Thermostable lyoprotectant-enhanced cell-free protein synthesis for on-demand endotoxin-free therapeutic production

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Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell‐free protein synthesis (CFPS) of FDA approved l‐asparaginase (crisantaspase) is demonstrated by employing an aspartate‐fed‐batch reactor format. A CFPS‐based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf‐stable and low‐cost Escherichia coli‐based CFPS reactions may be employed on‐demand to 1) synthesize biologics on‐site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not‐too‐distant future when shelf‐stable E. coli CFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Engineering Cell‐Free Protein Synthesis for High‐Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On‐Demand Treatment of Acute Lymphoblastic Leukemia

Hunt

Wilding

Barnett

et al. 2020

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Lyophilization of premixed COVID‐19 diagnostic RT‐qPCR reactions enables stable long‐term storage at elevated temperature

Hammerling

Warfel

Jewett

2021

Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) diagnostic tests for SARS‐CoV‐2 are the cornerstone of the global testing infrastructure. However, these tests require cold‐chain shipping to distribute, and the labor of skilled technicians to assemble reactions and interpret the results. Strategies to reduce shipping and labor costs at the point‐of‐care could aid in diagnostic testing scale‐up and response to the COVID‐19 outbreak, as well as in future outbreaks. In this study we test both lab‐developed and commercial SARS‐CoV‐2 diagnostic RT‐qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization. Fully assembled reactions were lyophilized and stored for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS‐CoV‐2 RNA. Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization. We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze‐dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days. We anticipate that the incorporation of these methods into SARS‐CoV‐2 diagnostic testing will improve testing pipelines by reducing labor at the testing facility and eliminating the need for cold‐chain shipping.

Mechanistic discoveries and simulation‐guided assay optimization of portable hormone biosensors with cell‐free protein synthesis

Hunt

Galiardi

Free

et al. 2021

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Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.