Govind Rao

Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From 1,231 different reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 ± 0.3 g/L of protein product at the 15-µL scale (~$60/g _protein ) and 3.7 ± 0.2 g/L (~$39/g _protein ) at the 4-mL scale with oxygen supplementation. This provides an average 95% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nitrogenous bases and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of more than 20 different proteins. For example, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

Shake flasks are widely used in early‐stage bioprocess development but are limited by their inability to monitor and control key gas‐transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real‐time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time‐resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen‐sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor‐like environment in a high‐throughput, low‐cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor‐like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.

The future of healthcare depends on leveraging state‐of‐the‐art advancements in biopharmaceutical manufacturing across the world. A near end‐to‐end distributed biomanufacturing setup will enable production to occur closer to points‐of‐care or points‐of‐need, thereby reducing lead times, improving adaptability to unseasonal demands and ensuring accessibility in rural and resource‐limited settings. However, the current distributed biomanufacturing systems typically produce lower volumes compared to conventional facilities. This perspective discusses the present status of distributed biomanufacturing and envisions futuristic models on portable platforms providing high mobility and production capacity. This conceptual framework for deployable biomanufacturing could facilitate an impartial global distribution of biotherapeutics, improved pandemic preparedness, reduced production costs, provide medical tourism and even utilization in space exploration. By integrating advanced technologies such as continuous manufacturing, machine learning, sensors, and real‐time process control, these platforms would enable rapid, agile, and efficient production of biologics. Furthermore, we discuss key considerations such as regulatory pathways, quality management systems and secure cloud‐based data handling for one such mobile platform housed on a ship. This vision for distributed biomanufacturing aims to spark innovation and drive efforts toward the futuristic goal of providing affordable and high‐quality biopharmaceuticals with equitable access around the world.

The SARS‐CoV‐2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell‐free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a 1‐L cell‐free gene expression (CFE) reaction that achieves over 90% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell‐free reaction conditions, such as temperature, additives, purification tags, and agitation, to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than four orders of magnitude in reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.

Recent studies project that the prevalence of diabetes is expected to increase significantly and lead to escalating demand on the insulin supply chain. Despite being the first recombinant therapeutic approved by the FDA, insulin remains challenging to access for many around the globe. Here we report on advancements in manufacturing insulin using cell-free protein synthesis (CFPS) systems to rapidly produce mature desB30-insulin in less than a day. This is a major advance towards decentralizing insulin manufacturing and bringing production closer to the point-of-care, thereby improving diabetic patient accessibility. To this end, a purified cell-free extract, PUREfrex ^® 2.1, was utilized to synthesize a tagged proinsulin construct that can be captured and converted into mature insulin using an on-column affinity chromatography process. Notably, two chaperones, peptidyl prolyl isomerase (FkpA), and seven kilodalton protein (Skp) were observed to play a critical role during cell-free expression of proinsulin. The proinsulin was then immobilized on a Ni-NTA column where the purification and conversion of cell-free products were performed sequentially to yield desB30-insulin. Following further optimization, this method serves as a time and resource-efficient production process as compared to current methods. When applied simultaneously, the cell-free expression and on-column conversion methods reported here can be adopted to enable rapid on-demand insulin manufacturing in order to improve the accessibility of insulin and prevent future shortages.

The presence of nitrate (NO₃ ⁻) in surface water and groundwater used for potable supply needs to be closely monitored since in elevated amounts it can adversely affect aquatic life and human health by causing hypoxia and methemoglobinemia. Many of the existing EPA-certified sensors used for environmental monitoring are expensive, bulky, and labor-intensive. To address these concerns, we have successfully developed a low-cost microfluidic electrochemical impedimetric sensor, consisting of a nitrate-binding nickel complex within a polyaniline/carbon nanocomposite (Ni@Pani/C) enabling nitrate monitoring in field samples. Under optimized conditions, our sensor demonstrated a high sensitivity of 2.31 ± 0.09 Ω ppm⁻¹ cm⁻² across a wide nitrate concentration range (0.6–10 ppm). It also showed a desirable low detection limit of 0.015 ppm and a swift response time under 20 seconds. It maintained repeatability over a wide temperature range (5–65 °C) and exhibited consistent performance over an extended period (∼1 month). The sensor exhibited high specificity towards nitrate when tested against potential interferences (SO₄ ²⁻, C₂H₃O₂ ⁻, HCO₃ ⁻, NH₄ ⁺, Cl⁻) and showed good reproducibility for test water samples collected from various streams in Maryland, U.S.A. A statistical model was used to confirm the sensor's accuracy, which yielded a maximum standard deviation of ±0.6 ppm (absolute value). Our sensor was also benchmarked against a commercial SUNA device resulting in comparable performance.

This article was originally published online on 1 December 1999 without Govind Rao’s affiliation to University of Maryland, Baltimore County, Baltimore, Marylan This article was originally published online on 1 December 1999 without Govind Rao’s affiliation to University of Maryland, Baltimore County, Baltimore, Marylan

Although online monitoring of dissolved O₂, pH, and dissolved CO₂ is critical in bioprocesses, nearly all existing technologies require some level of direct contact with the cell culture environment, posing risks of contamination. This study addresses the need for an accurate, and completely noninvasive technique for simultaneous measurement of these analytes. A “non-contact” technique for simultaneous monitoring of dissolved O_2, pH, and dissolved CO₂ was developed. Instead of direct contact with the culture media, the measurements were made through permeable membranes via either a sampling port in the culture vessel wall or a flow cell. The efficacy of the “non-contact” technique was validated in Escherichia coli (E.coli), Chinese hamster ovary (CHO) culture processes, and dynamic environments created by sparging gases in cell culture medium. The measurements obtained through the developed techniques were comparable to those obtained through control methods. The noninvasive monitoring system can offer accurate, and contamination-minimized monitoring of critical process parameters including dissolved O₂, pH, and dissolved CO₂. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

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 (k_La) 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.