Formulations for drying bacterial cell extracts
Inventors
Chandler, Martin • Hayenga, Kirk • Heinsohn, Henry
Assignees
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Publication Number
US-12098406-B2
Publication Date
2024-09-24
Expiration Date
Abstract
This invention provides for an improved method of stabilizing freeze dried bacterial extracts with a carbohydrate lyoprotectant such that the extracts can be for use in cell free protein synthesis. Also provided herein are formulations for stable, freeze dried bacterial extracts that when stored at room temperature retain at least about 70% protein synthesis activity compared to undried frozen bacterial extracts.
Core Innovation
The invention relates to a freeze dried bacterial extract for cell free protein synthesis. The extract includes dried, lysed bacterial components that synthesize, upon rehydration, a target protein from a template nucleic acid encoding the target protein. The formulation further includes a carbohydrate composition that acts as a lyoprotectant for maintaining activity after storage and during rehydration.
A central aspect is a defined carbohydrate composition characterized by a carbohydrate to dried bacterial components ratio and a sucrose/non-sucrose carbohydrate profile. In one described embodiment, the carbohydrate composition is in about 0.8-1.5:1.0 (w/w) ratio of carbohydrate to dried bacterial components, with the carbohydrate composition constituting about 20-100% sucrose and a non-sucrose portion comprising mannitol, sorbitol, dextran, or mixtures thereof.
The disclosed extracts are characterized by retention of protein synthesis activity after long-term storage under specified temperature conditions and by low residual water. The document further describes embodiments with a bacterial source and an active oxidative phosphorylation system, and it describes freeze-drying approaches including conventional lyophilization and radiant-energy/microwave vacuum drying while measuring functional performance by cell-free protein synthesis activity.
Claims Coverage
The document contains three independent claims. Across them, the core inventive coverage centers on a freeze dried, lysed bacterial extract that synthesizes a target protein upon rehydration from a template nucleic acid, together with defined carbohydrate excipient compositions and ratios.
Freeze dried lysed bacterial extract able to synthesize from template nucleic acid upon rehydration
A freeze dried bacterial extract for cell free protein synthesis comprising dried, lysed bacterial components, wherein the extract is able to synthesize upon rehydration a target protein from a template nucleic acid encoding the target protein.
Carbohydrate composition ratio and sucrose/non-sucrose lyoprotectant profile
A carbohydrate composition in about 0.8-1.5:1.0 (w/w) ratio of carbohydrate to dried bacterial components, wherein the carbohydrate composition constitutes about 20-100% sucrose and a non-sucrose portion comprising mannitol, sorbitol, dextran or mixtures thereof.
Defined carbohydrate composition distribution for lyophilized bacterial extract
A carbohydrate composition in about 0.6-2.0:1.0 (w/w) ratio of carbohydrate to dried bacterial components, wherein the carbohydrate composition constitutes about 22% sucrose, 22% sorbitol, 46% mannitol, and 10% dextran.
Essentially all-sucrose carbohydrate composition
A carbohydrate composition in about 0.6-2.0:1.0 (w/w) ratio of carbohydrate to dried bacterial components, wherein the carbohydrate composition constitutes about 100% sucrose.
Collectively, the independent claims cover freeze dried lysed bacterial extracts for cell free protein synthesis that recover activity upon rehydration to synthesize a target protein from template nucleic acid, with inventive definition of the carbohydrate excipient via specified carbohydrate-to-bacterial ratios and defined sucrose and non-sucrose profiles, including specific multi-component distributions and an all-sucrose embodiment.
Stated Advantages
Retains about ≥70% protein synthesis activity after long-term storage, including 20-22°C or 4°C, compared with additive-free controls that show much lower activity.
Meets low residual water targets, including ≤1.5% w/w residual water and in some cases ≤2.6% w/w residual water.
Documented Applications
Use as a freeze dried bacterial extract to perform cell free protein synthesis (CFPS) of a target protein from a template nucleic acid encoding the target protein.
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