The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.
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J.C.S. acknowledges support from NIH/NCI F32 Postdoctoral Fellowship 1F32CA250324-01 and American Cancer Society Postdoctoral Fellowship PF-20-143-01-LIB. T.J. acknowledges support from the European Molecular Biology Organization Postdoctoral Fellowship 336-2021. K.F.W. acknowledges support from the National Defense Science and Engineering (NDSEG) Fellowship Program (ND-CEN-013-096). M.C.J. acknowledges support from the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, the Defense Threat Reduction Agency Grants HDTRA1-15-10052, HDTRA-12-11-0038 and HDTRA-12-01-0004, the Army Research Office Grants W911NF-20-1-0195, W911NF-18-1-0200 and W911NF-16-1-0372, the Army Contracting Command Contract W52P1J-21-9-3023 and DARPA Grant W911NF-23-2-0039.
M.C.J. is a cofounder of SwiftScale Biologics, Stemloop, Inc., Design Pharmaceuticals, and Pearl Bio. M.P.D. has interests in Glycobia Inc. and Versatope Inc. M.P.D. and M.C.J. have an interest in SwiftScale Biologics. M.C.J.’s and M.P.D.’s interests are reviewed and managed by Northwestern University and Cornell University, respectively, in accordance with their conflict of interest policies.
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Key references using this protocol
Stark, J. C. et al. Sci. Adv. 7, eabe9444 (2021): https://doi.org/10.1126/sciadv.abe9444
Jaroentomeechai, T. et al. Nat. Commun. 9, 2686 (2018): https://doi.org/10.1038/s41467-018-05110-x
Natarajan, A. et al. Nat. Chem. Biol. 16, 1062–1070 (2020): https://doi.org/10.1038/s41589-020-0595-9
Hershewe, J. M. et al. Nat. Commun. 12, 2363 (2021): https://doi.org/10.1038/s41467-021-22329-3
Warfel, K. F. et al. ACS Synth. Biol. 12, 95–107 (2023): https://doi.org/10.1021/acssynbio.2c00392
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Stark, J.C., Jaroentomeechai, T., Warfel, K.F. et al. Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates. Nat Protoc 18, 2374–2398 (2023). https://doi.org/10.1038/s41596-022-00799-z