Effect of Nitrogen Gas Plasma Generated by a Fast-Pulsed Power Supply Using a Static Induction Thyristor on Scrapie Prion.

Previous studies show that nitrogen gas plasma generated by a fast-pulsed power supply using a static induction thyristor has both virucidal and bactericidal effects. In this study, nitrogen gas plasma was further evaluated for its potential effects on prions, which are well known to be the most resistant pathogen to both chemical and physical inactivation. Aliquots (10 µL) of mouse brain homogenate infected with Chandler scrapie prion were spotted onto cover glasses and subjected to nitrogen gas plasma. Treated samples were recovered and subjected to further analyses. Control prion samples were prepared in exactly the same way but without plasma treatment. Protein misfolding cyclic amplification (PMCA) showed that nitrogen gas plasma treatment at 1.5 kilo pulse per second for 15 or 30 min caused a reduction in the in vitro propagation level of PrPres (proteinase K-resistant prion protein), which was used as an index of abnormal prion protein (PrPSc). Moreover, mice injected with prion treated with plasma for 30 min showed longer survival than mice injected with control prion, indicating that nitrogen gas plasma treatment decreased prion infectivity. Altogether, these results suggest that nitrogen gas plasma treatment can inactivate scrapie prions by decreasing the propagation activity and infectivity of PrPSc.

Ethylene Oxide Gas Sterilization of Medical Devices.

　Ethylene oxide gas is an agent in the sterilization of medical devices due to its effectiveness and compatibility with most materials. The advantages and disadvantages, as well as its recommended uses, are explored in this review article. The variables and their relevance on process optimization are described, the types of processing cycles are detailed and emphasis is given to the design and validation of the sterilization process.

Methods of Rapid Microbiological Assay and Their Application to Pharmaceutical and Medical Device Fabrication.

　There are several rapid microbiological methods becoming available that have useful applications in pharmaceutical and medical devices. They are ATP bioluminescence, fluorescent labeling, electrical resistance, and nucleic acid probes. In choosing to employ rapid methods, the microbiologist should examine their prospective performances against the specific requirements for that sector. Some methods may require expensive equipment and offer full automation, and others represent only a small investment. The regulatory view of these methods is changing and they still officially have not been approved in medical and pharmaceutical area, but it will still be up to the microbiologist to demonstrate that the method chosen is fit for the purpose intended.

Validation Study and Quality Assurance of Pharmaceutical Water, Waterborne Microorganisms and Endotoxins.

　Water for injection (WFI) and purified water are the most widely used and stringently regulated raw material in pharmaceutical manufacturing. WFI is utilized for a parenteral drug product. Water system is monitored at frequent and routine frequency for demonstrating the overall system control and stability of performance. The critical ports demonstrating systemic control should be monitored more frequently. For reducing the overall risk of microbial contamination or microbial build-up, it is important to develop appropriate alert and action levels. The assignment of alert and action levels should be performance-based, derived from the historic data and well below water specifications. These levels and overall excursion rates should be assessed annually. An action level should not be established at a level equivalent to the specification. Consecutive or multiple alert level excursions and each action level excursion should be comprehensively investigated with appropriate corrective and preventive action. It is important to analyze the efficacy of the corrective and preventive action to reduce the overall excursion rates.

Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. 　For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent. 　The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic has been extensively addressed at conferences and in published documents around the world. More recently, the use of alternative methods for control of the microbiological quality of pharmaceutical products and materials used in pharmaceutical production has been addressed by the compendia in an attempt to facilitate implementation of these technologies by pharmaceutical companies. The author presents some of the rapid method technologies under evaluation or in use by pharmaceutical microbiologists and the current status of the implementation of alternative microbial methods.

Inactivation of Bacterial Spore, Endotoxin, Lipid A, Normal Prion and Abnormal Prion by Exposures to Several Sorts of Gases Plasma.

This review discusses the application of several sorts of non-equilibrium gas plasma discharges for sterilization and disinfection treatments against spores or bioburden on/in the healthcare products or biological indicators. The basic properties of electrical discharges are briefly reviewed and thereafter the paper discusses the interactions of gas plasma with several sorts of biological systems such as bacteria, bacterial spores, endotoxins, lipid A and normal and abnormal prion proteins.

Sterilization Effect of Wet Oxygen Plasma in the Bubbling Method.

A new low-temperature sterilization method to replace the ethylene oxide gas sterilization is needed. Strong bactericidal effects of OH and O2H radicals are well known. The purpose of this study was to evaluate the sterilization effect of wet oxygen ("O2+H2O") plasma in the bubbling method, confirming the effect of humidity. Sterility assurance was confirmed by using a biological indicator (Geobacillus stearothermophilus ATCC7953, Namsa, USA). One hundred and eight samples (10(5) spores/carrier) were divided into three groups of 36 in each for treatment with a different type of gas (O2, O2+H2O, Air+H2O). Plasma processing was conducted using a plasma ashing apparatus (13.56 MHz, PACK-3(®), Y. A. C., Japan) under various gas pressures (13, 25, 50 Pa) and gas flows (50, 100, 200 sccm). Fixed plasma treatment parameters were power at 150 W, temperature of 60 ℃, treatment time of 10 min. The samples after treatment were incubated in trypticase soy broth at 58 ℃ for 72 h. The negative culture rate in the "O2+H2O" group was significantly (Mantel-Haenszel procedure, p<0.001) higher than in the other gas groups. It is suggested that the significant sterilization effect of the "O2+H2O" group depends on the bubbling method which is the method of introducing vapor into the chamber. The bubbling method seems able to generate OH and O2H radicals in a stable way.

Validation Studies for Microbial Contamination and Control of Contaminants.

Microbiological control is a regulatory requirement and one that can be defined as the continued interaction of science and applied technology with products, processes, materials, equipment, and personnel entering the manufacturing areas. In the ISO 14644-1, control of microbial contamination is addressed. A good microbiological control program starts with understanding the risks for microbial contamination in the manufacturing process and identification of possible types of contaminants. The results obtained from such risk assessment can be used in the design of facilities and equipment as well as when establishing protocols regarding equipment and personnel. Once possible sources of contamination have been identified, control and preventative measures can be implemented and qualified/validated.

Formation and elution of toxic compounds from γ-ray-sterilized medical products and the Ames test of eluted components.

No formation of N,N'-methylene dianiline (MDA) was observed in chain-extended thermoplastic polyurethane (PU) when sterilized by autoclaving or γ-ray irradiation. No formation of MDA was observed in nonchain-extended thermoplastic PU when sterilized by γ-ray irradiation. Less than 1 ppm of MDA was produced in nonchain-extended thermoplastic PU subjected to autoclave sterilization. Autoclave sterilization did not produce MDA in thermosetting PU potting material. MDA formation in potting material was promoted by γ-ray irradiation and increased with increasing irradiation doses at a quadratic equation of regression. MDA formation at 100 kGy irradiation amounted to a few ppm and less than one ppm at 25 kGy irradiation: therefore, the potential risk to human recipients was not significant. The elution of compounds other than MDA from potting material was more problematic. Solvent extracts from potting material showed mutagenicity in the absence of metabolic activity (S9Mix). MDA showed mutagenicity in the presence of metabolic activity, therefore MDA was not the major mutagenic candidate. The chemical and biological characteristics of the specific mutagens should be identified in a further study. The lack of MDA formation and a smaller presence of mutagens in autoclave-sterilized potting material indicated that autoclave sterilization was preferable if the material is able to tolerate heating.

Validation study on how to avoid microbial contamination during pharmaceutical production.

Every person involved in pharmaceutical product manufacturing has the responsibility to assure the quality of the product being produced. The aim of this paper is to validate quality assurance throughout the process of manufacturing pharmaceutical products. Additionally, within aseptic manufacturing, certain monitoring and information needs to be collected on a routine basis to continually assess the state of control of the complete operation. The basis for assessing the state of control is to have rigorous and defined information flow processes. Once the information is collected, quality assurance involves the ability to assess, evaluate, and make appropriate decisions to ensure the product has the required safety, identity, strength, quality, and purity. Quality assurance study is the process of bringing all of the information together, evaluating the information, making decisions, refining systems, and applying process knowledge. This process begins in the early stages of drug development when not a lot of specific process information about the process is known, but it is important to allow for development to progress, building knowledge about process. However, even in early development, sterility assurance requirements should be largely the same at all stages of development and routine commercial manufacturing.

Sterilization mechanism of nitrogen gas plasma: induction of secondary structural change in protein.

The mechanism of action on biomolecules of N2 gas plasma, a novel sterilization technique, remains unclear. Here, the effect of N2 gas plasma on protein structure was investigated. BSA, which was used as the model protein, was exposed to N2 gas plasma generated by short-time high voltage pulses from a static induction thyristor power supply. N2 gas plasma-treated BSA at 1.5 kilo pulses per second showed evidence of degradation and modification when assessed by Coomassie brilliant blue staining and ultraviolet spectroscopy at 280 nm. Fourier transform infrared spectroscopy analysis was used to determine the protein's secondary structure. When the amide I region was analyzed in the infrared spectra according to curve fitting and Fourier self-deconvolution, N2 gas plasma-treated BSA showed increased α-helix and decreased ß-turn content. Because heating decreased α-helix and increased ß-sheet content, the structural changes induced by N2 gas plasma-treatment of BSA were not caused by high temperatures. Thus, the present results suggest that conformational changes induced by N2 gas plasma are mediated by mechanisms distinct from heat denaturation.

Confirmation of the sterilization effect using a high concentration of ozone gas for the bio-clean room.

A high-level aseptic environment must be maintained in bio-cleanrooms used for the manufacture of sterile products. In the past, formaldehyde gas was most commonly used to sterilize bio-cleanrooms, but due to strict residual limitations there has been a need to develop a less toxic alternative choice. The authors have developed a revolutionary new sterilization system using a high concentration of ozone gas and used this system to sterilize an actual bio-cleanroom. This system integrates the ozone gas generator with the air conditioning system by proper control. The design specifications for the system included an ozone gas concentration of 200 ppm or more, relative humidity of 80% or more, and a sterilizing time of 120 min. Blow vents and suction ports were placed to ensure a uniform airflow which would extend through the entire room during ozone gas sterilization. Tests regarding long-term material exposure to ozone gas were conducted when the system was introduced to distinguish usable and unusable materials. In an actually constructed cleanroom, simulations were used to predict the evenness of the diffusion of ozone gas concentration and relative humidity during ozone gas sterilization, and measurements of the actual indoor ozone gas concentration, temperature and relative humidity during sterilization revealed that the ozone concentration and relative humidity needed for sterilization had been achieved generally throughout the entire environment. In addition, the CT value (mg/m(3) (=ppm) × min) , derived by multiplying the ozone gas concentration during ozone gas sterilization by the sterilization time, was equal to or greater than the target value of 24 × 10(3) (ppm·min) . When the results of sterilization in a cleanroom were confirmed using a biological indicator (BI) , negative results were obtained at all measurement points, demonstrating that sterilization was being performed effectively in the actual factory at which the ozone gas sterilization system had been introduced.

Dengue virus presence and surveillance in Okinawa (Review).

Recent reports have shown that the dengue virus (DENV) is a serious concern worldwide, especially in subtropical areas such as South-East Asia. With the development of transportation systems, the risk of DENV infection spreading is increasing. Since mosquitoes transmit DENV to humans, surveillance of DENV-infected mosquito vectors is the most effective approach for preventing DENV. Okinawa is the only prefecture located in a subtropical region in Japan and historically shows continuous importation of DENV-related mosquito vectors. In this review, we describe the current and historical status of DENV in Okinawa.

Validation study and routine control monitoring of moist heat sterilization procedures.

The proposed approach to validation of steam sterilization in autoclaves follows the basic life cycle concepts applicable to all validation programs. Understand the function of sterilization process, develop and understand the cycles to carry out the process, and define a suitable test or series of tests to confirm that the function of the process is suitably ensured by the structure provided. Sterilization of product and components and parts that come in direct contact with sterilized product is the most critical of pharmaceutical processes. Consequently, this process requires a most rigorous and detailed approach to validation. An understanding of the process requires a basic understanding of microbial death, the parameters that facilitate that death, the accepted definition of sterility, and the relationship between the definition and sterilization parameters. Autoclaves and support systems need to be designed, installed, and qualified in a manner that ensures their continued reliability. Lastly, the test program must be complete and definitive. In this paper, in addition to validation study, documentation of IQ, OQ and PQ concretely were described.

Validation of sterilization procedures and usage of biological indicators in the manufacture of healthcare products.

Healthcare product manufacturers strive to provide safe, sterile products by validating and controlling manufacturing procedures. Validation study is the result of a multidisciplinary team effort. This successful effort relies on each discipline understanding the fundamentals of each technical disciplines and applying those fundamentals in terms of their own technical background. This paper is to introduce the basic concepts of sterilization methodology which are involved in validating various technical methods of sterilization and biological indicators (BIs). These will mainly address sterilization procedures in general terms and highlight on their microbiological aspects. The proper starting point is defining what sterility is and how it can be achieved through the validation studies by using BI.

Methods of rapid microbiological assay and their application to pharmaceutical and medical device fabrication.

There are several well-developed rapid microbiological methods now becoming available that may have useful applications in pharmaceutical and medical devices. They are ATP bioluminescence, fluorescent labeling, electrical resistance, and nucleic acid probes. In choosing to employ rapid methods, the microbiologist should examine their prospective performances against the specific requirements for that sector. Some methods may require expensive equipment and offer full automation, and others represent only a small investment. The regulatory view of these methods is changing and they still officially have not been approved in medical and pharmaceutical area, but it will still be up to the microbiologist to demonstrate that the method chosen is fit for the purpose intended.

Fundamentals of prions and their inactivation (review).

Prion is an infectious particle composed of an abnormal isoform of the prion protein (PrPSc) and causes prion diseases such as bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) and scrapie. Host cells express cellular prion protein (PrPC), which plays roles in normal functions such as anti-oxidative stress. PrPSc is derived from PrPC and produced by conformational conversion. Prion is notorious as a resistant pathogen, being difficult to inactivate with conventional sterilization procedures. Therefore, to prevent prion-caused iatrogenic diseases, the use of appropriate procedures to inactivate prions is important. For examples, alcohol treatment, autoclave (121˚C, 20 min) and γ-ray irradiation, which are used for disinfection, antisepsis or sterilization of viruses and bacteria, are not effective against prion. This is a fundamental review of prions and methods of their inactivation.

Structure of the prion protein and its gene: an analysis using bioinformatics and computer simulation.

Prion protein (PrP) gene encodes cellular PrP (PrPC), a glycosylphosphatidylinositol (GPI)-anchored cell membrane protein indispensable for infections of prion, which causes Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Although PrPC is known to be converted into an abnormal isoform (PrPSc) upon prion infection and play an important role in prion diseases, the mechanisms involved remain unclear, partly due to the insolubility of PrPSc, which prevents experimental biochemical and biophysical analyses. Recently, with improvements in computer power and methods, computer analyses have been contributing more to prion studies. A comparison of PrP gene sequences revealed mutations and polymorphisms in the open reading frame (ORF) of the human PrP gene related to prion diseases. In contrast, little mutations or polymorphisms related to susceptibility to BSE were found in the ORF of the bovine PrP gene, though relationships between insertion/deletion (Ins/Del) polymorphisms of the PrP gene promoter and susceptibility to BSE have been found. Our results have shown that the specific protein 1 (Sp1) plays important role in the activity of PrP gene promoter, which is influenced by polymorphisms in the Sp1 binding sites. The potential structural dynamics of PrP have been simulated by computational methods such as molecular dynamics (MD) and quantum mechanics (QM). The proposed mechanisms of conversion have revealed new insights in prion diseases. In this review, we will introduce the gene structure, polymorphisms, and potential structural dynamics of PrP revealed by basic and advanced computational analyses. The possible contribution of these methods to elucidation of the pathogenicity of prion diseases and functions of PrPC is discussed.

Gas plasma sterilization of microorganisms and mechanisms of action.

The use of true gas plasmas for the inactivation of microorganisms is an area of dynamic research. Many types of gases are used as a source of plasma, and different plasma production methods have been applied. The antimicrobial mechanisms of oxygen-based gas plasmas may be due to an etching effect on microbial structures, particularly bacterial endospores resulting in shrinkage. By contrast, the definite mechanisms of actions of other gas plasma sources, such as N(2), He, Ne, Ar and Xe gases, have not been clearly defined and indeed may be distinct. The speculated mechanisms of these gas plasmas involve the direct attack of metastable (excited molecular), UV and/or VUV to microbial structures, specifically the inner membrane and DNA in the core of bacterial endospores. According to this speculation, sterilized spore figures would remain unchanged. However, these mechanisms remain to be clarified. Future perspectives on the use of gas plasma for sterilization are of interest, as it is possible that appropriate sterility assurance levels can be obtained in parallel with material and functional compatibility. Traditional sterilization methods are often limited in these requirements. Therefore, gas plasma sterilization may prove to be an appropriate alternative sterilization procedure.

Application of vapor phase hydrogen peroxide sterilization to endoscope.

An advantage of vapor phase hydrogen peroxide (VPHP) is that it can readily react to form reactive free radicals, which perform the sterilization and form water and hydrogen by catalyst. Absorptive hydrophilic materials such as cellulosics hinder penetration due to hydrogen bonding and necessitate the use of hydrophobic materials, i.e., polyethylene or polypropylene, as packaging materials. The 8h TWA (time-weight-average) is 1 ppm. Hydrogen peroxide sterilization is now being used for the sterilization of gloveboxes, freeze dryers, isolators and endoscopes and so on. This paper focuses on the application of VPHP to the sterilization of the endoscopes.