A Cell-Free Protein Expression System Derived from Human Primary Peripheral Blood Mononuclear Cells.

Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.

Manufacturing biological medicines on demand: Safety and efficacy of granulocyte colony-stimulating factor in a mouse model of total body irradiation.

Protein therapeutics, also known as biologics, are currently manufactured at centralized facilities according to rigorous protocols. The manufacturing process takes months and the delivery of the biological products needs a cold chain. This makes it less responsive to rapid changes in demand. Here, we report on technology application for on-demand biologics manufacturing (Bio-MOD) that can produce safe and effective biologics from cell-free systems at the point of care without the current challenges of long-term storage and cold-chain delivery. The objective of the current study is to establish proof-of-concept safety and efficacy of Bio-MOD-manufactured granulocyte colony-stimulating factor (G-CSF) in a mouse model of total body irradiation at a dose estimated to induce 30% lethality within the first 30 days postexposure. To illustrate on-demand Bio-MOD production feasibility, histidine-tagged G-CSF was manufactured daily under good manufacturing practice-like conditions prior to administration over a 16-day period. Bio-MOD-manufactured G-CSF improved 30-day survival when compared with saline alone (p = .073). In addition to accelerating recovery from neutropenia, the platelet and hemoglobin nadirs were significantly higher in G-CSF-treated animals compared with saline-treated animals (p < .05). The results of this study demonstrate the feasibility of consistently manufacturing safe and effective on-demand biologics suitable for real-time release.

Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields.

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.

Real-time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control.

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.

Wood Microfluidics.

As nonbiodegradable plastics continue to pollute our land and oceans, countries are starting to ban the use of single-use plastics. In this paper, we demonstrated the fabrication of wood-based microfluidic devices and their adaptability for single-use, point-of-care (POC) applications. These devices are made from easily sourced renewable materials for fabrication while exhibiting all the advantages of plastic devices without the problem of nonbiodegradable waste and cost. To build these wood devices, we utilized laser engraving and traditional mechanical methods and have adapted specific surface coatings to counter the wicking effect of wood. To demonstrate their versatility, wood microfluidic devices were adapted for (i) surface plasmon coupled enhancement (SPCE) of fluorescence for detection of proteins, (ii) T-/Y-geometry microfluidic channel mixers, and (iii) devices for rapid detection of microbial contamination. These provide proof of concept for the use of wooden platforms for POC applications. In this study, we measured the fluorescence intensities of recombinant green fluorescent protein (GFP) standards (ranging from 1.5-25 ng/µL) and 6XHis-G-CSF (ranging from 0.1-100 ng/µL) expressed in cell-free translation systems. All tested devices perform as well as or better than their plastic counterparts.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the µCols compared with conventional columns for similar protein yields.

Minimally invasive technique for measuring transdermal glucose with a fluorescent biosensor.

There is a need for blood glucose monitoring techniques that eliminate the painful and invasive nature of current methods, while maintaining the reliability and accuracy of established medical technology. This research aims to ultimately address these shortcomings in critically ill pediatric patients. Presented in this work is an alternative, minimally invasive technique that uses microneedles (MN) for the collection of transdermal glucose (TG). Due to their comparable skin properties, diffusion studies were performed on full thickness Yucatan miniature pig skin mounted to an in-line diffusion flow cell and on different skin sites of human subjects. Collected TG samples were measured with a L255C mutant of the E. coli glucose-binding protein (GBP) with an attached fluorescent probe. The binding constant (Kd = 0.67 µM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the animal and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN-treated human skin, a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30 s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In addition, the his-tagged optical biosensor used in this work can be immobilized and used with a portable sensing fluorometer device at the point of care (POC) making this minimally invasive technology more ideal for use in the pediatric intensive care unit. Graphical abstract ᅟ.

Rapid recombinant protein expression in cell-free extracts from human blood.

Several groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.

Development and characterization of a point-of care rate-based transcutaneous respiratory status monitor.

Blood gas measurements provide vital clinical information in critical care. The current "gold standard" for blood gas measurements involves obtaining blood samples, which can be painful and can lead to bleeding, thrombus formation, or infection. Mass transfer equilibrium-based transcutaneous blood gas monitors have been used since the 1970s, but they require heating the skin to ≥42 °C to speed up the transcutaneous gas diffusion. Thus, these devices have a potential risk for skin burns. Here we report a new generation of noninvasive device for respiratory status assessment. Instead of waiting for mass transfer equilibrium, the blood gas levels are monitored by measuring the transcutaneous diffusion rate, which is proportional to blood gas concentration. The startup time of this device is almost independent of skin temperature, so the measurement can be made at any body temperature. The test results show that this device can track the blood gas levels quickly even at normal body temperature.

Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system.

Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.

Cell-free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate.

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.

Point-of-care production of therapeutic proteins of good-manufacturing-practice quality.

Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.

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.

Measuring transdermal glucose levels in neonates by passive diffusion: an in vitro porcine skin model.

Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which require intermittent blood collection through skin-penetrating pricks on the heel or fingers. This procedure is painful and often not clinically conducive, which presents a need for a noninvasive method for monitoring glucose in neonates. Our motivation for this study was to develop an in vitro method for measuring passive diffusion of glucose in premature neonatal skin using a porcine skin model. Such a model will allow us to initially test new devices for noninvasive glucose monitoring without having to do in vivo testing of newborns. The in vitro model is demonstrated by comparing uncompromised and tape-stripped skin in an in-line flow-through diffusion apparatus with glucose concentrations that mimic the hypo-, normo-, and hyper-glycemic conditions in the neonate (2.0, 5.0, and 20 mM, respectively). Transepidermal water loss (TEWL) of the tape-stripped skin was approximately 20 g m-2 h-1, which closely mimics TEWL for neonatal skin at about 190 days post-conceptional age. The tape-stripped skin showed a >15-fold increase in glucose diffusion compared to the uncompromised skin. The very small concentrations of collected glucose were measured with a highly selective and highly sensitive fluorescent glucose biosensor based on the glucose binding protein (GBP). The demonstrated method of glucose determination is noninvasive and painless, which makes it especially desirable for glucose testing in neonates and children. This study is an important step towards an in vitro model for noninvasive real-time glucose monitoring that may be easily transferred to the clinic for glucose monitoring in neonates. Graphical Abstract Glucose diffusion through model skin was measured using an in-line flow-through diffusion apparatus with glucose solutions mimicking hypo-, normo- and hyperglycemia in the neonate. Phosphate buffered saline was added to the top chamber and the glucose that diffused through the model skin into the buffer was measured using a fluorescent glucose binding protein biosensor.

The effect of pH on the glucose response of the glucose-galactose binding protein L255C labeled with Acrylodan.

The glucose-galactose binding protein (GGBP) is used as an optical biosensor in medical and bioprocess applications. This paper investigates the effect of pH on the behavior of GGBP-L255C labeled with Acrylodan for the purpose of finding the optimum conditions for sensing purposes as well as for protein preparation, purification and storage. The Acrylodan-GGBP fluorescence response in absence and presence of glucose was measured under varying buffer and pH conditions. Dissociation constants (Kd) and Gibbs free energies (ΔG) for the protein-glucose binding were calculated. Binding was found to be energetically favored at slightly acidic to neutral conditions, specifically close to the pI of GBP (∼ 5.0). Minimal fluorescence response to glucose was exhibited at pH 3.0 accompanied by a blue shift in the steady state fluorescence spectrum. In contrast, an almost 45% response to glucose was shown at pH 4.5-9.0 with a 13-nm red shift. Frequency domain lifetime measurements and quenching with KI suggest that at highly acidic conditions both the glucose-free and the glucose-bound protein are in a conformation distinct from those observed at higher pH values.

Passive Diffusion of Transdermal Glucose: Noninvasive Glucose Sensing Using a Fluorescent Glucose Binding Protein.

The motivation for this study was to determine if a statistically significant correlation exists between blood glucose (BG) and transdermal glucose (TG) collected by passive diffusion. A positive outcome will indicate that noninvasive passive TG diffusion is a painless alternative to collecting blood through a break on the skin. Sampling involves placing a small volume of buffer solution on the surface of membrane or skin for 5 minutes. The sample is then assayed with fluorescent GBP. In vitro testing was done on regenerated cellulose and a porcine skin model to determine diffusion of standard glucose solutions. In vivo testing was done on a healthy subject and a subject with type 2 diabetes. Glucose diffused readily through the regenerated cellulose membrane with good correlation between surface and internal glucose concentrations (R 2 = .997). But the porcine skin model required a surface prewash to achieve the same good correlation R 2 = .943). Based on this, an optimum prewash step was determined for the in vivo studies. The resulting correlation coefficients between TG and BG after a 15-minute prewash in a healthy subject and type 2 subject were .87 and .93, respectively. Removal of the extraneous glucose in the skin by prewashing was an important step in achieving good correlation between TG and BG. The results suggest that passive collection of TG is a noninvasive alternative to current practice of breaking the skin. Further studies are under way to determine the lag time between TG and BG and for the sampling protocol to be more amenable to point-of-care application.

Designing a Low-Cost Multifunctional Infant Incubator.

Every year, an unacceptably large number of infant deaths occur in developing nations, with premature birth and asphyxia being two of the leading causes. A well-regulated thermal environment is critical for neonatal survival. Advanced incubators currently exist, but they are far too expensive to meet the needs of developing nations. We are developing a thermodynamically advanced low-cost incubator suitable for operation in a low-resource environment. Our design features three innovations: (1) a disposable baby chamber to reduce infant mortality due to nosocomial infections, (2) a passive cooling mechanism using low-cost heat pipes and evaporative cooling from locally found clay pots, and (3) insulated panels and a thermal bank consisting of water that effectively preserve and store heat. We developed a prototype incubator and visited and presented our design to our partnership hospital site in Mysore, India. After obtaining feedback, we have determined realistic, nontrivial design requirements and constraints in order to develop a new prototype incubator for clinical trials in hospitals in India.

Portable system for the detection of micromolar concentrations of glucose.

Glucose in non-invasively collected biofluids is generally in the micromolar range and thus, requires sensing methodologies capable of measuring glucose at these levels. Here, we present a small fluorometer system that can quantify glucose in the range of 0-5 µM with resolution of ~0.07 µM. It relies on the glucose binding protein (GBP) fluorescently labeled with two fluorophores. Fluorescence signals from the dual-labeled GBP are utilized in a ratiometric mode, making the measurements insensitive to variations in protein concentration and other systematic errors. Fluorescence is quantified by a miniature, dedicated ratiometric fluorometer that is powered via USB. Concentration is calculated using an ultra-mobile personal computer (UMPC). The whole system is designed to be pocket sized suitable for point-of-care or bedside applications. Test results suggest that the system is a promising tool for accurate measurements of low glucose concentrations (0.1-10 µM) in biological samples.

Glucose monitoring in neonates: need for accurate and non-invasive methods.

Neonatal hypoglycaemia can lead to devastating consequences. Thus, constant, accurate and safe glucose monitoring is imperative in neonatal care. However, point-of-care (POC) devices for glucose testing currently used for neonates were originally designed for adults and do not address issues specific to neonates. This review will address currently available monitoring options and describe new methodologies for non-invasive glucose monitoring in newborns.