Introduction
Safety of the researcher, environment, manufacturer and the consumer is of utmost importance in each of the processes from strain development experiments to manufacturing and packaging of the final product. To ensure the same, consideration of all the precautions and safety measures must be taken to make the work environment, manufacturing environment and consumer product safe and to prevent any hazardous situations. Material safety data sheets for all reagents should be kept handy (List of reagents used have been given in the handbook).
Bacteria
Figure 1: Safe chassis bacteria
Our entire project requires a Biosafety Level 1 (BSL-1) laboratory. This is because our project has proposed the use of Biosafety level 1 bacteria (Escherichia coli DH5alpha and Escherichia coli Nissle 1917) as our probiotic chassis iGEM - safety risk groups and therefore we consider our project to be harmless to researchers in this aspect. None of the designed experiments are expected to increase the pathogenicity of the bacteria.
The bacteria Escherichia coli Nissle 1917 is an extremely well studied probiotic strain with no known negative effects on health Pradhan, S., & Weiss, A. A., 2020. It does not have the ability to become an aerosol and has no known pathogenic effects on animals and plants as well. It is responsible for producing fitness factors called ‘colicins' that are toxic to other Escherichia coli strains. In our case, it would be an advantage as we want our bacteria to compete while it stays in the gut. Furthermore, its ability to form biofilms increases its long-term persistence in the gut.
We would not reveal important information like activation mechanisms, etc to ensure that dual use does not occur (unless it has all the required clearances).
Biosafety & Biosecurity
Figure 2: Dual Use
Team Bielefeld-CeBiTec 2015 proposed a detailed analysis of biosecurity, particularly the dual use issue, with respect to the iGEM competition in which they have mentioned several aspects and questions to be considered Bielefeld-CeBiTec, 2015. Some of the questions which we have answered are given below (More can be found in the handbook).
Can you imagine any malevolent use of the knowledge and sequences published on your team's wiki? Could the knowledge you provide be used for the creation of products or organisms that pose a danger to humans or the environment?
Our project involves the release of an anti-inflammatory cytokine (IL-10) whose release is regulated by a control mechanism involving SoxS and SoxR genes. The SoxR gene is also regulated to be released only in the presence of methylmercury using the MerR-PmerT mercury responsive genes.
Selective mutation of any of these control mechanisms could lead to release of IL-10. This could lead to reduced immune response to pathogens. IL-10 also down regulates transcription and secretion of IL-1β, IL-6, IL-8, TNF-α, and G-CSF by activated monocytes and macrophages. This could lead to cancer Schreiber S, 1997. IL-10 upregulates ICAM-1 within neural tissues. This promotes massive macrophage influx, demyelination due to inflammation and subsequent loss of neural tissue, resulting in muscle weakness and paralysis Dru S., 2009.
Who will use your product? If your product is successful, who will receive benefits and who will be harmed?
Our product if successful will be used by people who are at risk for methylmercury poisoning. We have made sure to not use any allergens or preservatives which could potentially harm consumers.
WHO Questionnaire
WHO also has a self-assessment questionnaire to ensure that good quality, ethical research activities are conducted in safe and secure facilities. Some of the important question relevant to biosecurity and biosafety aspects have been answered below (more in handbook) WHO - Responsible life sciences research for global health security :
| Pillar 3: BIOSAFETY AND LABORATORY BIOSECURITY | |
|---|---|
| An assessment of the risk associated with research activities is conducted | Yes. The team has documented a Safety Handbook that covers various safety aspects and a detailed risk assessment for each experiment |
| Risk assessments are able to identify requirements for risk reduction measures including the level of containment required | Yes. The Safety Handbook covers not only the risk reduction measures but also the level of containment required for each stage of the research that will be conducted. |
| Legislation/regulations regarding hazardous waste disposal are followed | Yes. The team will strictly follow the regulations pertaining to hazardous waste disposal. (No Lab Experiments were performed this year) |
| Valuable biological material is safely and securely stored | Yes. The team will ensure that the valuable biological material is safely and securely stored for future use. (No Lab Experiments were performed this year) |
Toxic chemicals
Methylmercury
Figure 3: Methylmercury is extremely toxic.
All forms of mercury, both organic and inorganic are highly toxic. Exposure to mercury even in small amounts can causes severe health issues and can even affect the development of the child in utero and early in life WHO - Mercury and health.
- To avoid mercury poisoning by accidental inhalation, ingestion, injection or absorption through the skin while working with it, the use of appropriate type of gloves (Nitrile (8 mil) over Silver Shield) and other essential lab attire like Chemical goggles/face shield, lab coat with full sleeves, full-length pants, and closed toe shoes must be ensured.
- Mercury spill kits must be kept handy in the lab in case of an emergency.
- All mercury containing waste must be disposed of separately by packing in a container with a hazardous waste label and chemical name on it.
- We have taken into consideration all the hazardous waste disposal laws in India and have also enlisted the ones in the US for reference in our safety manual.
Paraquat
In the USA: Paraquat is categorized under ‘restricted use' by the US Environmental Protection Agency. Only those with a license are allowed to use this chemical. A blue dye and sharp odor are added as a warning, and another agent is added to induce vomiting in case of ingestion Paraquat dichloride-EPA.
In India: CIBRC (Central Insecticide Board and Registration Committee) has categorized paraquat dichloride as highly toxic Conditions of paraquat use in India - Pan-India.
To ensure safety while handling paraquat protective clothing, including gloves, safety glasses, respiratory equipment, full-sleeved lab coat, full-length pants, and closed-toe shoes must be worn. During disposal, suitable incineration and chemical treatment in accordance with local regulations must be followed (by a licensed professional).
Experimentation
Wet Lab
Figure 4: Gloves
To maintain aseptic conditions, all experiments must be performed inside the laminar flow chamber while ensuring the UV light doesn't cause any problems to the person performing the experiment.
Precautions must be taken while working with the autoclave and handling of hot autoclaved equipment must be done using appropriate heat proof gloves. Aseptic conditions must be maintained in order to prevent contamination by unwanted DNA or organisms. Gloves must be used not only for handling hot equipment and chemicals but also for cold samples taken out of the freezer to avoid cold burns. The stains and other hazardous substances should be handled with care during gel electrophoresis, sonication and gas chromatography.
Labs must be equipped with an emergency shower, eyewash station, and a first aid kit and workstation must be cleaned. Disposal of all waste materials must be done properly.
SAUL
Isolation of all electronic components from any conducting media should be ensured. All wiring connections should be insulated properly, and care must be taken to avoid spilling or leakage of chemicals or fluids onto the motors.
After experiments which involve methylmercury and paraquat, the dialysis tube must be discarded and other equipment components should be thoroughly cleaned following the handling protocols. Any leakage into the incubator should be prevented by placing the entire set up in a closed air tight container. All of the components used in the design are checked for temperature and pH resistance.
Figure 5: Specialized disposal
Product Development
All the experiments designed are relatively safe. The bacteria used should be highly tested for any safety concerns. However, any unexpected results or modifications must be handled with extreme care.
Growth Media experiments which utilize hazardous substances like mercuric oxide, concentrated HCl etc. must be handled in the fume hood in rooms with adequate ventilation. Protective equipment must be worn at all times. Disposal of inorganic peroxides and oxidants as well as bromine and iodine should be done by rendering them harmless by reducing it with acidic thiosulphate solution. Disposal of contaminants should be done separately with adequate labeling.
Implementation
Initially, we identified the worst cases in pharmaceutical cleaning validation Identification of worst case in cleaning validation- Pharmaceutical guidelines.
- Potency of product
- Solubility of Active Pharmaceutical Ingredient (API) in water
- Toxicity of API
- Concentration of API
- Contact surface area
- Product excipients
- Manufacturing process
The quality and quantity of the API (bacteria) must be maintained at recommended safe levels (CFU count) ensuring optimal potency. The water released during drying or other process must be sterilized before discarding. The API in our case is Escherichia coli Nissle 1917 which is a non-pathogenic known probiotic strain. The API concentration must be at recommended levels for a probiotic. The equipment used must have minimum surface area and should be sterilized regularly with non-harmful substances. The excipients used must not contain any known allergens as well as preservatives. Contamination of the product must be prevented by repeated cleaning and sterilization of all components including air, equipment, media etc.
Powder Design
Since the volume of the microorganisms present in bioreactors is much larger, extreme care must be taken to prevent the contamination of the bioreactor itself and also the surrounding environment.
There are possibilities of liquid spills and aerosol formation for some widely used bacterial chassis. However, the bacteria we have proposed is Escherichia coli Nissle 1917 which is a risk 1 non-pathogenic organism and does not have the capability to form aerosols.
The proximity of large volumes of liquid and mains electrical equipment (eg, from heaters, aerators, sensors, etc) presents a hazard. Commercially-designed equipment from a reliable source should prevent access to live conductors.
In case of any gas production, the gases must be vented out to prevent high pressure build up, and it should be kept away from fire.
The quality and safety of the manufactured product should be a key concern in order to ensure the client's safety Prevention of microbial contamination during manufacturing- Pharmaceutical guidelines.
Plant Hygiene
- Air filtration and air change rates must be set and done regularly.
- In case of contamination in one area, the plant should have a HVAC system which would remove and prevent contaminants from spreading to other areas.
- Ventilation dampers should also be positioned away from the production area for maintenance purposes.
Personnel Hygiene
Figure 6: Personal Protective Equipment
- The personnel handling the production must be trained on the importance of hygiene and it should be ensured that they would wear protective clothing such as hair cover, overshoes, over garments, beard covers, etc. to prevent any sort of contamination.
- It is required to ensure that direct contact with the product or an equipment which comes in contact with the product is avoided.
Cleaning
- Cleaning agents must be of a suitable grade to minimize any health risks. Cleaning and disinfection should be done regularly. Rotation of different disinfectants should be done to prevent any resistance development. The disinfectant must be sterilized using a 0.2 micron membrane filter in sterile conditions. The hold time of the disinfectants must also be validated.
- The equipment for cleaning should not have direct contact with the pharmaceutical product.
- The cleaning practice must be validated.
Equipment maintenance and cleanliness must be ensured and all guidelines must be followed.
Process Design
A controlled area for pharmaceutical manufacturing would be maintained. Any harm to the manufacturers due to dust particles or micro-organisms should be avoided by providing quality equipment, adequate hygiene training and regular cleaning and sterilization. The capsule integrity and sterility must be ensured during filling. Quality control of capsules should be done at each key stage. Different processes must be performed at different sections of the plant to prevent contamination.
There are four different levels at which a sterile pharmaceutical company should grade its control production: A B C and D. Grade A should be the zone for high risk operations like filling zone and zone for making aseptic connections; this zone should have laminar airflow systems with homogeneous airspeeds. Grade B is the background environment for grade A that allows for aseptic preparation and filling. Grade C and D are classified areas that handle the less critical stages of SPM Controlled areas in sterile manufacturing - pharmaceutical guidelines.
Packaging
Figure 7: Packaging
Blister punching machine is closed at the manufacturing part, thus remaining tamper resistant. The packaging must maintain the integrity and safety of the product.
Waste Management & Recycling
Pharmaceutical waste is categorized into 2 categories An overview of waste management in pharmaceutical industry - pharma journal
- Hazardous waste (any contaminants or chemicals).
- Non-hazardous waste
Waste Minimization
An important method of waste management is the prevention of waste material being created, also known as waste reduction. Before processing, any unnecessary waste generation must be checked and adequate measures should be taken.
Reuse
Re-use means the use of a product on more than one occasion, either for the same purpose or for a different purpose, without the need for reprocessing. Re-use avoids discarding a material to a waste stream when its initial use has concluded. The integrity of the product or equipment must not be sacrificed.
Recycling
Recycling involves the treatment or reprocessing of a discarded waste material to make it suitable for subsequent reuse either for its original form or for other purposes. Unused or haphazardly filled capsules should be recycled keeping in mind sterility. Discard water must be sterilized and used for some other purposes.
Handbook
Please refer to the full safety handbook for all details.
The Safety Handbook includes detailed and complete safety considerations of every reagent, process, experiment, device, equipment, etc described in the other handbooks as well as biosecurity and biosafety issues of our project.
To download this document, click here.