Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.
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Choi, E. J. & Ling, G. S. Battlefield medicine: paradigm shift for pharmaceuticals manufacturing. PDA J. Pharm. Sci. Technol. 68, 312 (2014).
Schellekens, H., Aldosari, M., Talsma, H. & Mastrobattista, E. Making individualized drugs a reality. Nat. Biotechnol. 35, 507–514 (2017).
Editorial. Patient-centered drug manufacture. Nat. Biotechnol. 35, 485 (2017).
Adamo, A. et al. On-demand continuous-flow production of pharmaceuticals in a compact, reconfigurable system. Science 352, 61–67 (2016).
Smith, M. T., Wilding, K. M., Hunt, J. M., Bennett, A. M. & Bundy, B. C. The emerging age of cell-free synthetic biology. FEBS Lett. 588, 2755–2761 (2014).
Zemella, A., Thoring, L., Hoffmeister, C. & Kubick, S. Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. ChemBioChem 16, 2420–2431 (2015).
Tran, K. et al. Cell-free production of a therapeutic protein: expression, purification, and characterization of recombinant streptokinase using a CHO lysate. Biotechnol. Bioeng. 115, 92–102 (2018).
Martin, R. W. et al. Development of a CHO-based cell-free platform for synthesis of active monoclonal antibodies. ACS Synth. Biol. 21, 1370–1379 (2017).
Boles, K. S. et al. Digital-to-biological converter for on-demand production of biologics. Nat. Biotechnol. 35, 672–675 (2017).
Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259 (2016).
Shukla, A. A., Hubbard, B., Tressel, T., Guhan, S. & Low, D. Downstream processing of monoclonal antibodies—application of platform approaches. Anal. Technol. Biomed. Life Sci. 15, 28–39 (2007).
Hammerling, U., Kroon, R. & Sjödin, L. In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor. J. Pharm. Biomed. Anal. 13, 9–20 (1995).
Brödel, A. K., Sonnabend, A. & Kubick, S. Cell-free protein expression based on extracts from CHO cells. Biotechnol. Bioeng. 111, 25–36 (2014).
Peñalber-Johnstone, C. et al. Optimizing cell-free protein expression in CHO: assessing small molecule mass transfer effects in various reactor configurations. Biotechnol. Bioeng. 114, 1478–1486 (2017).
Tiangco, C. et al. Measuring transdermal glucose levels in neonates by passive diffusion: an in vitro porcine skin model. Anal. Bioanal. Chem. 409, 3475–3482 (2017).
Collier, R. J. Multi-mutant diphtheria toxin vaccines. US patent 7115725 B2 (2006).
Giannini, G., Rappuoli, R. & Ratti, G. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 12, 4063–4069 (1984).
Leader, B., Baca, Q. J. & Golan, D. E. Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Discov. 7, 21–39 (2008).
Gurramkonda, C. et al. Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system. Biotechnol. Bioeng. 115, 1253–1264 (2018).
Rathore, A. S. & Winkle, H. Quality by design for biopharmaceuticals. Nat. Biotechnol. 27, 26–34 (2009).
Swartz, J. R. Transforming biochemical engineering with cell-free biology. AIChE J. 58, 5–13 (2012).
Buntru, M., Vogel, S., Stoff, K., Spiegel, H. & Schillberg, S. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates. Biotechnol. Bioeng. 112, 867–78 (2015).
Burgenson, D. et al. Rapid recombinant protein expression in cell-free extracts from human blood. Sci. Rep. https://doi.org/10.1038/s41598-018-27846-8 (2018).
Ding, W., Madsen, G., Mahajan, E., O’Connor, S. & Wong, K. Standardized extractables testing protocol for single-use systems in biomanufacturing. Pharm. Eng. 34, 1–11 (2014).
We thank DARPA Biologically-derived Medicines on Demand (Bio-MOD) Project Grant (N66001-13-C-4023, ‘CASTing Biologics on Demand’) for financial support. G. Ling (Retd. US Army Colonel and former DARPA Battlefield Medicine Program Director) conceived the vision for Bio-MOD based on his difficulties in securing medicines to treat patients during deployments in Iraq and Afghanistan. This paper is dedicated to him. We thank E. Choi, J. Lewin, A. Bryon, M. Zamisch, T. McQuade, B. Ringeisen, G. Kost, K. Pankratz, R. Cecil, B. Webb, A. Bose, B. Junker, W.-L. Ling, M. McGinnis, P. Latham and R. Gopinath for various discussions, encouragement and support, and I. Shaffer of the Molecular Characterization and Analysis Complex, University of Maryland Baltimore County for sample analysis of leachables and extractables. We thank E. Gutierrez for his assistance with the illustrations. S.V. acknowledges Y. Xia of the MNMR Structural Biology Centre, University of Minnesota for technical help in the NMR spectroscopy. We thank the FDA Emerging Technology Team for helpful guidance and discussions. Disclaimer: This work was conducted while S.V. was employed at the University of Maryland School of Pharmacy. The opinions expressed in the article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. No endorsement of this work by the Food and Drug Administration, National Institutes of Health, the Department of Health and Human Services, DARPA, Department of Defense or the United States government is implied.
G.R., Y.K., L.T., X.G. and D.F. are listed as inventors on the United States patents 9,388,373 ‘Microscale Bioprocessing System and Method for Protein Manufacturing’ and 9,982,227 ‘System and Method for Production of On-Demand Proteins in a Portable Unit for Point of Care Delivery’.
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Adiga, R., Al-adhami, M., Andar, A. et al. Point-of-care production of therapeutic proteins of good-manufacturing-practice quality. Nat Biomed Eng 2, 675–686 (2018). https://doi.org/10.1038/s41551-018-0259-1
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