title: Development of a CHO-Based Cell-Free Platform for Synthesis of Active Monoclonal Antibodies.
abstract: Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. Here, we have developed a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the cell-free synthesis of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high-throughput prediction of mAb expression ranking to accelerate design-build-test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Rey W
authors.identifier: 0000-0002-9613-6951
authors.initials: RW
authors.lastname: Martin
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Natalia I
authors.initials: NI
authors.lastname: Majewska
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Cindy X
authors.initials: CX
authors.lastname: Chen
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Albanetti
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Rod Brian C
authors.initials: RBC
authors.lastname: Jimenez
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Albert E
authors.initials: AE
authors.lastname: Schmelzer
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.affiliation: Department of Chemical and Biological Engineering, ‡Chemistry of Life Processes Institute, and §Interdisciplinary Biological Sciences Program, Northwestern University , Evanston, Illinois 60208, United States.|Cell Culture and Fermentation Sciences and ⊥Analytical Sciences, MedImmune LLC , Gaithersburg, Maryland 20878, United States.|Member, Robert H. Lurie Comprehensive Cancer Center and ∇Member, Simpson Querrey Institute, Northwestern University , Chicago, Illinois 60611, United States.
authors.forename: Varnika
authors.initials: V
authors.lastname: Roy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D016466:CHO Cells; D002474:Cell-Free System; D003412:Cricetulus; D014176:Protein Biosynthesis
chemical_list: D000911:Antibodies, Monoclonal
keywords: Chinese Hamster Ovary; antibody ranking tool; cell-free protein synthesis; monoclonal antibody; synthetic biology