Team:BrownStanfordPrinctn/Design
Our Astropharmacy can be divided into three subprojects: Diagnosis, Drug Production, and Product Purification. Below are further details on design corresponding to each subproject.
Diagnosis
Our fusion proteins revolve around three functional domains - a dimeric-cellulose binding domain, a fluorescent protein, and a hydrophobic domain.
By coating cellulose with proteins made up of different combinations of these domains, we were able to achieve the first steps towards a hydrophobic, protein-based “printer ink” that is able to guide fluids on the microfluidic scale.
During the development of our ink fusion proteins, we successfully created four constructs that utilized our highlighted functional domains. These included:
With these fusion proteins we were not only able to create functional microfluidic channels using low cost materials, but we were also able to integrate that process into a commercial office printer.
More information can be found on the Results page.
Drug Production
As discussed in Description, we chose hG-CSF and teriparatide primarily for their extremely relevant medical applications. Other factors making them good candidates include: (1) Small dosage - each drug requires only a small amount of the active ingredient per dosage (in the micrograms). This is crucial in order to scale it down for microfluidics. (2) Small size - the drugs are simple peptides rather than enormous proteins that would be more difficult to synthesize. (3) No post-translational modifications - this is important for compatibility with an E. coli cell-free system. (4) Commercially synthesized in E.coli - the U.S. manufacturers of these drugs synthesize them transgenically, meaning there are existing protocols to produce these drugs in large quantities. We consulted with Dr. David Loftus from NASA when deciding between our peptide-based drugs and weighing these factors.
When designing the constructs, we isolated the biologically functional region of the protein (e.g. teriparatide consists of only the first 34 amino acids of human parathyroid hormone). We added solubility factors [1][2] that have previously been shown to prevent the formation of inclusion bodies when the protein is expressed. We also added a 6X His-tag for eventual Ni-NTA purification. Finally, we inserted an enterokinase or a TEV protease cut site into each drug, generating 4 final constructs for expression.
- 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.
Cell-Free
First, we tried to develop a cell-free system. At its most basic, a cell-free system requires the user to culture and extract cell lysate containing all the molecules necessary for transcription and translation. Then, the lysate is combined with supplemental solutions like buffers, energy regeneration systems, and DNA encoding the protein of interest (in our case plasmids encoding teriparatide, hGCSF, and insulin). More reagents can be added or subtracted based on the proteins we want to produce - the highly manipulative nature of a cell-free systems enables a variety of drugs to be manufactured--even with non-canonical amino acids [2].
The lysate can be obtained from different organisms. Common platforms include Escherichia coli, wheat germ, and rabbit reticulocyte [1, 3]. For the purposes of our project, we decided to develop a prokaryotic system from E. coli because there is extensive literature on E. coli based cell-free systems, and it tends to have higher protein yields than cell-free systems based off of other organisms. For our eukaryotic cell-free system, we also choose to use Saccharomyces cerevisiae because it can perform the post-translational modifications required for the production of insulin [1].
Working with our advisor, Dr. Kate Adamala, we were able to establish protocols for our cell-free reactions. In addition, we used the commercial cell-free system PURExpress from NEB to test our biobricks. More information about our procedures for commercial and lab-grown cell-free systems can be found under our experimental procedures.
After making all the mixes and solutions for the cell-free reactions, we began testing our system using the biobrick BBa_J04450 (RFP Coding Device) provided by iGEM. First, we ran a preliminary trial to see if the reaction worked. Of the six trials run, two of them showed significant red fluorescence under UV, while the rest did not. With the indication that some cell-free reactions were working, we continued experimentation while trying to troubleshoot why some reactions were not working. More information about our process can be found in our lab notebooks.
- 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).
Expression Chamber
In order to generate a usable dosage of a given drug, we designed a compact, 3D-printed expression chip. The expression chip has dimensions 2.0” X 4.0” X .5” and has an internal volume of just over 2 mL. In essence, the chip is a long, serpentine channel (with 1600 micron diameter) encased by a solid volume of bio-compatible resin, beginning at three collinear intake ports and terminating at a single outtake port on the opposite side of the device. The goal of this device is to properly mix the precursors to the cell-free system (or cell-system) and to facilitate full expression of the protein of interest. For a cell-free system, these precursors are cell lysate, amino acid mix and energy mix. For a cellular system, these precursors are Bacillus subtillus cells, and detergent used to lyse these cells.
The serpentine structure of the channel (which facilitates mixing) was inspired by past literature [1], but has traditionally been implemented on PDMS-based microfluidic chips. These chips impose challengingly small constraints on the radius and length of channels, and given the quantity of protein demanded by the nature of the project, it was determined that using a PDMS-based chip as the locus for cell-expression was not sensible. Our 3D-printed design is significantly cheaper, easier and faster to produce than traditional microfluidics, with the caveat of being bulkier (though this is to some extent unavoidable, due to the large quantities of solution which require time on-chip to express suitable amounts of protein).
This design also fits with the spirit of our modularly designed purification system, which is another significant departure that has been made from past literature. The expression chamber is designed to interface directly with the purification modules via PEEK and latex tubing, which is a means of interconnection inspired by very recent microfluidic literature [2].
Flow through the expression chip is achieved using three syringe pumps, operating at a very low flow rate (12 microliters/min). Each cell-free precursor is steadily flowed into the winding channels, where they slowly combine until a uniform mixing is achieved. The residence time for a cross-section of liquid at the intake is approximately 3 hours, and after 6 hours (two full internal volumes of the expression chip) the desired amount of protein will have been generated.
- 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.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.
Our fusion proteins revolve around three functional domains - a dimeric-cellulose binding domain, a fluorescent protein, and a hydrophobic domain.
By coating cellulose with proteins made up of different combinations of these domains, we were able to achieve the first steps towards a hydrophobic, protein-based “printer ink” that is able to guide fluids on the microfluidic scale.
During the development of our ink fusion proteins, we successfully created four constructs that utilized our highlighted functional domains. These included:
With these fusion proteins we were not only able to create functional microfluidic channels using low cost materials, but we were also able to integrate that process into a commercial office printer.
More information can be found on the Results page.
Drug Production
As discussed in Description, we chose hG-CSF and teriparatide primarily for their extremely relevant medical applications. Other factors making them good candidates include: (1) Small dosage - each drug requires only a small amount of the active ingredient per dosage (in the micrograms). This is crucial in order to scale it down for microfluidics. (2) Small size - the drugs are simple peptides rather than enormous proteins that would be more difficult to synthesize. (3) No post-translational modifications - this is important for compatibility with an E. coli cell-free system. (4) Commercially synthesized in E.coli - the U.S. manufacturers of these drugs synthesize them transgenically, meaning there are existing protocols to produce these drugs in large quantities. We consulted with Dr. David Loftus from NASA when deciding between our peptide-based drugs and weighing these factors.
When designing the constructs, we isolated the biologically functional region of the protein (e.g. teriparatide consists of only the first 34 amino acids of human parathyroid hormone). We added solubility factors [1][2] that have previously been shown to prevent the formation of inclusion bodies when the protein is expressed. We also added a 6X His-tag for eventual Ni-NTA purification. Finally, we inserted an enterokinase or a TEV protease cut site into each drug, generating 4 final constructs for expression.
- 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.
Cell-Free
First, we tried to develop a cell-free system. At its most basic, a cell-free system requires the user to culture and extract cell lysate containing all the molecules necessary for transcription and translation. Then, the lysate is combined with supplemental solutions like buffers, energy regeneration systems, and DNA encoding the protein of interest (in our case plasmids encoding teriparatide, hGCSF, and insulin). More reagents can be added or subtracted based on the proteins we want to produce - the highly manipulative nature of a cell-free systems enables a variety of drugs to be manufactured--even with non-canonical amino acids [2].
The lysate can be obtained from different organisms. Common platforms include Escherichia coli, wheat germ, and rabbit reticulocyte [1, 3]. For the purposes of our project, we decided to develop a prokaryotic system from E. coli because there is extensive literature on E. coli based cell-free systems, and it tends to have higher protein yields than cell-free systems based off of other organisms. For our eukaryotic cell-free system, we also choose to use Saccharomyces cerevisiae because it can perform the post-translational modifications required for the production of insulin [1].
Working with our advisor, Dr. Kate Adamala, we were able to establish protocols for our cell-free reactions. In addition, we used the commercial cell-free system PURExpress from NEB to test our biobricks. More information about our procedures for commercial and lab-grown cell-free systems can be found under our experimental procedures.
After making all the mixes and solutions for the cell-free reactions, we began testing our system using the biobrick BBa_J04450 (RFP Coding Device) provided by iGEM. First, we ran a preliminary trial to see if the reaction worked. Of the six trials run, two of them showed significant red fluorescence under UV, while the rest did not. With the indication that some cell-free reactions were working, we continued experimentation while trying to troubleshoot why some reactions were not working. More information about our process can be found in our lab notebooks.
- 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).
Expression Chamber
In order to generate a usable dosage of a given drug, we designed a compact, 3D-printed expression chip. The expression chip has dimensions 2.0” X 4.0” X .5” and has an internal volume of just over 2 mL. In essence, the chip is a long, serpentine channel (with 1600 micron diameter) encased by a solid volume of bio-compatible resin, beginning at three collinear intake ports and terminating at a single outtake port on the opposite side of the device. The goal of this device is to properly mix the precursors to the cell-free system (or cell-system) and to facilitate full expression of the protein of interest. For a cell-free system, these precursors are cell lysate, amino acid mix and energy mix. For a cellular system, these precursors are Bacillus subtillus cells, and detergent used to lyse these cells.
The serpentine structure of the channel (which facilitates mixing) was inspired by past literature [1], but has traditionally been implemented on PDMS-based microfluidic chips. These chips impose challengingly small constraints on the radius and length of channels, and given the quantity of protein demanded by the nature of the project, it was determined that using a PDMS-based chip as the locus for cell-expression was not sensible. Our 3D-printed design is significantly cheaper, easier and faster to produce than traditional microfluidics, with the caveat of being bulkier (though this is to some extent unavoidable, due to the large quantities of solution which require time on-chip to express suitable amounts of protein).
This design also fits with the spirit of our modularly designed purification system, which is another significant departure that has been made from past literature. The expression chamber is designed to interface directly with the purification modules via PEEK and latex tubing, which is a means of interconnection inspired by very recent microfluidic literature [2].
Flow through the expression chip is achieved using three syringe pumps, operating at a very low flow rate (12 microliters/min). Each cell-free precursor is steadily flowed into the winding channels, where they slowly combine until a uniform mixing is achieved. The residence time for a cross-section of liquid at the intake is approximately 3 hours, and after 6 hours (two full internal volumes of the expression chip) the desired amount of protein will have been generated.
- 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.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.
As discussed in Description, we chose hG-CSF and teriparatide primarily for their extremely relevant medical applications. Other factors making them good candidates include: (1) Small dosage - each drug requires only a small amount of the active ingredient per dosage (in the micrograms). This is crucial in order to scale it down for microfluidics. (2) Small size - the drugs are simple peptides rather than enormous proteins that would be more difficult to synthesize. (3) No post-translational modifications - this is important for compatibility with an E. coli cell-free system. (4) Commercially synthesized in E.coli - the U.S. manufacturers of these drugs synthesize them transgenically, meaning there are existing protocols to produce these drugs in large quantities. We consulted with Dr. David Loftus from NASA when deciding between our peptide-based drugs and weighing these factors.
When designing the constructs, we isolated the biologically functional region of the protein (e.g. teriparatide consists of only the first 34 amino acids of human parathyroid hormone). We added solubility factors [1][2] that have previously been shown to prevent the formation of inclusion bodies when the protein is expressed. We also added a 6X His-tag for eventual Ni-NTA purification. Finally, we inserted an enterokinase or a TEV protease cut site into each drug, generating 4 final constructs for expression.
- 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.
Cell-Free
First, we tried to develop a cell-free system. At its most basic, a cell-free system requires the user to culture and extract cell lysate containing all the molecules necessary for transcription and translation. Then, the lysate is combined with supplemental solutions like buffers, energy regeneration systems, and DNA encoding the protein of interest (in our case plasmids encoding teriparatide, hGCSF, and insulin). More reagents can be added or subtracted based on the proteins we want to produce - the highly manipulative nature of a cell-free systems enables a variety of drugs to be manufactured--even with non-canonical amino acids [2].
The lysate can be obtained from different organisms. Common platforms include Escherichia coli, wheat germ, and rabbit reticulocyte [1, 3]. For the purposes of our project, we decided to develop a prokaryotic system from E. coli because there is extensive literature on E. coli based cell-free systems, and it tends to have higher protein yields than cell-free systems based off of other organisms. For our eukaryotic cell-free system, we also choose to use Saccharomyces cerevisiae because it can perform the post-translational modifications required for the production of insulin [1].
Working with our advisor, Dr. Kate Adamala, we were able to establish protocols for our cell-free reactions. In addition, we used the commercial cell-free system PURExpress from NEB to test our biobricks. More information about our procedures for commercial and lab-grown cell-free systems can be found under our experimental procedures.
After making all the mixes and solutions for the cell-free reactions, we began testing our system using the biobrick BBa_J04450 (RFP Coding Device) provided by iGEM. First, we ran a preliminary trial to see if the reaction worked. Of the six trials run, two of them showed significant red fluorescence under UV, while the rest did not. With the indication that some cell-free reactions were working, we continued experimentation while trying to troubleshoot why some reactions were not working. More information about our process can be found in our lab notebooks.
- 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).
Expression Chamber
In order to generate a usable dosage of a given drug, we designed a compact, 3D-printed expression chip. The expression chip has dimensions 2.0” X 4.0” X .5” and has an internal volume of just over 2 mL. In essence, the chip is a long, serpentine channel (with 1600 micron diameter) encased by a solid volume of bio-compatible resin, beginning at three collinear intake ports and terminating at a single outtake port on the opposite side of the device. The goal of this device is to properly mix the precursors to the cell-free system (or cell-system) and to facilitate full expression of the protein of interest. For a cell-free system, these precursors are cell lysate, amino acid mix and energy mix. For a cellular system, these precursors are Bacillus subtillus cells, and detergent used to lyse these cells.
The serpentine structure of the channel (which facilitates mixing) was inspired by past literature [1], but has traditionally been implemented on PDMS-based microfluidic chips. These chips impose challengingly small constraints on the radius and length of channels, and given the quantity of protein demanded by the nature of the project, it was determined that using a PDMS-based chip as the locus for cell-expression was not sensible. Our 3D-printed design is significantly cheaper, easier and faster to produce than traditional microfluidics, with the caveat of being bulkier (though this is to some extent unavoidable, due to the large quantities of solution which require time on-chip to express suitable amounts of protein).
This design also fits with the spirit of our modularly designed purification system, which is another significant departure that has been made from past literature. The expression chamber is designed to interface directly with the purification modules via PEEK and latex tubing, which is a means of interconnection inspired by very recent microfluidic literature [2].
Flow through the expression chip is achieved using three syringe pumps, operating at a very low flow rate (12 microliters/min). Each cell-free precursor is steadily flowed into the winding channels, where they slowly combine until a uniform mixing is achieved. The residence time for a cross-section of liquid at the intake is approximately 3 hours, and after 6 hours (two full internal volumes of the expression chip) the desired amount of protein will have been generated.
- 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.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.
First, we tried to develop a cell-free system. At its most basic, a cell-free system requires the user to culture and extract cell lysate containing all the molecules necessary for transcription and translation. Then, the lysate is combined with supplemental solutions like buffers, energy regeneration systems, and DNA encoding the protein of interest (in our case plasmids encoding teriparatide, hGCSF, and insulin). More reagents can be added or subtracted based on the proteins we want to produce - the highly manipulative nature of a cell-free systems enables a variety of drugs to be manufactured--even with non-canonical amino acids [2].
The lysate can be obtained from different organisms. Common platforms include Escherichia coli, wheat germ, and rabbit reticulocyte [1, 3]. For the purposes of our project, we decided to develop a prokaryotic system from E. coli because there is extensive literature on E. coli based cell-free systems, and it tends to have higher protein yields than cell-free systems based off of other organisms. For our eukaryotic cell-free system, we also choose to use Saccharomyces cerevisiae because it can perform the post-translational modifications required for the production of insulin [1].
Working with our advisor, Dr. Kate Adamala, we were able to establish protocols for our cell-free reactions. In addition, we used the commercial cell-free system PURExpress from NEB to test our biobricks. More information about our procedures for commercial and lab-grown cell-free systems can be found under our experimental procedures.
After making all the mixes and solutions for the cell-free reactions, we began testing our system using the biobrick BBa_J04450 (RFP Coding Device) provided by iGEM. First, we ran a preliminary trial to see if the reaction worked. Of the six trials run, two of them showed significant red fluorescence under UV, while the rest did not. With the indication that some cell-free reactions were working, we continued experimentation while trying to troubleshoot why some reactions were not working. More information about our process can be found in our lab notebooks.
- 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).
Expression Chamber
In order to generate a usable dosage of a given drug, we designed a compact, 3D-printed expression chip. The expression chip has dimensions 2.0” X 4.0” X .5” and has an internal volume of just over 2 mL. In essence, the chip is a long, serpentine channel (with 1600 micron diameter) encased by a solid volume of bio-compatible resin, beginning at three collinear intake ports and terminating at a single outtake port on the opposite side of the device. The goal of this device is to properly mix the precursors to the cell-free system (or cell-system) and to facilitate full expression of the protein of interest. For a cell-free system, these precursors are cell lysate, amino acid mix and energy mix. For a cellular system, these precursors are Bacillus subtillus cells, and detergent used to lyse these cells.
The serpentine structure of the channel (which facilitates mixing) was inspired by past literature [1], but has traditionally been implemented on PDMS-based microfluidic chips. These chips impose challengingly small constraints on the radius and length of channels, and given the quantity of protein demanded by the nature of the project, it was determined that using a PDMS-based chip as the locus for cell-expression was not sensible. Our 3D-printed design is significantly cheaper, easier and faster to produce than traditional microfluidics, with the caveat of being bulkier (though this is to some extent unavoidable, due to the large quantities of solution which require time on-chip to express suitable amounts of protein).
This design also fits with the spirit of our modularly designed purification system, which is another significant departure that has been made from past literature. The expression chamber is designed to interface directly with the purification modules via PEEK and latex tubing, which is a means of interconnection inspired by very recent microfluidic literature [2].
Flow through the expression chip is achieved using three syringe pumps, operating at a very low flow rate (12 microliters/min). Each cell-free precursor is steadily flowed into the winding channels, where they slowly combine until a uniform mixing is achieved. The residence time for a cross-section of liquid at the intake is approximately 3 hours, and after 6 hours (two full internal volumes of the expression chip) the desired amount of protein will have been generated.
- 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.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.
In order to generate a usable dosage of a given drug, we designed a compact, 3D-printed expression chip. The expression chip has dimensions 2.0” X 4.0” X .5” and has an internal volume of just over 2 mL. In essence, the chip is a long, serpentine channel (with 1600 micron diameter) encased by a solid volume of bio-compatible resin, beginning at three collinear intake ports and terminating at a single outtake port on the opposite side of the device. The goal of this device is to properly mix the precursors to the cell-free system (or cell-system) and to facilitate full expression of the protein of interest. For a cell-free system, these precursors are cell lysate, amino acid mix and energy mix. For a cellular system, these precursors are Bacillus subtillus cells, and detergent used to lyse these cells.
The serpentine structure of the channel (which facilitates mixing) was inspired by past literature [1], but has traditionally been implemented on PDMS-based microfluidic chips. These chips impose challengingly small constraints on the radius and length of channels, and given the quantity of protein demanded by the nature of the project, it was determined that using a PDMS-based chip as the locus for cell-expression was not sensible. Our 3D-printed design is significantly cheaper, easier and faster to produce than traditional microfluidics, with the caveat of being bulkier (though this is to some extent unavoidable, due to the large quantities of solution which require time on-chip to express suitable amounts of protein).
This design also fits with the spirit of our modularly designed purification system, which is another significant departure that has been made from past literature. The expression chamber is designed to interface directly with the purification modules via PEEK and latex tubing, which is a means of interconnection inspired by very recent microfluidic literature [2].
Flow through the expression chip is achieved using three syringe pumps, operating at a very low flow rate (12 microliters/min). Each cell-free precursor is steadily flowed into the winding channels, where they slowly combine until a uniform mixing is achieved. The residence time for a cross-section of liquid at the intake is approximately 3 hours, and after 6 hours (two full internal volumes of the expression chip) the desired amount of protein will have been generated.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.
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 .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]. 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.
To purify small doses of protein using a minimum mass of equipment, we created a microfluidic protein purification system. Microfluidics is the manipulation of fluids in channels on the order of microns (10^-6 m) [4]. These channels are contained in microfluidic devices or chips. These chips can be made using a number of techniques, including using photolithography to create chips made of polydimethylsiloxane (PDMS), 3D printing, and making channels out of wax on paper. Microfluidics offers a number of benefits as a platform for chemical processes. The first of these is control. On the micro scale, fluids move with very little turbulence in a laminar flow regime, which when combined with computer controlled pumps allows for very precise manipulation of any fluids on a chip [4]. Further, this small scale is very resource and time efficient, as microfluidics typically hold under 1ml of reagent at a time, and the high surface area to volume ratio found at small scales promotes very fast chemical reactions. Finally, these chips take up very little space, with most PDMS chips fitting on a microscope slide and having almost insignificant weight. All these factors make microfluidics an ideal platform for a low mass system for small scale protein purification.
Our purification system consists of two microfluidic chromatography modules, which can be directly connected to allow easy flow between the system. The first module used is an IMAC (immobilized metal affinity chromatography) column. The biopharmaceuticals are synthesized with a positively charged His tag, which is attracted to the negatively charged Ni-NTA complex that makes up the beads in this column. When the protein attaches to the beads, water can be flowed through the module, removing everything but the protein of interest. A His tagged protease can then be flowed into the column, which recognizes an amino acid sequence linking the drug and His tag and cuts the protein of interest free from the column. The His tagged protease is then left behind in the column while the native biopharmaceutical flows out of the IMAC column.
The second step is to run a buffer exchange on the protein, removing it from the protease buffer and placing it in a human friendly protein buffer. In our proof of concept drug, insulin, this buffer consists of water, glycerol, m-cresol and zinc. This exchange is carried out in our second module, a SEC (size exclusion chromatography) column. This column contains beads with small holes of varying sizes, which connect to long winding channels within the beads. Small molecules are more likely to get trapped in these channels, so they take longer to pass through the column than large molecules, which cannot fit in as many holes and bypass the beads entirely. Before use, the desired buffer solution is fully run through the column. As the protein and original buffer run through the column, the protein moves faster than the small molecules in the original buffer, and is collected out in several fractions, in a solution containing the desired buffer.
- 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)
- Whitesides, G. The origins and the future of microfluidics. Nature Volume 442 368-373 (2006)
Lyophilization
We freeze-dried lycoprotectant compounds (i.e. trehalose, sucrose, skim milk) at different concentration levels with our potential cellular synthesis systems: B. subtilis , E. coli. and VmaxTM. The lyophilized cells were left at room temperature. The cells were rehydrated at different time points, and serial dilutions were preformed to test cell viability. Besides testing how we could enhance the storability of the cellular systems using lycorpotectants to increase the accessibility of the Astropharmacy in different environments and over long periods of time, we also wanted to increase the transportability of the Astropharmacy and considered the potential of a paper-based Astropharmacy. To this end, we attempted to lyophilize cellular systems on paper (filter paper and printer paper), and quantify viability. More information can be found in our Results page.