title: A cell-free expression and purification process for rapid production of protein biologics.
abstract: Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: Challise J
authors.initials: CJ
authors.lastname: Sullivan
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Pendleton
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: Henri H
authors.initials: HH
authors.lastname: Sasmor
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: William L
authors.initials: WL
authors.lastname: Hicks
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Farnum
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Machiko
authors.initials: M
authors.lastname: Muto
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Amendt
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Schoborg
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Rey W
authors.initials: RW
authors.lastname: Martin
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Lauren G
authors.initials: LG
authors.lastname: Clark
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Anderson
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Alaksh
authors.initials: A
authors.lastname: Choudhury
authors.affiliation: Department of Electrical and Computer Engineering, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Fior
authors.affiliation: Department of Electrical and Computer Engineering, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Yu-Hwa
authors.initials: YH
authors.lastname: Lo
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Griffey
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Chappell
authors.affiliation: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Mauro
authors.affiliation: CBRN Division, Leidos, Inc., San Diego, CA, USA. john.dresios@leidos.com.
authors.forename: John
authors.initials: J
authors.lastname: Dresios
mesh_terms: D001688:Biological Products / Q000302:isolation & purification / Q000378:metabolism*; D019149:Bioreactors; D002474:Cell-Free System; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D060847:Metabolic Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D001688:Biological Products; D011994:Recombinant Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Cell-free protein synthesis; Granulocyte-macrophage colony-stimulating factor; Protein Biologics; Protein expression