Establishing and Monitoring an Aseptic Workspace

When are aseptic operations necessary? In order to meet certain bioburden requirements, some components must undergo dry heat microbial reduction (DHMR) or other sterilizing procedures. If sensitive surfaces must be re-exposed after DHMR, this could compromise the bioburden levels. Recontaminating sterilized surfaces could be costly both in time by requiring repeated DHMR and risk to the hardware, which may not be compatible with repeated high temperature bakes. In order to prevent recontamination of the sensitive surfaces, an aseptic environment and sterile technique must be employed. Aseptic environments mean working in a space with almost no detectable bioburden in the air or on surfaces. Ideally, DHMR happens as late as possible to avoid requiring aseptic operations, as it can be considered a high-risk operations. Preparing the cleanroom for aseptic operations Establishing an ISO (International Organization for Standardization) class 5 space to minimize airborne particles. Maintain low bioburden in the cleanroom by using biocidal cleaners. Using multiple biocidal techniques decreases the likelihood of selecting for resistant microorganisms. 70% Isopropyl Alcohol (IPA) denatures the proteins in a microorganism (note: 70% IPA is better at killing microorganisms than 100% IPA) 7% hydrogen peroxide: damages DNA and proteins through oxygen radical damage. Ultraviolet-C (UV-C) lamps: causes crosslinking in DNA which prevents replication. Monitor cleanroom regularly for bioburden trending: Standard bioassay: Swab or wipe samples of cleanroom surfaces processed for colony forming unity (viable or spore selected); Rapid bioassay: Adenosine triphosphate (ATP) or Limulus amebocyte lysate (LAL) for a bioburden snapshot. High levels can signal an immediate re-cleaning before standard bioassay samples are taken. Airborne monitoring: Active (pulling air through a filter) or passive (particle fallout) for bioburden. Verify bioburden levels just before aseptic operation. Test hardware and cleanroom surfaces and air 3 days before the planned aseptic operation. Rapid bioburden just before aseptic operation to ensure room was not re-contaminated. Preparing personnel and tools: Personnel training. Everyone in the cleanroom: Standard cleanroom certification Everyone on the team: 1 day Planetary Protection overview. Aseptic operators only: Half-day aseptic operations training. Covers sterile garmenting/gloves, Sterile handling with a focus on contact transfer risk, tool/GSE preparation, and two-operator system for opening sterilized tools/components. Tool sterilization: All tools to be used during an aseptic operation need to be identified. Compatible tools are sterilized by DHMR or Autoclave. Double wrapped so that the exterior bag can be handled by a non-sterile operator, and the sterile. Tools that are not compatible with high heat do not come in contact with sensitive surfaces: either substitutes are found, or tools are isolated by wrapping in sterile foil. During an aseptic operation. Pre-task to make sure everyone understands the operations, who is handling what, and when the most critical surfaces will be exposed. Monitoring during the operation. Bioburden: active and passive airborne bioburden sampling, glove-tip dabs onto a plate after completion of operation (3 days for results). Particles: real time particle counter constantly running, with alarm for exceeding ISO 5 conditions

Establishing and Monitoring an Aseptic Workspace for Building the MOMA Mass Spectrometer

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Aseptic Handling of the MOMA Mass Spectrometer After Dry Heat Microbial Reduction

Mars Organic Molecule Analyzer Mass Spectrometer (MOMA-MS) is an instrument in the larger MOMA instrument suite for the European Space Agency (ESA) ExoMars 2020 Rover. As a life-detection instrument on a Mars landing mission, MOMA-MS has very stringent Planetary Protection (PP) bioburden requirements. Within the MOMA instrument suite, the hardware surfaces of the sample path must be cleaned to a level of 0.03 spore/sq m. To meet this requirement, a process called Dry Heat Microbial Reduction (DHMR) is used to decrease the number of viable spores by 4 orders of magnitude. Before DHMR, the hardware is handled using standard cleanroom practices, while after DHMR, all sample path surfaces must be handled aseptically when exposed. Aseptic handling of the sample path involves a number of strategies and protocols including working only in an aseptic ISO class 5 work space, limiting the amount of time of exposure, using sterile garmenting with sterile gloves, and using sterile tools. Before work begins, the aseptic workspace will be tested for bioburden and particle fallout, and all tools that will contact sample path surfaces must be sterilized. During the exposure activity, sterile garments will be worn, sterile tools will be handled in a 2 person set up so that the operator touches only the sterile tool and not the exterior surfaces of the sterile pouch, and the environment will be monitored with active and passive fallout for bioburden and particle levels. Any breach in the planetary protection cleanliness can necessitate repeating DHMR, which not only has significant cost and schedule implications, it also become a risk to hardware that is not rated for repeated long exposures to high temperatures

Establishing and Monitoring an Aseptic Workspace for Building the MOMA Mass Spectrometer

Mars Organic Molecule Analyzer (MOMA) is an instrument suite on the ESA ExoMars 2018 Rover, and the Mass Spectrometer (MOMA-MS) is being built at Goddard Space Flight Center (GSFC). As MOMA-MS is a life-detection instrument and it thus falls in the most stringent category of Planetary Protection (PP) biological cleanliness requirements. Less than 0.03 sporem2 is allowed in the instrument sample path. In order to meet these PP requirements, MOMA-MS must be built and maintained in a low bioburden environment. The MOMA-MS project at GSFC maintains three cleanrooms with varying levels of bioburden control. The Aseptic Assembly Cleanroom has the highest level of control, applying three different bioburden reducing methods: 70 IPA, 7.5 Hydrogen Peroxide, and Ultra-Violet C light. The three methods are used in rotation and each kills microbes by a different mechanism, reducing the likelihood of microorganisms developing resistance to all three. The Integration and Mars Chamber Cleanrooms use less biocidal cleaning, with the option to deploy extra techniques as necessary. To support the monitoring of cleanrooms and verification that MOMA-MS hardware meets PP requirements, a new Planetary Protection lab was established that currently has the capabilities of standard growth assays for spore or vegetative bacteria, rapid bioburden analysis that detects Adenosine Triphosphate (ATP), plus autoclave and DHMR verification. The cleanrooms are monitored both for vegetative microorganisms and by rapid ATP assay, and a clear difference in bioburden is observed between the aseptic the other cleanroom

Aseptic Operations for Post DHMR Processing of MOMA Mass Spectrometer

Mars Organic Molecule Analyzer - Mass Spectrometer (MOMA-MS) is an instrument in the MOMA instrument suite for the European Space Agency (ESA) ExoMars 2020 Rover. The rover is Planetary Protection Mission Category IVb, the first IVb mission since the Viking missions in the 1970s. Within the sample path of the MOMA instrument suite, the hardware surfaces of the must be sanitized to a level of less than 0.03 spore/m sq. To meet this requirement, the MS sample path was subjected to Dry Heat Microbial Reduction (DHMR) to decrease the number of viable spores by 4 orders of magnitude from a measured 88 spores/m sq to 0.009 spores/m sq. Before DHMR, the hardware is handled using standard cleanroom practices. After DHMR, planetary protection filters protect the sample path for most of integration, but when sample path exposure is required, aseptic operations are instituted and exposure times are kept to an absolute minimum. The surface area of exposure is also taken into account to determine safe exposure times. Before work begins, the ISO class 5 aseptic workspace is cleaned and tested for surface and airborne bioburden, and all tools that will contact or be used near sample path surfaces are sterilized. During the exposure activity, sterile garments are worn, sterile gloves are changed as often as necessary, and the environment is monitored with active and passive fallout for bioburden and real time airborne particle counts. Sterile tools are handled by a two person team so that the operator touches only the tool and not the exterior surfaces of the sterilization pouch, and a sterile operating field is established as a safe place to organize tools or parts during the aseptic operations. In cases where aseptic operations are not feasible, localized DHMR is used after exposure. Any breach in the planetary protection cleanliness can necessitate repeating instrument level DHMR, which not only has significant cost and schedule implications, it also become a risk to hardware that is not rated for repeated long exposures to high temperatures

Developing the Cleanliness Requirements for an Organic-detection Instrument MOMA-MS

The cleanliness requirements for an organic-detection instrument, like the Mars Organic Molecule Analyzer Mass Spectrometer (MOMA-MS), on a Planetary Protection Class IVb mission can be extremely stringent. These include surface molecular and particulate, outgassing, and bioburden. The prime contractor for the European Space Agencys ExoMars 2018 project, Thales Alenia Space Italy, provided requirements based on a standard, conservative approach of defining limits which yielded levels that are unverifiable by standard cleanliness verification methods. Additionally, the conservative method for determining contamination surface area uses underestimation while conservative bioburden surface area relies on overestimation, which results in inconsistencies for the normalized reporting. This presentation will provide a survey of the challenge to define requirements that can be reasonably verified and still remain appropriate to the core science of the ExoMars mission

Development and Implementation of Aseptic Operations for the MOMA-Mass Spectrometer

The ExoMars 2020 Rover is a life detection mission, and is classified as Planetary Protection (PP) Mission Category IVb, the first IVb mission since the Viking missions. Mars Organic Molecule Analyzer Mass Spectrometer (MOMA-MS) is a life detection instrument for the rover. To meet the stringent bioburden requirement of 0.03 spore/m2, the MS is subjected to Dry Heat Microbial Reduction (DHMR) to decrease the bioburden from a measured 88 spores/m2 to 0.009 spores/m2. After DHMR, exposure of the sample path must be kept to an absolute minimum and requires aseptic operations. Aseptic operations include determining the safe exposure time based on the surface area of exposure and particle fallout expected in the aseptic ISO class 5 workspace, preparing an aseptic ISO class 5 workspace, and using sterile garments and tools. During the exposure activity the environment is monitored with active and passive fallout for bioburden and real time airborne particle counts. Sterile tools are handled by a two person team so the operator touches only the tool and not the exterior surfaces of the sterilization pouch, and a sterile operating field is established as a safe place to organize tools or parts during the aseptic operations. In cases where aseptic operations are not feasible, localized DHMR is used after exposure. Any breach in the PP cleanliness can necessitate repeating instrument level DHMR, which not only has significant cost and schedule implications, but also is a risk to hardware that is not rated for repeated long exposures to high temperatures

Measles Virus Infection of Primary Respiratory Epithelial Cells Derived from Rhesus Macaques

Measles remains a leading vaccine-preventable cause of child mortality globally. Although a live-attenuated vaccine against measles virus (MV) is available, measles has been difficult to control. MV is a respiratory infection typically spread by aerosol droplets which target respiratory epithelial cells as initial sites of viral entry and replication. Primary tracheal and nasal epithelial cells (rmTECs/NECs) derived from rhesus macaques serve as an ideal system to study MV infection in the respiratory tract, because: 1) rmTECs/NECs are polarized and differentiated to mimic respiratory epithelium in vivo and 2) rhesus macaques are the only susceptible host to MV infection other than humans. We have optimized a method for culturing well-differentiated polarized rmTECs/NECs and shown that both WT and vaccine strains of MV successfully infect cells from both apical and basolateral surfaces. Though no significant difference in viral infection was observed with an increased duration of infection, viral titers maintained high. Evidence of infection was characterized by observations of changes in cell morphology and titering of infectious virus in the supernatant. A working in vitro model of the respiratory system is important in bringing greater appreciation and understanding for the development of a respiratory vaccine against measles

Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are Critical for Production of Infectious Virus Particles▿

The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly

Measles Virus Infection of Primary Respiratory Epithelial Cells Derived from Rhesus Macaques

Measles remains a leading vaccine-preventable cause of child mortality globally. Although a live-attenuated vaccine against measles virus (MV) is available, measles has been difficult to control. MV is a respiratory infection typically spread by aerosol droplets which target respiratory epithelial cells as initial sites of viral entry and replication. Primary tracheal and nasal epithelial cells (rmTECs/NECs) derived from rhesus macaques serve as an ideal system to study MV infection in the respiratory tract, because: 1) rmTECs/NECs are polarized and differentiated to mimic respiratory epithelium in vivo and 2) rhesus macaques are the only susceptible host to MV infection other than humans. We have optimized a method for culturing well-differentiated polarized rmTECs/NECs and shown that both WT and vaccine strains of MV successfully infect cells from both apical and basolateral surfaces. Though no significant difference in viral infection was observed with an increased duration of infection, viral titers maintained high. Evidence of infection was characterized by observations of changes in cell morphology and titering of infectious virus in the supernatant. A working in vitro model of the respiratory system is important in bringing greater appreciation and understanding for the development of a respiratory vaccine against measles