fluorescently labeled multiple myeloma cells in vivo. Compared to our previous work, this design yields an improvement in detection signal-to-noise ratio of 10 dB, virtually eliminates problematic motion artifacts due to mouse breathing, and potentially allows operation in larger animals and limbs.

Versatile common instrumentation for optical detection of pH and dissolved oxygen.

The recent trend toward use of disposable and miniature bioreactors requires the use of appropriate sensors. pH and dissolved oxygen (DO) are often measured using optical chemical sensors due to their small form factor and convenience in use. These sensors are often interrogated using a specialized opto-electronic transducer that is designed around the optical sensor. In this contribution, we are presenting a new class of opto-electronic transducers that are usable with several different chemical sensors without the need to switch the optics or hardware when changing the type of the chemical sensor. This allows flexibility closer to the lab-grade devices while the size is closer to a dedicated sensor. This versatile instrumentation is capable of seamlessly switching between the pH and DO measurement modes and is capable of auto recognition of the sensor type. The principle of ratiometric fluorescence is used for pH measurements, and that of fluorescence lifetime for DO measurements. An approach to obtain identical calibrations between several devices is also presented. The described hardware constitutes common instrumentation for measuring either pH or DO and has been tested in actual bioprocesses. It has been found adequate for continuous bioprocess monitoring.