Design and validation of a GMP stem cell manufacturing protocol for MPSII hematopoietic stem cell gene therapy.

Hematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy for the treatment of neurodegenerative, metabolic disorders. The approach involves the ex vivo introduction of a missing gene into patients' own stem cells via lentiviral-mediated transduction (TD). Once transplanted back into a fully conditioned patient, these genetically modified HSCs can repopulate the blood system and produce the functional protein, previously absent or non-functional in the patient, which can then cross-correct other affected cells in somatic organs and the central nervous system. We previously developed an HSCGT approach for the treatment of Mucopolysaccharidosis type II (MPSII) (Hunter syndrome), a debilitating pediatric lysosomal disorder caused by mutations in the iduronate-2-sulphatase (IDS) gene, leading to the accumulation of heparan and dermatan sulfate, which causes severe neurodegeneration, skeletal abnormalities, and cardiorespiratory disease. In HSCGT proof-of-concept studies using lentiviral IDS fused to a brain-targeting peptide ApoEII (IDS.ApoEII), we were able to normalize brain pathology and behavior of MPSII mice. Here we present an optimized and validated good manufacturing practice hematopoietic stem cell TD protocol for MPSII in preparation for first-in-man studies. Inclusion of TEs LentiBOOST and protamine sulfate significantly improved TD efficiency by at least 3-fold without causing adverse toxicity, thereby reducing vector quantity required.

Design and Construction of a Radiochemistry Laboratory and cGMP-Compliant Radiopharmacy Facility.

The establishment of a compliant radiopharmacy facility within a university setting is crucial for supporting fundamental and preclinical studies, as well as for the production of high-quality radiopharmaceuticals for clinical testing in human protocols as part of Investigational New Drug (IND) applications that are reviewed and approved by the U.S. Food and Drug Administration (FDA). This manuscript details the design and construction of a 550 ft2 facility, which included a radiopharmacy and a radiochemistry laboratory, to support radiopharmaceutical development research and facilitate translational research projects. The facility was designed to meet FDA guidelines for the production of aseptic radiopharmaceuticals in accordance with current good manufacturing practice (cGMP). A modular hard-panel cleanroom was constructed to meet manufacturing classifications set by the International Organization of Standardization (ISO), complete with a gowning room and an anteroom. Two lead-shielded hot cells and two dual-mini hot cells, connected via underground trenches containing shielded conduits, were installed to optimize radioactive material transfer while minimizing personnel radiation exposure. Concrete blocks and lead bricks provided sufficient and cost-effective radiation shielding for the trenches. Air quality was controlled using pre-filters and high-efficiency particulate air (HEPA) filters to meet cleanroom ISO7 (Class 10,000) standards. A laminar-flow biosafety cabinet was installed in the cleanroom for preparation of sterile dose vials. Noteworthy was a laminar-flow insert in the hot cell that provided a shielded laminar-flow sterile environment meeting ISO5 (class 100) standards. The design included the constant control and monitoring of differential air pressures across the cleanroom, anteroom, gowning room, and controlled research space, as well as maintenance of temperature and humidity. The facility was equipped with state-of-the-art equipment for quality control and release testing of radiopharmaceuticals. Administrative controls and standard operating procedures (SOPs) were established to ensure compliance with manufacturing standards and regulatory requirements. Overall, the design and construction of this radiopharmacy facility exemplified a commitment to advancing fundamental, translational, and clinical applications of radiopharmaceutical research within an academic environment.

Environmental monitoring of current good manufacturing practices cleanroom facilities for manufacturing of cellular therapy products in an academic hospital setting.

As the field of cell and gene therapy (CGT) continues to grow, so too must the infrastructure and regulatory guidance supporting the manufacture of these potentially life-saving products-especially early-phase products manufactured at an increasing number of academic or hospital-based facilities providing decentralized (or point of care) manufacturing. An important component of current good manufacturing practices, including those regulating cell and gene therapies, is the establishment of an effective environmental monitoring (EM) program. While several guidelines for establishing an EM program are available, these guidelines do not specifically address the unique aspects of manufacturing CGT products and they do not provide real-world evidence demonstrating the effectiveness of the program. Here, we describe the establishment and evolution of an EM program in a cell therapy manufacturing facility at an academic hospital. With 10 years of EM data, we analyze the effectiveness for identifying trends in environmental conditions and highlight important findings, with the aim of providing practical evidence and guidance for the development of future early-phase EM programs.

Whole-genome sequencing of Paenibacillus phoenicis isolated from the Phoenix Mars Lander spacecraft assembly facility.

The genome of Paenibacillus phoenicis, a spore-forming bacterium isolated from the spacecraft assembly facility of the Phoenix mission, was generated via hybrid assembly by merging short and long reads. Examining this genome may shed light on strategies to minimize the risk of contaminating extraterrestrial environments with Earth-based microorganisms.

Development and Implementation of an Ultraviolet-Dye-Based Qualification Procedure for Hand Washing and Disinfection to Improve Quality Assurance of Pharmacy Preparations and Compounding, Especially in Cleanrooms: A Pilot Study.

Even though, nowadays, most medicines are manufactured industrially, patients may have medical needs that can only be met by a tailor-made approach. This requires the availability of pharmacy preparations made under Good Manufacturing Practice (GMP) conditions. An efficient hand hygiene practice is essential herewith, especially if sterile products that are prepared in a cleanroom are concerned. The effectiveness of hand washing and hand disinfection procedures greatly relies on adequate training. We carried out an observational cross-sectional pilot study aimed at optimizing hand hygiene training with objective and measurable quality assessments using an ultraviolet (UV) dye. Practical acceptance criteria for qualifying personnel through this method were set and evaluated. In total, 25 GMP-qualified cleanroom operators washed and disinfected their hands with UV dye hand wash lotion and UV dye hand alcohol, respectively. To obtain a proof-of-concept, the results were judged based on adherence to the WHO six-step protocol and associated acceptance criteria. Commonly missed areas were brought to light, and the influence of procedure duration was investigated. UV-dye-based assessments appeared to be more valuable in hand disinfection than in hand washing. In both procedures, the back of the hands and the thumbs were frequently missed. This underpins the need for enhanced and repeated education on hand washing and disinfection. Additionally, a dry skin gave rise to extra cleaning challenges. From this pharmacy practice pilot study with a focus on pharmaceutical product care, it may be concluded that the application of UV-dye-based assessments offers valuable insights for pharmacists to optimize hand hygiene, thereby increasing the safety of tailor-made medicines and on-site preparations.

Culturable microbiological profile of a non-sterile drugs pharmaceutical production environment

Abstract For places where non-sterile drug production occurs, regulatory bodies recommend monitoring of the environmental bioburden. This procedure provides information regarding possible microbiological risks to which the products may be exposed, so that subsequent action measures may be implemented. The aim of the present work was to quantify and characterize the microorganisms present in Grade D (ISO 8) cleanrooms of a Brazilian pharmaceutical industry, identifying any possible seasonal climatic influences on these environments. Sampling was performed by surface and air monitoring, over 12 months during the year 2019, in rooms that were in operation. For both sampling methods, no statistically significant differences in bacteria and fungi counts were found between months or seasonal periods. Microorganisms that presented higher incidence included Staphylococcus epidermidis (15%) and Micrococcus spp. (13%), common to the human microbiota, and the fungi Cladosporium sp. (23%) and Penicillium sp. (21%), typical of the external environment. The results showed that microbial contamination in the Grade D cleanrooms was within the permissible maximum levels and remained similar throughout the year. Microbiological quality control in the clean areas of the pharmaceutical industry investigated was considered effective, with regular maintenance being necessary to keep bioburden levels controlled.

Quality management overview for the production of a tissue-engineered human skin substitute in Malaysia.

Treatments for skin injuries have recently advanced tremendously. Such treatments include allogeneic and xenogeneic transplants and skin substitutes such as tissue-engineered skin, cultured cells, and stem cells. The aim of this paper is to discuss the general overview of the quality assurance and quality control implemented in the manufacturing of cell and tissue product, with emphasis on our experience in the manufacturing of MyDerm®, an autologous bilayered human skin substitute. Manufacturing MyDerm® requires multiple high-risk open manipulation steps, such as tissue processing, cell culture expansion, and skin construct formation. To ensure the safety and efficacy of this product, the good manufacturing practice (GMP) facility should establish a well-designed quality assurance and quality control (QA/QC) programme. Standard operating procedures (SOP) should be implemented to ensure that the manufacturing process is consistent and performed in a controlled manner. All starting materials, including tissue samples, culture media, reagents, and consumables must be verified and tested to confirm their safety, potency, and sterility. The final products should also undergo a QC testing series to guarantee product safety, efficacy, and overall quality. The aseptic techniques of cleanroom operators and the environmental conditions of the facility are also important, as they directly influence the manufacturing of good-quality products. Hence, personnel training and environmental monitoring are necessary to maintain GMP compliance. Furthermore, risk management implementation is another important aspect of QA/QC, as it is used to identify and determine the risk level and to perform risk assessments when necessary. Moreover, procedures for non-conformance reporting should be established to identify, investigate, and correct deviations that occur during manufacturing. This paper provides insight and an overview of the QA/QC aspect during MyDerm® manufacturing in a GMP-compliant facility in the Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia.

Establishment of a Single Temperature Incubation Approach for Environmental Monitoring Samples with Focus on Mold Recoveries.

A robust environmental monitoring program is essential to properly estimate and identify microorganisms in cleanrooms, ensuring that microbial contamination remains acceptably low, and a good state of control is maintained in the manufacturing areas. The incubation conditions are important to support optimal microbial recoveries, considering that there is no single culture medium, temperature and incubation time that can recover all microorganisms. Particularly molds are quite sensitive microorganisms, and some species may have very specific nutritional and environmental needs. In this study, a two-phase approach was used to identify a single incubation-temperature approach that could recover most of the cleanroom microbial flora with focus on molds. Phase 1 included a growth promotion study performed in the laboratory using pharmacopeial and in-house strains, comparing different media (SDA and TSA) at single or dual incubation-temperature approaches for 5 or 6 days. Phase 2 was based on an in situ study where sampling was performed in different areas of a pharmaceutical facility, comparing the recoveries at different incubation conditions. In addition, extension studies of phase 1 and phase 2 were performed to get a better understanding of growth requirements for in-house molds. The results show that an incubation on Tryptone Soy Agar (TSA) at 25-30° for 3-4 days was able to recover most tested microorganisms in phase 1 and a large variety of microorganisms in phase 2, indicating that the single incubation-temperature is an optimal approach for the recovery of microorganisms in cleanrooms. Exceptions were noted for one strain of the species Cutibacterium acnes, a microaerophilic bacterium for which anaerobiosis and higher temperatures were needed, and two mold strains (Sistotrema brinkmannii and Stereum hirsutum), indicating that those molds required a specific media (Sabouraud Dextrose Agar, SDA) for their proliferation. The results showed that TSA incubated at single or dual incubation-temperature approach cannot compensate for the absence of SDA for some environmental molds that may be atypical in cleanrooms. Therefore, in addition to TSA, certain monitoring with SDA at e.g., cleanroom entrance points may be beneficial to recover molds with very specific nutritional requirements.

Identification and Archive of Mars 2020 Spacecraft Microbial Isolates.

To support NASA's Mars 2020 mission, bioassays were performed to ensure the biological cleanliness of the spacecraft, instruments, and hardware assembly areas. Bioassays began in May 2014, as the first components were assembled, and continued until their launch in July 2020. Over this 6-year period, 1811 bioassay sampling sessions were conducted. To understand the nature of microbiological presence on and around the spacecraft, an archive of organisms resulting from the bioassays was assembled. This archive included 4232 microbial specimens preserved as frozen stocks. To date, more than 3489 microbial isolates have been tested by MALDI-TOF mass spectrometry analysis. Identifications were based on high confidence level matches to known microorganisms in the reference spectra database where 39 distinct genera were identified. Gram-positive bacteria were isolated almost exclusively. Most, but not all, were spore-forming genera. The most prevalent genera isolated in order of frequency were Bacillus, Priestia, Paenibacillus, Staphylococcus, Micrococcus, and Streptomyces. Within the largely represented Bacillus-like genera, the five most prevalent species were cereus, licheniformis, horneckiae, subtilis, and safensis.

Characteristics of bacterial community and ARG profiles in the surface and air environments in a spacecraft assembly cleanroom.

Understanding the microbial communities and antibiotic resistance genes (ARGs) in spacecraft assembly cleanrooms is crucial for spacecraft microbial control and astronaut safety. However, there have been few reports of ARG profiles and their relationship with microbiomes in such environments. In the present study, we assessed the bacterial community and ARGs in the air dust and surface environments of a typical spacecraft assembly cleanroom. Our results show a significant difference in bacterial composition between surfaces and air dust, as they belong to two distinct ecostates. Bacillus and Acinetobacter were significantly enriched in the air samples. Bacterial community network analysis revealed lower topological parameters and robustness of bacterial networks in the air samples. We also observed different distribution patterns of some typical ARGs between surface and air dust samples. Notably, the ermB gene exhibited a relatively high copy number and was enriched in the surface environment, compared to that in the air. Overall, our study provides insight into the complex microbial community and the distribution and transfer of ARGs in spacecraft assembly cleanrooms, and offers important input for developing control strategies against ARGs.

Development of a microfluidic electroosmosis pump on a chip for steady and continuous fluid delivery.

Infusion therapy is the most common form of therapy used in health care. However, the existing infusion devices show higher flow discrepancies as flow rates decrease to a few nL min-1. As a result, dosing errors can contribute to the morbidity and mortality of patients. In the scope of project 18HLT08 MeDD II - Metrology for drug delivery, this investigation aims at the development of a silicon microchip flow pump capable of steadily and continuously dispense very low flow rates of a few nL min-1. The fabrication methodologies explored here use a combination of typical cleanroom micro/nanofabrication techniques and off-the-shelf equipment. Preliminary tests show flow rates as low as 45 nL min-1 can be obtained in this microfluidic electroosmotic pump. The experimental flow rates are in good agreement with results predicted by multiphysics simulation, with less than 8% deviation ratio. This cost effective electroosmotic micropump has the potential to act as a steady and continuous drug delivery system to neonatal patients as well as to organs on chip (OoC), determining the stability of the shear stress imposed on the cells or the right cell culture medium conditions.

Performance Challenges in a Newly Established Clean Room at Omid Hospital, Isfahan, Iran: A Descriptive Study.

Objective: This study aimed to comprehensively assess the challenges faced by a newly established clean room in the oncology center of Omid Hospital, Isfahan, Iran, one of the first of its kind in the country. The research also sought to identify the underlying causes of these challenges and propose potential solutions to address them. Methods: A 6-month cross-sectional study was conducted from December 2021 to May 2022. International guidelines such as British Columbia Cancer Agencies' guideline of hazardous drugs, the National Institute for Occupational Safety and Health guideline for working with hazardous drugs, and United States pharmacopeia related to cleanroom performance were studied, translated, and summarized into a checklist. The staff performance in Omid Hospital's clean room was compared to the data collection form, and all medication errors were documented and analyzed. The study also explained the underlying causes of these challenges and proposed potential solutions. Findings: Among 1005 chemotherapy regimens, 836 errors were detected, stemming from issues such as engineering and construction challenges, lack of human resources and essential equipment, and budgetary constraints. Conclusion: Despite the involvement of a trained oncology clinical pharmacist, Omid Hospital's cleanroom faces significant challenges within the medical and hospital system, leading to non-standard challenges. The study recommends multidisciplinary approaches in the hospital to mitigate these challenges and improve cleanroom performance.

Airborne Toluene Detection Using Metal-Organic Frameworks.

The pollution of indoor air is a major worldwide concern in our modern society for people's comfort, health, and safety. In particular, toluene, present in many substances including paints, thinners, candles, leathers, cosmetics, inks, and glues, affects the human health even at very low concentrations throughout its action on the central nervous system. Its prevalence in many workplace environments can fluctuate considerably, which led to firm regulation with exposure limits varying between 50 and 400 ppm depending on exposure time. This therefore requires the development of technologies for an accurate detection of this contaminant. Metal-organic frameworks have been proposed as promising candidates to detect and monitor a series of molecules at even extremely low concentrations owing to the high tunability of their functionality. Herein, a high-throughput Monte Carlo screening approach was devised to identify the best MOFs from the computation-ready, experimental (CoRE) metal-organic framework (MOF) density-derived electrostatic and chemical (DDEC) database for the selective capture of toluene from air at room temperature, with the consideration of a ternary mixture composed of extremely low-level concentration of toluene (10 ppm) in oxygen and nitrogen to mimic the composition of air. An aluminum MOF, DUT-4, with channel-like micropores was identified as an excellent candidate for the selective adsorption of toluene from air with a predicted adsorption uptake of 0.5 g/g at 10 ppm concentration and room temperature. The toluene adsorption behavior of DUT-4 at low equivalent concentrations, alongside its sensing performance, was further experimentally investigated by its incorporation in a quartz crystal microbalance sensor, confirming the promises of DUT-4. Decisively, the resulting high sensitivity and fast kinetics of our developed sensor highlight the applicability of this hand-in-hand computational-experimental methodology to porous material screening for sensing applications.

Recapitulating Tumor Hypoxia in a Cleanroom-Free, Liquid-Pinning-Based Microfluidic Tumor Model.

In tumors, the metabolic demand of cancer cells often outpaces oxygen supply, resulting in a gradient of tumor hypoxia accompanied with heterogeneous resistance to cancer therapeutics. Models recapitulating tumor hypoxia are therefore essential for developing more effective cancer therapeutics. Existing in vitro models often fail to capture the spatial heterogeneity of tumor hypoxia or involve high-cost, complex fabrication/handling techniques. Here, we designed a highly tunable microfluidic device that induces hypoxia through natural cell metabolism and oxygen diffusion barriers. We adopted a cleanroom-free, micromilling-replica-molding strategy and a microfluidic liquid-pinning approach to streamline the fabrication and tumor model establishment. We also implemented a thin-film oxygen diffusion barrier design, which was optimized through COMSOL simulation, to support both two-dimensional (2-D) and three-dimensional (3-D) hypoxic models. We demonstrated that liquid-pinning enables an easy, injection-based micropatterning of cancer cells of a wide range of parameters, showing the high tunability of our design. Human breast cancer and prostate cancer cells were seeded and stained after 24 h of 2-D and 3-D culture to validate the natural induction of hypoxia. We further demonstrated the feasibility of the parallel microfluidic channel design to evaluate dual therapeutic conditions in the same device. Overall, our new microfluidic tumor model serves as a user-friendly, cost-effective, and highly scalable platform that provides spatiotemporal analysis of the hypoxic tumor microenvironments suitable for high-content biological studies and therapeutic discoveries.

Outdoor environmental effects on cleanrooms - A study from a Swedish hospital pharmacy compounding unit.

In this study we examined how outdoor climate affects indoor conditions of a cleanroom used for the preparation of radiopharmaceuticals in the Uppsala university hospital pharmacy, Sweden. Further objectives were to identify associated risk factors to ensure a consistent extemporaneous manufacturing process. Data for two years from the facility monitoring system (with one minute resolution for temperature, relative humidity (%RH), differential pressure) were compared with meteorological outdoor data from Uppsala (Swedish Meteorological and Hydrological Institute, 60-minute mean data for temperature, relative humidity, wind speed and air pressure). The findings of this study indicate a linear relationship between indoor and outdoor temperature for the autumn, winter and spring seasons. The typical summer outdoor diurnal pattern is also seen for indoor temperature. During the study period, the minimum outdoor temperature was -17.5 °C and the maximum 31.4 °C. This wide temperature range also entails a wide range of air humidity from 10 %RH to 100 %RH indoors. Cleanroom temperature and %RH are factors that may affect the quality of medications, especially the risk of microbiological growth in aseptic processes, stability of medications during storage but also may affect handling of for example uncoated tablets or weighing of powder, especially at high %RH for hygroscopic drugs or at low %RH due to static electricity. Further the risk of damage on electrical equipment from electrostatic discharge at low %RH is discussed with a focus on the need for humidity control of cleanrooms and/or systems for mitigation of electrostatic discharge in climates with outdoor temperature in the wintertime below freezing point.

The Isolation and Characterization of Rare Mycobiome Associated With Spacecraft Assembly Cleanrooms.

Ensuring biological cleanliness while assembling and launching spacecraft is critical for robotic exploration of the solar system. To date, when preventing forward contamination of other celestial bodies, NASA Planetary Protection policies have focused on endospore-forming bacteria while fungi were neglected. In this study, for the first time the mycobiome of two spacecraft assembly facilities at Jet Propulsion Laboratory (JPL) and Kennedy Space Center (KSC) was assessed using both cultivation and sequencing techniques. To facilitate enumeration of viable fungal populations and downstream molecular analyses, collected samples were first treated with chloramphenicol for 24 h and then with propidium monoazide (PMA). Among cultivable fungi, 28 distinct species were observed, 16 at JPL and 16 at KSC facilities, while 13 isolates were potentially novel species. Only four isolated species Aureobasidium melanogenum, Penicillium fuscoglaucum, Penicillium decumbens, and Zalaria obscura were present in both cleanroom facilities, which suggests that mycobiomes differ significantly between distant locations. To better visualize the biogeography of all isolated strains the network analysis was undertaken and confirmed higher abundance of Malassezia globosa and Cyberlindnera jadinii. When amplicon sequencing was performed, JPL-SAF and KSC-PHSF showed differing mycobiomes. Metagenomic fungal reads were dominated by Ascomycota (91%) and Basidiomycota (7.15%). Similar to amplicon sequencing, the number of fungal reads changed following antibiotic treatment in both cleanrooms; however, the opposite trends were observed. Alas, treatment with the antibiotic did not allow for definitive ascribing changes observed in fungal populations between treated and untreated samples in both cleanrooms. Rather, these substantial differences in fungal abundance might be attributed to several factors, including the geographical location, climate and the in-house cleaning procedures used to maintain the cleanrooms. This study is a first step in characterizing cultivable and viable fungal populations in cleanrooms to assess fungal potential as biocontaminants during interplanetary explorations. The outcomes of this and future studies could be implemented in other cleanrooms that require to reduce microbial burden, like intensive care units, operating rooms, or cleanrooms in the semiconducting and pharmaceutical industries.

National Economic and Environmental Benefit of Reusable Textiles in Cleanroom Industry.

In cleanroom facilities, both disposable and reusable textile garments (coveralls, boots, hoods, and frocks) meet the particulate standards from the most rigorous to the most basic levels. However, the reusables clearly offer two other important benefits, lower annual cost and lower environmental impact. The objectives of this article are to now provide quantitative reusable product benefits on a U.S. national environmental and economic basis. This is the first quantitative, novel multi-user economic evaluation of selecting cleanroom reusables over disposables. For personal protection equipment (PPE), these cost and environmental benefits indicate there is also an improved environmental and economic aspect to the increased national demand for reusables related to coronavirus disease 2019 (COVID-19), while necessary cleaning with approved detergents is easily achieved. The current reusable cleanroom market (14.1 million packages) was estimated to be 60% nonsterile and 40% sterilized. The total market is about 50% reusable and 50% disposable. This research documents that there is an annual cost reduction of about 58% when selecting reusables over disposables, giving an economic savings to the U.S. cleanroom sector from reusables of about $1.2 billion in the next decade. This is also saving the total U.S. about 136 million MJ natural resource energy/year (38 million kWh) and about 8.4 million kg CO2eq annually (removal of about 1,650 cars/year). A maximum hypothetical case for reusables at 87.5% of the market (12.5% are mandatory Hazmat disposable) would yield a U.S. national savings of nearly $2.1 billion/decade to the cleanroom sector bottom line, as well as 2.4 billion MJ nre savings in energy or removal of about 29,000 cars/decade. These results indicate there are effective, verifiable, and easily obtained environmental and economic benefits by the basic transition by diverse cleanrooms in deciding to select reusable garments.

Effects of different air change rates on cleanroom 'in operation' status.

The objective of this experimental study is to analyze non-viable and viable particle loads in a pharmaceutical cleanroom under 'in operation' conditions using different air change rates (ACRs). Regulatory guidelines give limit values for particles/m3 and colony forming units (CFUs)/m3. A widely used ACR is 20 h-1 as this value is recommended by the Food and Drug Administration (FDA) in its guidance for industry on sterile drug products. However, this value may be too high, resulting in increased costs for energy. A typical pharmaceutical cleanroom was used for this study, and operations were simulated with a process unit and two operators in the room. The experiments were conducted twice with four different ACRs and four different types of operator garments, resulting in 32 trials in total. Particle load and CFUs were measured by calibrated particle counters and microbial air samplers. The results give evidence that an ACR of 20 h-1 is not required. ACR 10 h-1 is sufficient without compromising the demanded air quality. Furthermore, it was found that regulatory agencies should reevaluate the expected limits as these currently give a high buffer between the required and actual values, which potentially cover up problems in aseptic manufacturing.

Pressure Optimized PowEred Respirator (PROPER): A miniaturized wearable cleanroom and biosafety system for aerially transmitted viral infections such as COVID-19.

The supply of Personal Protective Equipment (PPE) in hospitals to keep the Health Care Professionals (HCP) safe taking care of patients may be limited, especially during the outbreak of a new disease. In particular, the face and body protective equipment is critical to prevent the wearer from exposure to pathogenic biological airborne particulates. This situation has been now observed worldwide during the onset of the COVID-19 pandemic. As concern over shortages of PPE at hospitals grows, we share with the public and makers' community the Pressure Optimized PowEred Respirator (PROPER) equipment, made out of COTS components. It is functionally equivalent to a Powered Air Purifying Respirator (PAPR). PROPER, a hood-based system which uses open source and easily accessible components is low-cost, relatively passive in terms of energy consumption and mechanisms, and easy and fast to 3D print, build and assemble. We have adapted our experience on building clean room environments and qualifying the bioburden of space instruments to this solution, which is in essence a miniaturized, personal, wearable cleanroom. PROPER would be able to offer better protection than an N95 respirator mask, mainly because it is insensitive to seal fit and it shields the eyes as well. The PROPER SMS fabric is designed for single-use and not intended for reuse, as they may start to tear and fail but the rest of the parts can be disinfected and reused. We provide a set of guidelines to build a low-cost 3D printed solution for an effective PAPR system and describe the procedures to validate it to comply with the biosafety level 3 requirements. We have validated the prototype of PROPER unit for air flow, ISO class cleanliness level, oxygen and carbon-dioxide gas concentrations during exhalation, and present here these results for illustration. We demonstrate that the area inside the hood is more than 200 times cleaner than the external ambient without the operator and more than 175 times with the operator and in an aerosol exposed environment. We also include the procedure to clean and disinfect the equipment for reuse. PROPER may be a useful addition to provide protection to HCPs against the SARS-CoV-2 virus or other potential future viral diseases that are transmitted aerially.

Description of Chloramphenicol Resistant Kineococcus rubinsiae sp. nov. Isolated From a Spacecraft Assembly Facility.

A Gram-positive, coccoid, motile, aerobic bacterium, designated strain B12T was isolated from a Jet Propulsion Laboratory spacecraft assembly cleanroom, Pasadena, CA, United States. Strain B12T was resistant to chloramphenicol (100 µg/mL), and is a relatively slow grower (3-5 days optimal). Strain B12T was found to grow optimally at 28 to 32°C, pH 7 to 8, and 0.5% NaCl. Fatty acid methyl ester analysis showed that the major fatty acid of the strain B12T was anteiso C15 : 0 (66.3%), which is also produced by other Kineococcus species. However, arachidonic acid (C20 : 4 ω6,9,12,16c) was present in strain B12T and Kineococcus glutinatus YIM 75677T but absent in all other Kineococcus species. 16S rRNA analysis revealed that strain B12T was 97.9% similar to Kineococcus radiotolerans and falls within the Kineococcus clade. Low 16S rRNA gene sequence similarities (<94%) with other genera in the family Kineosporiaceae, including Angustibacter (93%), Kineosporia (94% to 95%), Pseudokineococcus (93%), Quadrisphaera (93%), and Thalassiella (94%) demonstrated that the strain B12T does not belong to these genera. Phylogenetic analysis of the gyrB gene show that all known Kineococcus species exhibited <86% sequence similarity with B12T. Multi-locus sequence and whole genome sequence analyses confirmed that B12T clades with other Kineococcus species. Average nucleotide identity of strain B12T were 75-78% with other Kineococcus species, while values ranged from 72-75% with species from other genera within family Kineosporiaceae. Average amino-acid identities were 66-72% with other Kineococcus species, while they ranged from 50-58% with species from other genera. The dDDH comparison of strain B12T genome with members of genera Kineococcus showed 20-22% similarity, again demonstrating that B12T is distantly related to other members of the genus. Furthermore, analysis of whole proteome deduced from WGS places strain B12T in order Kineosporiales, confirming that strain B12T is a novel member of family Kineosporiaceae. Based on these analyses and other genome characteristics, strain B12T is assigned to a novel species within the genus Kineococcus, and the name Kineococcus rubinsiae sp. nov., is proposed. The type strain is B12T (=FJII-L1-CM-PAB2T; NRRL B-65556T, DSM 110506T).