Team:BrownStanfordPrinctn/Safety
Introduction
If the Astropharmacy is made by people and used by people, it needs to be safely made and safely used. This means the risk our project holds to ourselves, the intended users, and the environment should be minimized and contained. Here we will describe the protective and preventive measures used in the laboratory, the safety considerations made in the design of the Astropharmacy, and the potential risks that arise between the Astropharmacy and the environment. More detailed information can be found on our Safety Form.
Lab Safety
Security and Training. To familiarize ourselves with the proceedings of the lab and reduce the risk our research imposes on ourselves and colleagues, all team members underwent lab safety training in-person and online as required by the NASA Ames Research Center. These courses covered the usage of personal protective equipment, hazardous waste management, laboratory safety, biosafety, chemical hygiene, and cybersecurity. When conducting lab work, we all wore personal protective equipment, and practiced sterile technique. As another safety measure, all research was conducted under the supervision of a senior member of the lab.
The lab is equipped with fume hoods, laminar flow biosafety hoods with UV sterilization, an autoclave, and safety showers. All waste that has been exposed to bacteria was autoclaved and disposed of separately from non-hazardous waste. There are also special containers and procedures we followed to dispose of hazardous chemicals. The lab has undergone multiple government-mandated inspections from NASA to enforce high safety standards.
Access to the lab is strictly controlled by passcode locks on doors, and all people who wish to enter the research center must show proper identification.
Strains. During the course of experimentation, and for the purposes of our project, we used a variety of standard bacteria strains that are commercially available:
- Escherichia coli DH5-alpha
- Escherichia coli T7-Express
- Escherichia coli BL21 (DE3)
- VmaxTM
- Bacillus subtilis 168
- Saccharomyces cerevisiae
All strains are considered safe and are classified Biosafety Level 1 based on Safety Data Sheets provided by the suppliers.
Protocol Safety. Many protocols have physical and chemical risks that can be prevented with the right protection and/or safer alternative. To create a safe laboratory environment, we took measures to make sure nobody was harmed.
One common procedure performed in the lab is gel electrophoresis. We used SYBR Safe instead of ethidium bromide to stain our gels because ethidium bromide is a toxic mutagen, and SYBR Safe is not.
Another procedure that can lead to physical harm happens during the cell lysate extraction. We lyse cells using sonication - releasing sound energy to disrupt the cell membrane. The frequency and amplitude we use to lyse cells is harmful to the human ear if exposed for too long. To prevent hearing damage, we employ ear protection at all times during the sonication process.
The SDS-Page protocol requires the use of acetic acid, a highly corrosive chemical. Whenever we require acetic acid for a procedure, we always wear gloves and handle the chemical under a fume hood. To dispose of acetic acid, we dilute it with water and pour it into a separate container.
Drug Safety
Pharmaceutical drugs need to have high purity to be effective and safe for users. This ensures that the product doesn’t have damaging side-effects that arise from contaminants or experience reduced potency. Discussions with Merck have informed us that drugs need to be at least 98% pure--an industry standard. To achieve a high level of purity, our purification chip is designed with chromatography columns that can bind our tagged protein-drugs; further purification and usability is generated by the consecutive buffer-exchange process.
Even so, the improper use of our manufactured drugs can be damaging. For example, hGSCF may lead to allergic reactions and damage to muscloskeletal skeletal system, respiratory system, hematological system, renal system, and spleen; more common side-effects include fever and bone pain [1, 2]. Teriparatide may lead to allergic reactions. Side-effects include nausea, leg-cramps, and infrequently, orthostatic hypotension [3, 4, 5]. Adverse effects of insulin include allergic reactions, hypoglycemia, lipohypertrophy, and weight gain [6, 7, 8]. However, these side-effects can be dependent on the patient and drug dosage. The Astropharmacy can easily be controlled to produce the desired dosage by manipulating reagent quantity due to its flexible cell-free or cellular synthesis platform. Additionally, the drugs we are manufacturing were chosen in consultation with Dr. Loftus and the drugs would be administered under the guidance of a trained medical professional.
In the lab, the amount of hGCSF, teriparatide, and insulin we are producing is on the microgram scale and less, so there is little risk of harm to the members on the team if exposed. We also wear protective equipment to minimize any contact with the drugs we are producing.
Microfluidic Chip Manufacturing Safety
The protocol for production of PDMS microfluidic devices our team followed required exposure to several potential safety hazards. Many of the materials used in device production, in particular SU-8 and SU-8 developer, can be toxic, and are flammable. This is a significant hazard in that these chemicals can vaporize when exposed to heat, and this protocol requires baking wafers coated in SU-8. The protocol also requires heating plates, which can cause burns if used improperly. Finally, the wafers in this protocol are exposed to intense UV light, which can damage the eyes if one were to look at the exposure process. These risks were mitigated by carrying out these procedures in proper facilities, under the direct guidance of graduate students with the Fordyce Lab at Stanford. They assisted in writing fabrication protocols for PDMS devices, and also gave us hands on instruction in how to properly make our chips. Their expertise, coupled with good general lab safety practices like wearing proper attire and exercising caution in the use of hot plates, allowed us to mitigate hazards and risks in running this protocol.
Risks of the Astropharmacy
User Safety. The Astropharmacy has Earth and outer space applications; the people using the Astropharmacy will not only be astronauts, but also everyday people with medical needs. Therefore, the device must be safe and easy to use. To account for this, the Astropharmacy either contains no living organisms (for cell-free) or non-pathogenic strains (VmaxTM , Bacillus subtilis). The device itself is made out of non-toxic polymers like PDMS, and is designed to connect conveniently and seamlessly between production and purification modules to stop any potentially toxic materials from leaking out and harming the user and the environment.
Accidental Release, Regulations, and Environmental Safety. Collaborating with the San Paulo iGEM team, we looked into the regulations surrounding GMO experimentation in the space and atmosphere. We reached out to EPA officers and various NASA employees working on biological research payloads. Based on our discussions with people from the EPA, NASA, and Biosentinel, we discovered that biological experimentation in space is a regulatory gray area, and that NASA evaluates the potential for biological hazard on a case-by-case basis. However, we were unable to determine what the exact criteria was.
Though we do not plan to release out project into the environment, the end product “Astropharmacy is meant to be used in a variety of environments. Hence, we wanted to ensure that our drug production system does not harm the environment. The Astropharmacy explores a cellular method of protein production using VmaxTM and B. subtilis. These organisms are meant to remain within the expression module, and not the environment where they can potentially interfere with the ecosystem. VmaxTM being a derivative of the halophile Vibrio natriegens, is sensitive to the salt content and temperature of the environment for optimal growth; given this vulnerability, it is unlikely that VmaxTM will be able to survive outside of the Astropharmacy. In comparison, B. subtilis is much more robust, and can survive under harsh environmental conditions due to its spore-forming capacity; to prevent its growth in the environment, the chosen B. subtilis strain is auxotrophic and requires tryptophan to grow.
References
- Granulocyte Colony Stimulating Factor (G-CSF) | Cancer Information | Cancer Research UK. https://www.cancerresearchuk.org/about-cancer/cancer-in-general/treatment/cancer-drugs/drugs/g-csf. Accessed 3 Oct. 2019.
- Mehta, Hrishikesh M., et al. “G-CSF and GM-CSF in Neutropenia.” Journal of Immunology (Baltimore, Md. : 1950), vol. 195, no. 4, Aug. 2015, pp. 1341–49. PubMed Central, doi:10.4049/jimmunol.1500861.
- TERIPARATIDE - National Library of Medicine HSDB Database. https://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+7367. Accessed 3 Oct. 2019.
- Teriparatide.. Accessed 3 Oct. 2019.
- Gundgurthi, Abhay, et al. “Insulin Poisoning with Suicidal Intent.” Indian Journal of Endocrinology and Metabolism, vol. 16, no. Suppl1, Mar. 2012, pp. S120–22. PubMed Central, doi:10.4103/2230-8210.94254.
- “Insulin Overdose: Dosage, Symptoms, and Treatment.” Medical News Today, https://www.medicalnewstoday.com/articles/317300.php. Accessed 3 Oct. 2019.
- Insulin Side Effects. https://www.diabetes.co.uk/insulin/insulin-side-effects.html. Accessed 3 Oct. 2019.
- What Are the Side Effects of Taking Insulin? https://www.webmd.com/diabetes/qa/what-are-the-side-effects-of-taking-insulin. Accessed 3 Oct. 2019.