Team:BrownStanfordPrinctn/Description
Project Description
Listen to our very own Cale describe the Astropharmacy!
The Astropharmacy was broken down into three sub-projects: Diagnostics, Drug Production and Drug Purification.
Diagnostics with Fusion Protein-Based Microfluidics
In the mid 20th century, microfluidics was identified as the ability to process liquids on the micrometer-scale using low cost, reproducible components [1]. However, it wasn’t until the early 2000’s that George Whitesides of Harvard University recognized their sweeping potential to satisfy a number of research and commercial needs [6]. Microfluidic devices have since been pinpointed and developed for a wide range of medical diagnostics, from single step urinalyses to screen for potential diabetes patients to multiplex lateral flow assays for chemical contaminants [5]. Overall, the benefits of microfluidics have been largely realized through two popular materials among others: cellulose and PDMS, or polydimethylsiloxane.
Microfluidic paper-based analytical devices (uPADs) revolve around painting or printing the outlines of channels using wax on laboratory-grade cellulose [2]. This technique is relatively low cost and simple to replicate, but the 300um [3] resolution of channels is not comparable to other microfluidic fabrication methods, as photolithography produced PDMS microfluidic channels are able to reach a width of less than 10 um. So, while the nature of paper-based microfluidics is extremely economical, this benefit can’t be applied to microfluidic chip designs that require more precision. Likewise, while PDMS printing can yield extremely thin channels, it’s increased production cost limits the production of precise microfluidic devices for widespread use [4].
To address this disconnect between affordability and precision, we developed fusion proteins that can mimic the precision of photolithography, but exist on a low cost membrane and be as a whole cost effective. We replaced the chemically-imposed hydrophobic wax channels on paper with cellulose-binding, hydrophobic fusion proteins, and then produced devices using these proteins using low cost reagents.
.
- “History of Microfluidics.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/history-of-microfluidics/.
- “Paper Microfluidic Devices : A Review 2017.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/paper-microfluidic-devices-a-review-2017/.
- Strong, E. Brandon, et al. “Fabrication of Miniaturized Paper-Based Microfluidic Devices (MicroPADs).” Nature News, Nature Publishing Group, 9 Jan. 2019, www.nature.com/articles/s41598-018-37029-0.
- Sher, Mazhar, et al. “Paper-Based Analytical Devices for Clinical Diagnosis: Recent Advances in the Fabrication Techniques and Sensing Mechanisms.” Expert Review of Molecular Diagnostics, U.S. National Library of Medicine, Apr. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5529145.
- Shi Z.Z., Lu Y., Yu L. (2017) Microfluidic Paper-Based Analytical Devices for Point-of-Care Diagnosis. In: Chandra P., Tan Y., Singh S. (eds) Next Generation Point-of-care Biomedical Sensors Technologies for Cancer Diagnosis. Springer, Singapore
- Whitesides , George M. “The Origins and the Future of Microfluidics.” Nature.com, July 2006.
Drug Production
Drug Choice
For this prototypical project, we are manufacturing 3 peptide-based drugs: insulin, teriparatide, and hG-CSF. Insulin, chosen for its implications on Earth, could be used to aid the 425 million people worldwide with diabetes, particularly those without access to treatment. For our second two drugs, we consulted with astronaut physicians at NASA to choose drugs that are applicable for long-term space missions and that they anticipate will be in constant demand. We chose hG-CSF (human granulocyte colony-stimulating factor) and teriparatide for our prototype.
Both hG-CSF and teriparatide are great candidates for this system for several reasons. Microgravity-induced bone degradation due to the lack of physical stress associated with a microgravity environment can cause osteoporosis and renal stones. Teriparatide is a peptide drug shown to counteract these effects in space by stimulating bone deposition by osteoblasts. hG-CSF is an acute radiation poisoning drug that is useful in preventing radiation damage to astronauts coming from Solar Particle Events (SPEs), which are unpredictable, deadly, and may strike astronauts at times when they do not have access to proper shielding (in transit to or from Mars. Both drugs are essential to have on long space-missions, teriparatide for maintenance and hG-CSF for SPE, especially for the unprecedentedly long Mars missions. The longest single-term space mission was 14 months long, whereas the shortest possible Mars mission will be at least 22-24 months. Unfortunately, both drugs are unstable peptide drugs and have shown to only be viable for months to a year even with refrigeration. This means any peptide drugs with similar stability brought from Earth will only last about 25% of Mars mission length, clearly not suitable given the essentiality of these drugs and the unprecedented health challenge to Mars-bound astronauts. Commercially, both drugs have been synthesized in cell-free and E. coli, meaning they can be optimized to be synthesized in both a cellular and cell-free system. Both systems can synthesize peptide-based drugs on demand, allowing us to bypass shelf-life, which will become necessary when long-term space flight becomes a reality. In addition, in situ resource utilization on location would reduce the necessary mass of drugs required to be shipped, reducing valuable mass during space missions.
- Song, Jong-Am, Kyung-Yeon Han, Jin-Seung Park, Hyuk-Seong Seo, Keum-Young Ahn, and Jeewon Lee. “Human G-CSF Synthesis Using Stress-Responsive Bacterial Proteins.” FEMS Microbiology Letters296, no. 1 (2009): 60–66. https://doi.org/10.1111/j.1574-6968.2009.01616.x.
- Bakhtiari, Nahid et al. “Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.” Avicenna journal of medical biotechnology vol. 9,1 (2017): 19-22.
- Suzuki, Y et al. “High-level production of recombinant human parathyroid hormone 1-34.” Applied and environmental microbiology vol. 64,2 (1998): 526-9.
Synthesis Systems
We utilize both cellular and cell-free systems for drug manufacturing.
Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins.
Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
Listen to our very own Cale describe the Astropharmacy!
The Astropharmacy was broken down into three sub-projects: Diagnostics, Drug Production and Drug Purification.
Diagnostics with Fusion Protein-Based Microfluidics
In the mid 20th century, microfluidics was identified as the ability to process liquids on the micrometer-scale using low cost, reproducible components [1]. However, it wasn’t until the early 2000’s that George Whitesides of Harvard University recognized their sweeping potential to satisfy a number of research and commercial needs [6]. Microfluidic devices have since been pinpointed and developed for a wide range of medical diagnostics, from single step urinalyses to screen for potential diabetes patients to multiplex lateral flow assays for chemical contaminants [5]. Overall, the benefits of microfluidics have been largely realized through two popular materials among others: cellulose and PDMS, or polydimethylsiloxane.
Microfluidic paper-based analytical devices (uPADs) revolve around painting or printing the outlines of channels using wax on laboratory-grade cellulose [2]. This technique is relatively low cost and simple to replicate, but the 300um [3] resolution of channels is not comparable to other microfluidic fabrication methods, as photolithography produced PDMS microfluidic channels are able to reach a width of less than 10 um. So, while the nature of paper-based microfluidics is extremely economical, this benefit can’t be applied to microfluidic chip designs that require more precision. Likewise, while PDMS printing can yield extremely thin channels, it’s increased production cost limits the production of precise microfluidic devices for widespread use [4].
To address this disconnect between affordability and precision, we developed fusion proteins that can mimic the precision of photolithography, but exist on a low cost membrane and be as a whole cost effective. We replaced the chemically-imposed hydrophobic wax channels on paper with cellulose-binding, hydrophobic fusion proteins, and then produced devices using these proteins using low cost reagents.
.
- “History of Microfluidics.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/history-of-microfluidics/.
- “Paper Microfluidic Devices : A Review 2017.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/paper-microfluidic-devices-a-review-2017/.
- Strong, E. Brandon, et al. “Fabrication of Miniaturized Paper-Based Microfluidic Devices (MicroPADs).” Nature News, Nature Publishing Group, 9 Jan. 2019, www.nature.com/articles/s41598-018-37029-0.
- Sher, Mazhar, et al. “Paper-Based Analytical Devices for Clinical Diagnosis: Recent Advances in the Fabrication Techniques and Sensing Mechanisms.” Expert Review of Molecular Diagnostics, U.S. National Library of Medicine, Apr. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5529145.
- Shi Z.Z., Lu Y., Yu L. (2017) Microfluidic Paper-Based Analytical Devices for Point-of-Care Diagnosis. In: Chandra P., Tan Y., Singh S. (eds) Next Generation Point-of-care Biomedical Sensors Technologies for Cancer Diagnosis. Springer, Singapore
- Whitesides , George M. “The Origins and the Future of Microfluidics.” Nature.com, July 2006.
Drug Production
Drug Choice
For this prototypical project, we are manufacturing 3 peptide-based drugs: insulin, teriparatide, and hG-CSF. Insulin, chosen for its implications on Earth, could be used to aid the 425 million people worldwide with diabetes, particularly those without access to treatment. For our second two drugs, we consulted with astronaut physicians at NASA to choose drugs that are applicable for long-term space missions and that they anticipate will be in constant demand. We chose hG-CSF (human granulocyte colony-stimulating factor) and teriparatide for our prototype.
Both hG-CSF and teriparatide are great candidates for this system for several reasons. Microgravity-induced bone degradation due to the lack of physical stress associated with a microgravity environment can cause osteoporosis and renal stones. Teriparatide is a peptide drug shown to counteract these effects in space by stimulating bone deposition by osteoblasts. hG-CSF is an acute radiation poisoning drug that is useful in preventing radiation damage to astronauts coming from Solar Particle Events (SPEs), which are unpredictable, deadly, and may strike astronauts at times when they do not have access to proper shielding (in transit to or from Mars. Both drugs are essential to have on long space-missions, teriparatide for maintenance and hG-CSF for SPE, especially for the unprecedentedly long Mars missions. The longest single-term space mission was 14 months long, whereas the shortest possible Mars mission will be at least 22-24 months. Unfortunately, both drugs are unstable peptide drugs and have shown to only be viable for months to a year even with refrigeration. This means any peptide drugs with similar stability brought from Earth will only last about 25% of Mars mission length, clearly not suitable given the essentiality of these drugs and the unprecedented health challenge to Mars-bound astronauts. Commercially, both drugs have been synthesized in cell-free and E. coli, meaning they can be optimized to be synthesized in both a cellular and cell-free system. Both systems can synthesize peptide-based drugs on demand, allowing us to bypass shelf-life, which will become necessary when long-term space flight becomes a reality. In addition, in situ resource utilization on location would reduce the necessary mass of drugs required to be shipped, reducing valuable mass during space missions.
- Song, Jong-Am, Kyung-Yeon Han, Jin-Seung Park, Hyuk-Seong Seo, Keum-Young Ahn, and Jeewon Lee. “Human G-CSF Synthesis Using Stress-Responsive Bacterial Proteins.” FEMS Microbiology Letters296, no. 1 (2009): 60–66. https://doi.org/10.1111/j.1574-6968.2009.01616.x.
- Bakhtiari, Nahid et al. “Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.” Avicenna journal of medical biotechnology vol. 9,1 (2017): 19-22.
- Suzuki, Y et al. “High-level production of recombinant human parathyroid hormone 1-34.” Applied and environmental microbiology vol. 64,2 (1998): 526-9.
Synthesis Systems
We utilize both cellular and cell-free systems for drug manufacturing.
Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins.
Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
In the mid 20th century, microfluidics was identified as the ability to process liquids on the micrometer-scale using low cost, reproducible components [1]. However, it wasn’t until the early 2000’s that George Whitesides of Harvard University recognized their sweeping potential to satisfy a number of research and commercial needs [6]. Microfluidic devices have since been pinpointed and developed for a wide range of medical diagnostics, from single step urinalyses to screen for potential diabetes patients to multiplex lateral flow assays for chemical contaminants [5]. Overall, the benefits of microfluidics have been largely realized through two popular materials among others: cellulose and PDMS, or polydimethylsiloxane.
Microfluidic paper-based analytical devices (uPADs) revolve around painting or printing the outlines of channels using wax on laboratory-grade cellulose [2]. This technique is relatively low cost and simple to replicate, but the 300um [3] resolution of channels is not comparable to other microfluidic fabrication methods, as photolithography produced PDMS microfluidic channels are able to reach a width of less than 10 um. So, while the nature of paper-based microfluidics is extremely economical, this benefit can’t be applied to microfluidic chip designs that require more precision. Likewise, while PDMS printing can yield extremely thin channels, it’s increased production cost limits the production of precise microfluidic devices for widespread use [4].
To address this disconnect between affordability and precision, we developed fusion proteins that can mimic the precision of photolithography, but exist on a low cost membrane and be as a whole cost effective. We replaced the chemically-imposed hydrophobic wax channels on paper with cellulose-binding, hydrophobic fusion proteins, and then produced devices using these proteins using low cost reagents.
.- “History of Microfluidics.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/history-of-microfluidics/.
- “Paper Microfluidic Devices : A Review 2017.” Elveflow, www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/paper-microfluidic-devices-a-review-2017/.
- Strong, E. Brandon, et al. “Fabrication of Miniaturized Paper-Based Microfluidic Devices (MicroPADs).” Nature News, Nature Publishing Group, 9 Jan. 2019, www.nature.com/articles/s41598-018-37029-0.
- Sher, Mazhar, et al. “Paper-Based Analytical Devices for Clinical Diagnosis: Recent Advances in the Fabrication Techniques and Sensing Mechanisms.” Expert Review of Molecular Diagnostics, U.S. National Library of Medicine, Apr. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5529145.
- Shi Z.Z., Lu Y., Yu L. (2017) Microfluidic Paper-Based Analytical Devices for Point-of-Care Diagnosis. In: Chandra P., Tan Y., Singh S. (eds) Next Generation Point-of-care Biomedical Sensors Technologies for Cancer Diagnosis. Springer, Singapore
- Whitesides , George M. “The Origins and the Future of Microfluidics.” Nature.com, July 2006.
Drug Production
Drug Choice
For this prototypical project, we are manufacturing 3 peptide-based drugs: insulin, teriparatide, and hG-CSF. Insulin, chosen for its implications on Earth, could be used to aid the 425 million people worldwide with diabetes, particularly those without access to treatment. For our second two drugs, we consulted with astronaut physicians at NASA to choose drugs that are applicable for long-term space missions and that they anticipate will be in constant demand. We chose hG-CSF (human granulocyte colony-stimulating factor) and teriparatide for our prototype.
Both hG-CSF and teriparatide are great candidates for this system for several reasons. Microgravity-induced bone degradation due to the lack of physical stress associated with a microgravity environment can cause osteoporosis and renal stones. Teriparatide is a peptide drug shown to counteract these effects in space by stimulating bone deposition by osteoblasts. hG-CSF is an acute radiation poisoning drug that is useful in preventing radiation damage to astronauts coming from Solar Particle Events (SPEs), which are unpredictable, deadly, and may strike astronauts at times when they do not have access to proper shielding (in transit to or from Mars. Both drugs are essential to have on long space-missions, teriparatide for maintenance and hG-CSF for SPE, especially for the unprecedentedly long Mars missions. The longest single-term space mission was 14 months long, whereas the shortest possible Mars mission will be at least 22-24 months. Unfortunately, both drugs are unstable peptide drugs and have shown to only be viable for months to a year even with refrigeration. This means any peptide drugs with similar stability brought from Earth will only last about 25% of Mars mission length, clearly not suitable given the essentiality of these drugs and the unprecedented health challenge to Mars-bound astronauts. Commercially, both drugs have been synthesized in cell-free and E. coli, meaning they can be optimized to be synthesized in both a cellular and cell-free system. Both systems can synthesize peptide-based drugs on demand, allowing us to bypass shelf-life, which will become necessary when long-term space flight becomes a reality. In addition, in situ resource utilization on location would reduce the necessary mass of drugs required to be shipped, reducing valuable mass during space missions.
- Song, Jong-Am, Kyung-Yeon Han, Jin-Seung Park, Hyuk-Seong Seo, Keum-Young Ahn, and Jeewon Lee. “Human G-CSF Synthesis Using Stress-Responsive Bacterial Proteins.” FEMS Microbiology Letters296, no. 1 (2009): 60–66. https://doi.org/10.1111/j.1574-6968.2009.01616.x.
- Bakhtiari, Nahid et al. “Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.” Avicenna journal of medical biotechnology vol. 9,1 (2017): 19-22.
- Suzuki, Y et al. “High-level production of recombinant human parathyroid hormone 1-34.” Applied and environmental microbiology vol. 64,2 (1998): 526-9.
Synthesis Systems
We utilize both cellular and cell-free systems for drug manufacturing.
Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins.
Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
Drug Choice
For this prototypical project, we are manufacturing 3 peptide-based drugs: insulin, teriparatide, and hG-CSF. Insulin, chosen for its implications on Earth, could be used to aid the 425 million people worldwide with diabetes, particularly those without access to treatment. For our second two drugs, we consulted with astronaut physicians at NASA to choose drugs that are applicable for long-term space missions and that they anticipate will be in constant demand. We chose hG-CSF (human granulocyte colony-stimulating factor) and teriparatide for our prototype.
Both hG-CSF and teriparatide are great candidates for this system for several reasons. Microgravity-induced bone degradation due to the lack of physical stress associated with a microgravity environment can cause osteoporosis and renal stones. Teriparatide is a peptide drug shown to counteract these effects in space by stimulating bone deposition by osteoblasts. hG-CSF is an acute radiation poisoning drug that is useful in preventing radiation damage to astronauts coming from Solar Particle Events (SPEs), which are unpredictable, deadly, and may strike astronauts at times when they do not have access to proper shielding (in transit to or from Mars. Both drugs are essential to have on long space-missions, teriparatide for maintenance and hG-CSF for SPE, especially for the unprecedentedly long Mars missions. The longest single-term space mission was 14 months long, whereas the shortest possible Mars mission will be at least 22-24 months. Unfortunately, both drugs are unstable peptide drugs and have shown to only be viable for months to a year even with refrigeration. This means any peptide drugs with similar stability brought from Earth will only last about 25% of Mars mission length, clearly not suitable given the essentiality of these drugs and the unprecedented health challenge to Mars-bound astronauts. Commercially, both drugs have been synthesized in cell-free and E. coli, meaning they can be optimized to be synthesized in both a cellular and cell-free system. Both systems can synthesize peptide-based drugs on demand, allowing us to bypass shelf-life, which will become necessary when long-term space flight becomes a reality. In addition, in situ resource utilization on location would reduce the necessary mass of drugs required to be shipped, reducing valuable mass during space missions.
- Song, Jong-Am, Kyung-Yeon Han, Jin-Seung Park, Hyuk-Seong Seo, Keum-Young Ahn, and Jeewon Lee. “Human G-CSF Synthesis Using Stress-Responsive Bacterial Proteins.” FEMS Microbiology Letters296, no. 1 (2009): 60–66. https://doi.org/10.1111/j.1574-6968.2009.01616.x.
- Bakhtiari, Nahid et al. “Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.” Avicenna journal of medical biotechnology vol. 9,1 (2017): 19-22.
- Suzuki, Y et al. “High-level production of recombinant human parathyroid hormone 1-34.” Applied and environmental microbiology vol. 64,2 (1998): 526-9.
Synthesis Systems
We utilize both cellular and cell-free systems for drug manufacturing.
Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins.
Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
For this prototypical project, we are manufacturing 3 peptide-based drugs: insulin, teriparatide, and hG-CSF. Insulin, chosen for its implications on Earth, could be used to aid the 425 million people worldwide with diabetes, particularly those without access to treatment. For our second two drugs, we consulted with astronaut physicians at NASA to choose drugs that are applicable for long-term space missions and that they anticipate will be in constant demand. We chose hG-CSF (human granulocyte colony-stimulating factor) and teriparatide for our prototype.
Both hG-CSF and teriparatide are great candidates for this system for several reasons. Microgravity-induced bone degradation due to the lack of physical stress associated with a microgravity environment can cause osteoporosis and renal stones. Teriparatide is a peptide drug shown to counteract these effects in space by stimulating bone deposition by osteoblasts. hG-CSF is an acute radiation poisoning drug that is useful in preventing radiation damage to astronauts coming from Solar Particle Events (SPEs), which are unpredictable, deadly, and may strike astronauts at times when they do not have access to proper shielding (in transit to or from Mars. Both drugs are essential to have on long space-missions, teriparatide for maintenance and hG-CSF for SPE, especially for the unprecedentedly long Mars missions. The longest single-term space mission was 14 months long, whereas the shortest possible Mars mission will be at least 22-24 months. Unfortunately, both drugs are unstable peptide drugs and have shown to only be viable for months to a year even with refrigeration. This means any peptide drugs with similar stability brought from Earth will only last about 25% of Mars mission length, clearly not suitable given the essentiality of these drugs and the unprecedented health challenge to Mars-bound astronauts. Commercially, both drugs have been synthesized in cell-free and E. coli, meaning they can be optimized to be synthesized in both a cellular and cell-free system. Both systems can synthesize peptide-based drugs on demand, allowing us to bypass shelf-life, which will become necessary when long-term space flight becomes a reality. In addition, in situ resource utilization on location would reduce the necessary mass of drugs required to be shipped, reducing valuable mass during space missions.
- Song, Jong-Am, Kyung-Yeon Han, Jin-Seung Park, Hyuk-Seong Seo, Keum-Young Ahn, and Jeewon Lee. “Human G-CSF Synthesis Using Stress-Responsive Bacterial Proteins.” FEMS Microbiology Letters296, no. 1 (2009): 60–66. https://doi.org/10.1111/j.1574-6968.2009.01616.x.
- Bakhtiari, Nahid et al. “Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.” Avicenna journal of medical biotechnology vol. 9,1 (2017): 19-22.
- Suzuki, Y et al. “High-level production of recombinant human parathyroid hormone 1-34.” Applied and environmental microbiology vol. 64,2 (1998): 526-9.
Synthesis Systems
We utilize both cellular and cell-free systems for drug manufacturing.
Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins.
Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
We utilize both cellular and cell-free systems for drug manufacturing. Our cellular system will use Vibrio natriegens (VmaxTM) and spores of Bacillus subtilis. We selected VmaxTM and B. subtilis as cellular chassis because VmaxTM is a fast-dividing bacterium with a doubling time of <14 minutes, and because B. subtilis spores have been shown to be viable after several years in Earth’s orbit [1, 2]. The peptide-drugs will be produced using traditional methods of protein expression adapted to a microfluidic chip.
Part of the Astropharmacy will utilize the abilities of a cell-free system to synthesize our drugs of choice, teriparatide, hGCSF, and insulin. A cell-free system extracts transcription and translation machinery (polymerases, ribosomes, and tRNAs) from within cells and combines them with chemical supplements and the DNA sequence of interest to manufacture proteins. Cell-free systems are an attractive method of synthesis for our purposes because...
...It’s dead. Unlike a cellular system where a constant supply of nutrition is required to maintain bacterial protein production, a cell-free system is non-living; this means energy is not diverted to forming proteins necessary for survival. Instead, all resources are funneled to creating the protein of interest, leading to higher protein yields than cellular systems [3]. Without the pressure to maintain cell viability, cell-free systems can also produce a broader range of products like difficult-to-synthesize and toxic proteins - both of which are potential applications of the Astropharmacy.
...It’s flexible. With the inner parts of the cell exposed to the outside, a cell-free system can be directly manipulated by the user. Whether it’s scaling the reaction up or down, setting up different reaction formats, or altering the chemical composition of the reaction for scientific investigation, it can all be done [3]. This means the Astropharmacy could produce customized quantities of a drug, and assemble more than one drug at a time. The ability to immediately produce a myriad of protein-based drugs within 24 hours, without having to have bacteria transformed with the particular plasmid, achieves the adaptability astronauts need in times of emergency.
- Competent Vibrio Natriegens Cells. https://www.biocat.com/genomics/competent-vibrio-natriegens-cells. Accessed 20 Oct. 2019.
- Microbes and Molecules Get a Space-Stress Test | Science Mission Directorate. https://science.nasa.gov/science-news/science-at-nasa/2010/22oct_ooreos. Accessed 20 Oct. 2019.
- Gregorio, N. E., Levine, M. Z. & Oza, J. P. A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols 2, 24 (2019).
- Seki, E., Yanagisawa, T. & Yokoyama, S. Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. in Noncanonical Amino Acids: Methods and Protocols (ed. Lemke, E. A.) 49–65 (Springer New York, 2018). doi:10.1007/978-1-4939-7574-7_3.
- Promega. Discover Reliable Tools for Protein Analysis. Madison: Promega Corporation. Electronic.
- Krinsky, N. et al. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE 11, e0165137 (2016).
- Kwon, Y.-C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5, 8663 (2015).
- Linder, M. B. et al. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems †. Biochemistry 43, 11873–11882 (2004).
- Liu, D. V., Zawada, J. F. & Swartz, J. R. Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis. Biotechnol Progress 21, 460–465 (2008).
- Pardee, K. et al. Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248-259.e12 (2016).
Drug Expression Chamber
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
In designing our integrated system, we decided to manufacture a 3D-printed expression chamber. This expression chamber is capable of facilitating cell-expression by thoroughly mixing cells or cell lysate with other precursors to express a protein of interest. The chamber itself is composed of a winding serpentine channel, with three inlets and one outlet, encased in a bio-compatible resin. Similar designs have been implemented on microfluidic chips in the past [1], however because of the large volume of product our system is designed to produce, we instead decided on a more robust 3D-printed chamber, which is capable of holding much larger quantities of liquid and supports higher flow rates. The three inlets interface with a syringe pump via PEEK and latex tubing [2], which is capable of flowing the precursors to cell-expression at a very low and consistent flow rate. The terminal end of the expression chip interfaces directly with the purification module of the Astropharmacy, which takes the impure mixture (including the expressed protein of interest) and purifies it into a ready-to-use product.
- Murphy TW, Sheng J, Naler LB, Feng X, Lu C. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng. 2019;25(5):13. https://doi.org/10.1038/s41378-019-0051-8.
- Song, In-Hyouk & Park, Taehyun. (2019). Connector-Free World-to-Chip Interconnection for Microfluidic Devices. Micromachines. 10. 166. 10.3390/mi10030166.
Drug Purification
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].
- ATCC - Methods for Freezing and Freeze-Drying Bacteria-140. https://www.atcc.org/support/faqs/840a5/Methods+for+freezing+and+freeze-drying+bacteria-140.aspx. Accessed 16 Oct. 2019.
- Zhang, Miao, et al. “Freeze-Drying of Mammalian Cells Using Trehalose: Preservation of DNA Integrity.” Scientific Reports, vol. 7, no. 1, July 2017, pp. 1–10. www-nature-com.revproxy.brown.edu, doi:10.1038/s41598-017-06542-z.
- Smith, Mark Thomas, and Scott D. Berkheimer. “Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage.” BioTechniques, vol. 56, no. 4, Apr. 2014. DOI.org (Crossref), doi:10.2144/000114158.
- Pardee, Keith, Alexander A. Green, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no. 4, Nov. 2014, pp. 940–54. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.004.
- Labconco FreeZone 2.5L -84C Benchtop Freeze Dryers, 115V US Models Includes. p. 2.
- Leslie, Samuel B., et al. “Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.” APPL. ENVIRON. MICROBIOL., vol. 61, 1995, p. 6.
- Lopatkin, Allison J., and Lingchong You. “Synthetic Biology Looks Good on Paper.” Cell, vol. 159, no. 4, Nov. 2014, pp. 718–20. DOI.org (Crossref), doi:10.1016/j.cell.2014.10.003.
- Lu, Yuan. “Cell-Free Synthetic Biology: Engineering in an Open World.” Synthetic and Systems Biotechnology, vol. 2, no. 1, Mar. 2017, pp. 23–27. DOI.org (Crossref), doi:10.1016/j.synbio.2017.02.003.
- Ogonah, Olotu W., et al. “Cell Free Protein Synthesis: A Viable Option for Stratified Medicines Manufacturing?” Current Opinion in Chemical Engineering, vol. 18, Nov. 2017, pp. 77–83. DOI.org (Crossref), doi:10.1016/j.coche.2017.10.003.
- Pardee, Keith. “Perspective: Solidifying the Impact of Cell-Free Synthetic Biology through Lyophilization.” Biochemical Engineering Journal, vol. 138, Oct. 2018, pp. 91–97. DOI.org (Crossref), doi:10.1016/j.bej.2018.07.008.
- Pardee, Keith, Shimyn Slomovic, et al. “Portable, On-Demand Biomolecular Manufacturing.” Cell, vol. 167, no. 1, Sept. 2016, pp. 248-259.e12. DOI.org (Crossref), doi:10.1016/j.cell.2016.09.013.
While the ability to synthesize small quantities of protein on demand has many potential applications, using our system to create protein based drugs requires significant downstream processing of raw products. Downstream processing of protein based drugs is typically broken into four steps, removal of protein from cell lysate, separation of the drug from other proteins, separation of the drug from non proteins, and removal of hazardous materials from the drug buffer solution [1]. On an industrial scale this is typically done in many steps, using specialized heavy equipment. Common processes carried out in protein purification include centrifugation, various types of chromatography, electrophoresis, and filtration [1]. The goal of this purification is to get protein purity upwards of 98%, with no single contaminant making up more than 0.5% of the remaining 2% impurity, which is the current industry purity standard [2]. For major pharmaceutical production facilities on Earth, it is optimal to put products through unique production processes, each of which relies on different heavy machinery not designed or suitable for space environments. This system is not suitable in a space environment due to upmass and feasibility issues ($1400/kg to launch a payload to the L1 lagrange point), which is why drugs are brought from Earth into space and kept stable until needed.[3]. As such, we designed a microfluidic system to both produce and purify medicine. Using a microfluidic system negates the need for heavy equipment, and fits our need to only produce small doses of medicine. This system is capable of running two different types of chromatography, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). IMAC separates our his tagged protein from most other large contaminants using a negatively charged resin which binds to the positive his tag. We can then cut the tag off with a protease, which is itself his tagged allowing it to be removed using the same column, and giving us native protein. Finally, SEC is used to remove small contaminants from our system and place our medicine in the proper protein buffer. Our system aims to produce the drugs on demand in space environments when needed, thus saving cost in upmass and refrigeration. Additionally, some drugs are unstable over several years and must be made on-site if astronauts are to conduct multi-year missions.
Prior research exists on the subject of on-chip protein purification. Scientists at the Oak Ridge National Laboratory published a paper in 2015 on designing microfluidic chromatography modules that formed the basis for our design [4]. They were able to create and test modules for Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC), which were the two purification techniques used in our system. Their paper gave us information on packing microfluidic modules with chromatography media, optimal dimensions for our column, and a simple set of processes that yield a high degree of protein purity. A further paper by researchers at Virginia Tech in 2019 created an IMAC module which connected to a separate chip which could express proteins using a cell free system, which formed the idea behind our expression chip [5]. We made several modifications to the systems outlined in these papers. Our expression chip was made using a 3D printer instead of photolithography, which allowed us to dramatically increase fluid capacity and produce full doses of protein. Our expression chamber is designed to hold 2ml of fluid, so under optimal conditions it should be able to produce over 1mg of target protein. We also shifted away from an integrated purification system, instead chaining together our 3 modules using tubing, which made it possible for our purification system to run with just one pump, although the expression chip can use up to three. Finally, by using his tagged protease for on column cutting, we should be able to produce native proteins instead of eluting his tagged proteins. This is crucial for biopharmaceutical production because tagged protein would likely activate an immune response in anyone who took the medicine, and would not be safe for use. Our project modifies existing research in on chip protein purification to better fit our purposes so that we can produce and purify quantities of protein approaching actual doses of medicine, use a more modular and simple system, and produce native proteins usable as medicine.
Our iGEM project is not the first to create a protein purification system, and is certainly not the first to use microfluidics. Our project has a very similar goal to that of UCopenhagen 2018, who proposed a system for producing and purifying drugs that used proteins tagged with an injectisome secretion signal, which causes bacteria to inject tagged proteins through a membrane which could potentially allow for creation of pure drugs for use in space. Our project builds on theirs by proposing an alternate purification to what they use. Rather than relying on secretion tags to purify in a single step, we rely on various types of chromatography to purify our proteins of interest. This multi step purification process adds some complexity to our system, but also allows for tag cutting with a protease. This allows our system to produce native proteins which could be used as medicine, rather than tagged protein. Our work is also similar to BostonU 2017, who designed a repository of microfluidic device designs that have synthetic biology applications. Our protein purification modules were built on PDMS in a very similar style to the chips they designed, and while our hardware achieves different goals from theirs, our chips would certainly be a useful contribution to their repository. Uppsala 2016 used 3D printed microfluidics to carry out on chip transformations, and produced their chips using a Form2 SLA printer, which was the machine we chose to use for our 3D printed expression chip. Our chips had similar manufacturing techniques, which helped us in producing our expression chip even if the devices had different purposes.
- Berthold, W & Walter, J. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals Volume 22 135-150 (1994)
- Interview with Merck executive
- Jones, H. The Recent Large Reduction in Space Launch Cost. 48th International Conference on Environmental Systems (2018)
- Millet, L.J. et al. Modular microfluidics for point-of-care protein purifications. Lab on a Chip Volume 15 1799-1811 (2015)
- Murphy, T. et al. On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsystems and Nanoengineering Volume 5 Article 13 (2019)
Lyophilization
In addition to the three main subsections of our project, we also wanted the Astropharmacy to be viable at ambient temperature for long-term space missions, so we decided to lyophilize our cell-free and cellular platforms. Lyophilization, or freeze-drying, is the process of low-temperature dehydration: the product meant to be lyophilized is frozen, and water is removed by sublimation. The lyophilized product retains its original properties after long periods of time during its dried and rehydrated state, which is ideal for extending the shelf-life of biological products. To increase transportability of the Astropharmacy, we wanted to see if we could also implement our cellular and cell-free synthesis systems on paper.
Lycoprotectants are often used to preserve the active ingredients in lyophilized samples, allowing more cells to remain viable. Common protectants include sucrose, skim milk, and trehalose [1, 2]. It is also observed that preservation of freeze-dried products is longest when they are at cold temperatures; keeping freeze-dried samples at temperatures at or above room temperature lead to decreased lyophilization efficacy [3]. Much cell-free lyophilization is focused on developing paper-based systems that are stable at room temperature for its potential applications as highly transportable, easily accessible protein manufacturing systems [4].