Pharmaceutical Cleanrooms Research Articles

Probabilistic optimal design of cleanroom air-conditioning systems facilitating optimal ventilation control under uncertainties

Buildings with spaces requiring strict temperature and humidity controls, such as pharmaceutical cleanrooms and semiconductor/microchip factories, have been growing very quickly in terms of total floor area and energy consumption. In such buildings, much of the energy is unnecessarily wasted due to the incoordination of system design and operation/control, especially under “off-design” and ever-changing ambient and load conditions. This paper, therefore, proposes a probabilistic optimal design method for cleanroom air-conditioning systems facilitating optimal ventilation control under uncertainties. To consider the effects of asynchronous loads in different zones/spaces with reduced computation demand, a probabilistic diversity factor method is proposed which is a simplified method to quantify the effects of uncertainties of space load diversities in multiple zones/spaces using diversity factors. The proposed design method is implemented and validated in the design optimization of air-conditioning systems for implementing four different ventilation control strategies considering possible and uncertain off-design conditions. The energy and economic performance as well as service satisfaction of the air-conditioning systems are also evaluated and compared. Results show that the proposed design method can obtain the optimal air-conditioning systems with minimum life-cycle cost and superior satisfaction of service.

Energy demand reduction in pharmaceutical cleanrooms through optimization of ventilation

The high Air Change Rates (ACRs) required for cleanrooms makes them energy intensive. This research elaborates on three strategies for energy efficient ventilation in pharmaceutical cleanrooms: Fine-tuning, Demand Controlled Filtration (DCF), and optimizing airflow pattern. To study the possibilities for fine-tuning and DCF, two case studies were investigated and simulations were performed to assess the potential of both options. Experiments in a demonstration cleanroom were used to examine how an ideal airflow pattern may be achieved in the cleanroom, resulting in a high contaminant removal efficiency. Results showed that DCF could lead to substantial energy savings, up to 93.6% in the specific case study facilities. Besides this, DCF based on occupancy could be implemented with negligible effect on the environmental cleanliness requirements. Fine-tuning, based on particle concentration, required representative measurement of the concentration in the cleanroom. It was more difficult to implement in practice. With respect to contaminant removal efficiency, best results (within the experiments performed, ACR in the range of 16h−1 – 38h−1), were obtained when air was supplied without a diffuser above the product area and when the work position was located close to the air extraction grilles.

Survey of particle production rates from process activities in pharmaceutical and biological cleanrooms

The lack of measurement data on particle generation rates from process activities for companies involved in the life sciences industry, such as pharmaceutical and biotechnology firms, has the potential to undermine cleanroom design, particulate concentration control, and energy efficiency. Based on this situation, this paper surveys the particle production rates from process activities in pharmaceutical and biological cleanrooms using field measurements for the particle sizes of interest (0.3 µm, 0.5 µm, and 1 µm). The measured and collected data were correlated to the surveyed activities to obtain a range of particle generation rates for different cleanroom operations. In terms of the recommended ACH values and measured ACH values, the results indicate that although most cleanrooms operate below the recommended ranges, in most cases they satisfy or exceed the required ISO standard cleanliness levels. In addition, the particle generation rates are process-specific and that suggests process-specific guidelines for different cleanrooms will help engineers, owners and contractors arrive at a better design solution. It is worth mentioning that nonperfect mixing of airflow and particles was observed in several cleanrooms, resulting in large variances in the measurements at different sampling locations. This observation suggests that a correction factor might be needed for the ventilation effectiveness for Class 7 and 8 cleanrooms. Although the determination of the correction factor is beyond the study of this paper, the data presented in this paper are critical to understand the severity of the non-perfect mixing in Class 7 and 8 cleanrooms and thus provides insights on the determination of the correction factor. The research reported in this paper was conducted through the ASHRAE 1399 research project, with a focus on developing scientific cleanroom design guidelines that can assist engineers, owners, and contractors to arrive at appropriately sized and efficient cleanroom systems.

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes-special notes on pharmaceutical environmental isolates.

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry for identification of bacteria isolated from pharmaceutical clean rooms

IntroductionDuring the manufacturing of sterile drugs, it is of the utmost importance to meet the minimum requirements for asepsis recommended by the legislations on good manufacturing practices-based efficient environmental monitoring.Aims and methodsThe availability of relatively simple to use matrix-assisted laser desorption ionization–time of flight mass spectromtomy (MALDI-TOF MS) devices in the last years has changed the laboratory workflows for the microbial identification, mainly in the clinical area. Thus, the objective of this work was to evaluate the suitability of the MALDI-TOF MS technique for the identification of bacteria isolated from the environment of clean rooms used in some stages of the production of a viral vaccine. Eighteen known bacterial species commonly isolated from clean rooms studied were identified by MALDI-TOF technique and by a biochemical technique (BBL Crystal® System).ResultsPerformance of MALDI-TOF MS was better than biochemical technique for correct species identifications (88.89% and 38.89%, respectively) and produced less unreliable identification (5.55% and 22.22%).ConclusionMALDI-TOF MS can be implemented for routine identification of bacteria in a pharmaceutical quality control laboratory, but as a database-dependent system, maybe some isolated not identified by this technique must be additionally studied and, if appropriate, added to an in-house database.

Multicenter Study on Incubation Conditions for Environmental Monitoring and Aseptic Process Simulation.

Microbiological environmental monitoring and aseptic process simulation confirm that pharmaceutical cleanrooms are in an appropriate hygienic condition for manufacturing of sterile drug products. Guidance documents from different health authorities or expert groups differ regarding recommendation of the applied incubation time and incubation temperature, leading to variable manufacturers practices. Some recent publications have demonstrated that laboratory studies are not relevant to determine the best incubation regime and that in situ manufacturing site studies should be used. To solve any possible bias coming from laboratory studies or single-site in situ studies, we conducted a multicenter study at four manufacturing sites with a significant amount of real environmental monitoring samples collected directly from the environment in pharmaceutical production during manufacturing operations with four solid and liquid nutrient media. These samples were then incubated under four different conditions suggested in the guidance documents. We believe that the results of our multicenter study confirming recent other single-site in situ studies could be the basis of the strategy to determine the best incubation regime for both viable environmental monitoring and aseptic process simulation in any manufacturing facility.

Evaluation of the Recovery Rate of Different Swabs for Microbial Environmental Monitoring.

Environmental microbiological monitoring provides information on the hygiene condition of pharmaceutical clean rooms and equipment for manufacturing of drug products. Different methods can be used to recover microorganisms. For surfaces, normally contact plates (e.g., RODAC or dipslides) are used; however, when surfaces are uneven, swabs should be used. In the present study three different swabs were evaluated for their ability to recover microorganisms from different surfaces. Thereby two methods and two approaches were evaluated. Swab samples were either directly stroked on agar or the swab was eluted, membrane-filtrated, and the filter placed on an agar plate. Experimentally, artificial inoculated surfaces typically found in clean rooms (in vitro study) and naturally inoculated floors (in situ study) were sampled. Thus with this approach the most convenient swab and the most suitable recovery methods under laboratory as well as real clean room conditions were evaluated. With this set-up, we found the most suitable swab for our environmental monitoring not only by using artificial inoculated surfaces but also under more realistic clean room conditions, which is most important for microbiological environmental monitoring sampling.

Synergistic Effect of Biogenic Silver-nanoparticles with β. lactam Cefotaxime against Resistant Staphylococcus arlettae AUMC b-163 Isolated from T3A Pharmaceutical Cleanroom, Assiut, Egypt

The aim of this study was to biosynthesis silver nanoparticles (AgNPs) from Staphylococcus arlettae AUMC b-163 isolated from T3A pharmaceutical company cleanroom, its antimicrobial activity, and the synergistic effect of AgNPs in combination with commonly used antibiotic Cefotaxime sodium against resistant bacteria. The synthesized AgNPs from bacterial were characterized by using UV-VS spectrophotometer analysis, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD) and Transmission Electron Microscopy (TEM).UV-VS spectrophotometer analysis showed a peak at 420 nm corresponding to the Plasmon absorbance of silver nanoparticles and FTIR analysis showed the potential biomolecule responsible for the reduction of silver. The structural properties of silver nanoparticles were confirmed using XRD technique, while TEM micrographs revealed that the silver nanoparticles are dispersed and aggregated, and mostly having spherical shape within the size range between 8 and 35 nm. The synthesized silver nanoparticles exhibited a varied growth inhibition activity against the tested pathogenic bacteria. A significant increase in area of growth inhibition was observed when a combination of silver nanoparticles and Cefotaxime antibiotics was applied. The current results revealed that the synthesized silver nanoparticles produced by the bacterial strain Staphylococcus arlettae AUMC b-163 is a promising to be used in medical therapy due to their broad spectrum against some pathogenic bacteria, fungi and resistant tested bacteria.

In vitro fungicidal activity of biocides against pharmaceutical environmental fungal isolates.

To determine the minimum inhibitory concentrations (MIC) of a range of cleanroom fungi against three disinfectants common to the pharmaceutical and healthcare sectors: biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide). The in vitro fungicidal activities of the three biocides were studied against 112 cleanroom fungal isolates using broth microdilution technique (CLSI M38-A2 standard). Minimum inhibitory concentration (MIC) for all three biocides against hyaline fungi showed results of not more than 16 μg ml(-1). Alternaria showed <32 μg ml(-1) and other dematiaceous fungi reported that 8-16 μg ml(-1) for biguanides and QACs. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring programme may be periodically subjected to broth microdilution testing with cleanroom disinfectant agents used in the disinfection programme confirm their sensitivity profile. No large collection of data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. This is the first study report with large collection of cleanroom fungal isolates tested against common biocides using the broth microdilution antifungal susceptibility testing to determine the MIC value. The data presented support a quality control procedure for cleanroom disinfection.

Bacterial diversity in the indoor air of pharmaceutical environment

To monitor bacterial diversity of ISO Class 8 pharmaceutical clean room environment using conventional culture-based methods and pyrosequencing analysis. Bacterial isolates were obtained through viable particulate air monitoring, passive air monitoring and surface-monitoring procedures. A total of 157 bacterial isolates were obtained and assigned to four different phyla, Actinobacteria, Firmicutes, Proteobacteria and Deinococcus-Thermus, encompassing 52 species of 24 genera based on 16S rRNA gene sequence analysis. The genera Micrococcus and Staphylococcus were found as the main bacterial groups among the isolates. However, a big discrepancy was found between the culture based and pyrosequencing results. A total of 11409 quality reads were obtained from the pyrosequencing analysis, and the subsequent phylogenetic analysis indicated that Proteobacteria was the most abundant group at phylum level, followed by Actinobacteria and Firmicutes. Bacillus, Propionibacterium and Acinetobacter were identified as the most abundant genera by the pyrosequencing analysis. The culture-based results were in line with previous reports on the airborne bacterial composition of various environments, but the pyrosequencing analysis revealed a unique diversity of bacteria in this case. No significant pathogens above Riskgroup 2 were found from either culture based or pyrosequencing studies. The presence of various bacterial taxa including a number of groups, whose presence in air is previously unknown, was confirmed through this analysis. The main source of bacteria in the indoor air environment of pharmaceutical processes is likely human, but no significant primary pathogens were detected. Culture-based analysis may give limited information on the bacterial diversity of air environment.

Domibacillus robiginosus gen. nov., sp. nov., isolated from a pharmaceutical clean room

A novel red-pigmented bacterial strain, designated WS 4628(T), was isolated from a pharmaceutical clean room of a vaccine-producing company and was investigated in a taxonomic study using a polyphasic approach. The strain was Gram-stain-positive, strictly aerobic, motile, catalase-positive and produced spherical to slightly ellipsoidal endospores in rods. The genomic DNA G+C content was 44.1 mol%. The major fatty acids were anteiso-C15:0, iso-C15:0 and anteiso-C17:0 and the predominant quinone was MK-6. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phosphoglycolipid and an unidentified phospholipid. meso-diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan and the major whole-cell sugars were glucose and ribose. The closest phylogenetic neighbours were identified as Bacillus badius ATCC 14574(T) (95.8% 16S rRNA gene sequence similarity), Bacillus indicus Sd/3(T) (94.8%), Jeotgalibacillus alimentarius YKJ-13(T) (94.8%) and Bacillus cibi JG-30(T) (94.8%). Phylogenetic, physiological, biochemical and morphological differences between strain WS 4628(T) and its closest relatives in the families Bacillaceae and Planococcaceae suggest that this strain represents a novel species in a new genus in the family Bacillaceae for which the name Domibacillus robiginosus gen. nov., sp. nov. is proposed; the type strain of the type species is WS 4628(T) (=DSM 25058(T)=LMG 26645(T)).

In vitro Antifungal Efficacy of Biguanides and Quaternary Ammonium Compounds against Cleanroom Fungal Isolates

In vitro antifungal activities of three biocides including one biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide) were studied against eight cleanroom fungal isolates by using a microbiological broth dilution technique as per Clinical and Laboratory Standards Institute (CLSI) M38-A guidelines. No data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. Minimum inhibitory concentrations (MICs) for all three biocides against species of Aspergillus and Penicillium species ranged between 4 and 16 μg/mL. MICs of Curvularia, Cladosporium, and Alternaria species also showed less than 16 μg/mL. To date, susceptibility breakpoints have not been established for biocides, and this is the first study using the CLSI broth microdilution antifungal susceptibility testing to determine the MIC value of biocides. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring program may be periodically subjected to microbroth dilution testing with cleanroom disinfectant agents used in the disinfection program to confirm their sensitivity profile. Further work is needed in this field to increase our understanding of biocides against different fungal isolates and to enable to the design of more efficient disinfection and contamination control programs. Increased trend of fungal growth is observed in pharmaceutical and medical device cleanrooms. It is essential to have knowledge of choosing effective disinfectants for minimizing fungal occurrence in cleanrooms. The present study establish minimum cut-offs for specific fungal isolates that are problematic in cleanrooms against commonly used disinfectants (quaternary ammonium compounds and biguanides). Further studies based on this minimum inhibitory concentration of disinfectants, effective time, and cleaning methods could prevent fungal occurrence.

Micrococcus cohnii sp. nov., isolated from the air in a medical practice

Three Gram-reaction-positive bacteria, isolated from the air in a medical practice (strains WS4601(T), WS4602) or a pharmaceutical clean room (strain WS4599), were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences of the three novel strains showed that they formed a distinct lineage within the genus Micrococcus, sharing 16S rRNA gene sequence similarities of 96.1-98.0 % with other species of this genus. Chemotaxonomic features also supported the classification of the three novel strains within the genus Micrococcus. The major cellular fatty acids of strain WS4601(T) were anteiso-C(15 : 0) and iso-C(15 : 0), the cell-wall peptidoglycan was of type A3α (L-Lys-L-Ala), and the predominant respiratory quinones were MK-7(H(2)) and MK-8(H(2)). The polar lipid profile contained diphosphatidylglycerol and phosphatidylglycerol, but no phosphatidylinositol. The G+C content of the genomic DNA was 70.4 mol%. Numerous physiological properties were found that clearly distinguished strains WS4599, WS4601(T) and WS4602 from established members of the genus Micrococcus. Based on the phenotypic and phylogenetic data, strains WS4599, WS4601(T) and WS4602 are considered to represent three different strains of a novel species of the genus Micrococcus, for which the name Micrococcus cohnii sp. nov. is proposed. The type strain is WS4601(T) (=DSM 23974(T)=LMG 26183(T)).

Naumannella halotolerans gen. nov., sp. nov., a Gram-positive coccus of the family Propionibacteriaceae isolated from a pharmaceutical clean room and from food

Four Gram-stain-positive, aerobic bacterial strains isolated from a pharmaceutical clean room (strain WS4616(T)), a dessert milk product (strain WS4617) and from raw milk (strains WS4623 and WS4624) were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences showed that they formed a distinct lineage within the family Propionibacteriaceae. Similarity values between 16S rRNA gene sequences of the four novel strains and the type species of all genera belonging to the family Propionibacteriaceae were 89.2-94.1%. The major cellular fatty acid was anteiso-C(15:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Respiratory quinones were MK-8(H(4)) and MK-9(H(4)). The cell-wall peptidoglycan of type A3γ contained ll-diaminopimelic acid, alanine, glycine and glutamic acid. The G+C content of the genomic DNA of strain WS4616(T) was 67.7 mol%. The whole-cell sugar pattern contained ribose, mannose, arabinose, glucose and galactose. On the basis of phenotypic and genetic data, strains WS4616(T), WS4617, WS4623 and WS4624 are classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Naumannella halotolerans gen. nov., sp. nov. is proposed. The type strain is WS4616(T) ( = DSM 24323(T) = LMG 26184(T)) and three additional strains are WS4617, WS4623 and WS4624.

Towards single-microorganism detection using surface-enhanced Raman spectroscopy

The identification and discrimination of microorganisms is important not only for clinical reasons but also for pharmaceutical clean room production and food-processing technology. Vibrational spectroscopy such as IR, Raman, and surface-enhanced Raman scattering (SERS) can provide a rapid ‘fingerprint’ on the chemical structure of molecules and is used to obtain a ‘fingerprint’ from microorganisms as well. Because of the requirement that a single bacterium cell and noble metal nanoparticles must be in close contact and the lack of a significant physical support to hold nanoparticles around the single bacterium cell, the acquisition of SERS spectra for a single bacterium using colloidal nanoparticles could be a challenging task. The feasibility of SERS for identification down to a single bacterium is investigated. A Gram-negative bacterium, Escherichia coli, is chosen as a model for the investigation. Because the adsorption of silver nanoparticles onto the bacterial cell is an exclusive way for locating nanoparticles close to the bacterium cell, the absorption characteristics of silver nanoparticles with different surface charges are investigated. It is demonstrated that the citrate-reduced colloidal silver solution generates more reproducible SERS spectra. It is found that E. coli cells aggregate upon mixing with silver colloidal solution, and this may provide an additional benefit in locating the bacterial cell under a light microscope. It is also found that a laser wavelength in the UV region could be a better choice for the study due to the shallow penetration depth. It is finally shown that it is possible to obtain SERS spectra from a single cell down to a few bacterial cells, depending on the aggregation properties of bacterial cells for identification and discrimination.

Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: application to clean-room-relevant biological contaminations.

Microorganisms, such as bacteria, which might be present as contamination inside an industrial food or pharmaceutical clean room process need to be identified on short time scales in order to minimize possible health hazards as well as production downtimes causing financial deficits. Here we describe the first results of single-particle micro-Raman measurements in combination with a classification method, the so-called support vector machine technique, allowing for a fast, reliable, and nondestructive online identification method for single bacteria.

Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions

Microbial contamination is not only a medical problem, but also plays a large role in pharmaceutical clean room production and food processing technology. Therefore many techniques were developed to achieve differentiation and identification of microorganisms. Among these methods vibrational spectroscopic techniques (IR, Raman and SERS) are useful tools because of their rapidity and sensitivity. Recently we have shown that micro-Raman spectroscopy in combination with a support vector machine is an extremely capable approach for a fast and reliable, non-destructive online identification of single bacteria belonging to different genera. In order to simulate different environmental conditions we analyzed in this contribution different Staphylococcus strains with varying cultivation conditions in order to evaluate our method with a reliable dataset. First, micro-Raman spectra of the bulk material and single bacterial cells that were grown under the same conditions were recorded and used separately for a distinct chemotaxonomic classification of the strains. Furthermore Raman spectra were recorded from single bacterial cells that were cultured under various conditions to study the influence of cultivation on the discrimination ability. This dataset was analyzed both with a hierarchical cluster analysis (HCA) and a support vector machine (SVM).