Abstract
Protein biologics have emerged as a safe and effective group of drug products that can be used in a variety of medical disorders and clinical settings, including treatment of orphan diseases, personalized medicine, and point-of-care applications. However, the full potential of protein biologics for such applications will not be realized until there are methods available for rapid and cost-effective production of small scale products for individual needs. Here, we describe a modular and scalable method for rapid and adaptable production of protein-based medical products at small doses. The method includes cell-free synthesis of the protein target in a reactor module followed by a fluidic process for protein purification. As a proof of concept, we describe the application of this method for expression and purification of a bioactive pharmaceutically relevant protein biologic, recombinant human erythropoietin, at a single dose within 24 h. This method can be applied toward the development of automated platforms for rapid and adaptive production of protein biologics at the point of care in response to specific medical needs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22:538–545
Gilbert M, Albala JS (2002) Accelerating code to function: sizing up the protein production line. Curr Opin Chem Biol 6:102–105
Mei Q, Fredrickson CK, Simon A, Khnouf R, Fan ZH (2007) Cell-free protein synthesis in microfluidic array devices. Biotechnol Prog 23:1305–1311
Sullivan CJ, Pendleton ED, Sasmor HH, Hicks WL, Farnum JB, Muto M, Amendt EM, Schoborg JA, Martin RW, Clark LG, Anderson MJ, Choudhury A, Fior R, Lo YH, Griffey RH, Chappell SA, Jewett MC, Mauro VP, Dresios J (2016) A cell-free expression and purification process for rapid production of protein biologics. Biotechnol J 11:238–248
Goerke AR, Swartz JR (2008) Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol Bioeng 99:351–367
Kanter G, Yang J, Voloshin A, Levy S, Swartz JR, Levy R (2007) Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines. Blood 109:3393–3399
Yang J, Kanter G, Voloshin A, Michel-Reydellet N, Velkeen H, Levy R, Swartz JR (2005) Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system. Biotechnol Bioeng 89:503–511
Hodgman CE, Jewett MC (2013) Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis. Biotechnol Bioeng 110:2643–2654
Kim DM, Swartz JR (2001) Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotechnol Bioeng 74:309–316
Katzen F, Chang G, Kudlicki W (2005) The past, present and future of cell-free protein synthesis. Trends Biotechnol 23:150–156
Swartz J (2006) Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol 33:476–485
Shirokov VA, Kommer A, Kolb VA, Spirin AS (2007) Continuous-exchange protein-synthesizing systems. Methods Mol Biol 375:19–55
Jewett MC, Calhoun KA, Voloshin A, Wuu JJ, Swartz JR (2008) An integrated cell-free metabolic platform for protein production and synthetic biology. Mol Syst Biol 4:220
Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ (2011) Microscale to manufacturing scale-up of cell-free cytokine production—a new approach for shortening protein production development timelines. Biotechnol Bioeng 108:1570–1578
Carlson ED, Gan R, Hodgman CE, Jewett MC (2012) Cell-free protein synthesis: applications come of age. Biotechnol Adv 30:1185–1194
Stech M, Brödel AK, Quast RB, Sachse R, Kubick S (2013) Cell-free systems: functional modules for synthetic and chemical biology. Adv Biochem Eng Biotechnol 137:67–102
Schoborg JA, Hodgman CE, Anderson MJ, Jewett MC (2014) Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis. Biotechnol J 9:630–640
Gan R, Jewett MC (2014) A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthesis. Biotechnol J 9:641–651
Mortimer RK, Johnston JR (1986) Genealogy of principal strains of the yeast genetic stock center. Genetics 113:35–43
Kitamura T, Tojo A, Kuwaki T, Chiba S, Miyazono K, Urabe A, Takaku F (1989) Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1. Blood 73:375–380
Dresios J. Sullivan CJ, Pendleton ED, Sasmor HH, Hicks WL, Amendt EM, Griffey RH. Portable fluidic platform for rapid cell-free production of protein biologics. USPTO 2016/0230203A1
Brödel AK, Sonnabend A, Kubick S (2014) Cell-free protein expression based on extracts from CHO cells. Biotechnol Bioeng 111:25–36
Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13
Acknowledgments
This work was supported by funding from the Defense Advanced Research Projects Agency (N66001-13-C-4024). The views expressed are those of the authors and do not reflect the official policy or position of the Department of Defense or the US Government.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Sullivan, C.J., Pendleton, E.D., Dresios, J. (2018). Cell-Free Production of Protein Biologics Within 24 H. In: Picanço-Castro, V., Swiech, K. (eds) Recombinant Glycoprotein Production. Methods in Molecular Biology, vol 1674. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7312-5_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7312-5_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7311-8
Online ISBN: 978-1-4939-7312-5
eBook Packages: Springer Protocols