Evaluation of the Effect of the Volume Throughput and Maximum Flux of Low-Surface-Tension Fluids on Bacterial Penetration of 0.2 Micron-Rated Filters during Process-Specific Filter Validation Testing.

UNLABELLED: Approximately 97% of filter validation tests result in the demonstration of absolute retention of the test bacteria, and thus sterile filter validation failure is rare. However, while Brevundimonas diminuta (B. diminuta) penetration of sterilizing-grade filters is rarely detected, the observation that some fluids (such as vaccines and liposomal fluids) may lead to an increased incidence of bacterial penetration of sterilizing-grade filters by B. diminuta has been reported. The goal of the following analysis was to identify important drivers of filter validation failure in these rare cases. The identification of these drivers will hopefully serve the purpose of assisting in the design of commercial sterile filtration processes with a low risk of filter validation failure for vaccine, liposomal, and related fluids. Filter validation data for low-surface-tension fluids was collected and evaluated with regard to the effect of bacterial load (CFU/cm(2)), bacterial load rate (CFU/min/cm(2)), volume throughput (mL/cm(2)), and maximum filter flux (mL/min/cm(2)) on bacterial penetration. The data set (∼1162 individual filtrations) included all instances of process-specific filter validation failures performed at Pall Corporation, including those using other filter media, but did not include all successful retentive filter validation bacterial challenges. It was neither practical nor necessary to include all filter validation successes worldwide (Pall Corporation) to achieve the goals of this analysis. The percentage of failed filtration events for the selected total master data set was 27% (310/1162). Because it is heavily weighted with penetration events, this percentage is considerably higher than the actual rate of failed filter validations, but, as such, facilitated a close examination of the conditions that lead to filter validation failure. In agreement with our previous reports, two of the significant drivers of bacterial penetration identified were the total bacterial load and the bacterial load rate. In addition to these parameters, another three possible drivers of failure were also identified: volume throughput, maximum filter flux, and pressure. Of the data for which volume throughput information was available, 24% (249/1038) of the filtrations resulted in penetration. However, for the volume throughput range of 680-2260 mL/cm(2), only 9 out of 205 bacterial challenges (∼4%) resulted in penetration. Of the data for which flux information was available, 22% (212/946) resulted in bacterial penetration. However, in the maximum filter flux range from 7 to 18 mL/min/cm(2), only one out of 121 filtrations (0.6%) resulted in penetration. A slight increase in filter failure was observed in filter bacterial challenges with a differential pressure greater than 30 psid. When designing a commercial process for the sterile filtration of a low-surface-tension fluid (or any other potentially high-risk fluid), targeting the volume throughput range of 680-2260 mL/cm(2) or flux range of 7-18 mL/min/cm(2), and maintaining the differential pressure below 30 psid, could significantly decrease the risk of validation filter failure. However, it is important to keep in mind that these are general trends described in this study and some test fluids may not conform to the general trends described here. Ultimately, it is important to evaluate both filterability and bacterial retention of the test fluid under proposed process conditions prior to finalizing the manufacturing process to ensure successful process-specific filter validation of low-surface-tension fluids. LAY ABSTRACT: An overwhelming majority of process-specific filter validation (qualification) tests result in the demonstration of absolute retention of test bacteria by sterilizing-grade membrane filters. As such, process-specific filter validation failure is rare. However, while bacterial penetration of sterilizing-grade filters during process-specific filter validation is rarely detected, some fluids (such as vaccines and liposomal fluids) have been associated with an increased incidence of bacterial penetration. The goal of the following analysis was to identify important drivers of process-specific filter validation failure. The identification of these drivers will possibly serve to assist in the design of commercial sterile filtration processes with a low risk of filter validation failure. Filter validation data for low-surface-tension fluids was collected and evaluated with regard to bacterial concentration and rates, as well as filtered fluid volume and rate (Pall Corporation). The master data set (∼1160 individual filtrations) included all recorded instances of process-specific filter validation failures but did not include all successful filter validation bacterial challenge tests. This allowed for a close examination of the conditions that lead to process-specific filter validation failure. As previously reported, two significant drivers of bacterial penetration were identified: the total bacterial load (the total number of bacteria per filter) and the bacterial load rate (the rate at which bacteria were applied to the filter). In addition to these parameters, another three possible drivers of failure were also identified: volumetric throughput, filter flux, and pressure. When designing a commercial process for the sterile filtration of a low-surface-tension fluid (or any other penetrative-risk fluid), targeting the identified bacterial challenge loads, volume throughput, and corresponding flux rates could decrease, and possibly eliminate, the risk of validation filter failure. However, it is important to keep in mind that these are general trends described in this study and some test fluids may not conform to the general trends described here. Ultimately, it is important to evaluate both filterability and bacterial retention of the test fluid under proposed process conditions prior to finalizing the manufacturing process to ensure successful filter validation of low-surface-tension fluids.

The Development of a Microbial Challenge Test with Acholeplasma laidlawii To Rate Mycoplasma-Retentive Filters by Filter Manufacturers.

Mycoplasma are bacteria that can penetrate 0.2 and 0.22 µm rated sterilizing-grade filters and even some 0.1 µm rated filters. Primary applications for mycoplasma filtration include large scale mammalian and bacterial cell culture media and serum filtration. The Parenteral Drug Association recognized the absence of standard industry test parameters for testing and classifying 0.1 µm rated filters for mycoplasma clearance and formed a task force to formulate consensus test parameters. The task force established some test parameters by common agreement, based upon general industry practices, without the need for additional testing. However, the culture medium and incubation conditions, for generating test mycoplasma cells, varied from filter company to filter company and was recognized as a serious gap by the task force. Standardization of the culture medium and incubation conditions required collaborative testing in both commercial filter company laboratories and in an Independent laboratory (Table I). The use of consensus test parameters will facilitate the ultimate cross-industry goal of standardization of 0.1 µm filter claims for mycoplasma clearance. However, it is still important to recognize filter performance will depend on the actual conditions of use. Therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application. LAY ABSTRACT: Mycoplasma are small bacteria that have the ability to penetrate sterilizing-grade filters. Filtration of large-scale mammalian and bacterial cell culture media is an example of an industry process where effective filtration of mycoplasma is required. The Parenteral Drug Association recognized the absence of industry standard test parameters for evaluating mycoplasma clearance filters by filter manufacturers and formed a task force to formulate such a consensus among manufacturers. The use of standardized test parameters by filter manufacturers, including the preparation of the culture broth, will facilitate the end user's evaluation of the mycoplasma clearance claims provided by filter vendors. However, it is still important to recognize filter performance will depend on the actual conditions of use; therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application.

Sterilizing filtration of liposome and related lipid-containing solutions: enhancing successful filter qualification.

Bacterial penetration of integral sterilizing-grade 0.2 µm rated filters, although rare, is not a new phenomenon in the biopharmaceutical industry. It is recognized by both the Parenteral Drug Association and the Food and Drug Administration via recommended bacterial retention qualification (also commonly called filter validation) performed in relevant product fluids or suitable surrogates when necessary (1-3). As noted in recent work, formulations such as some adjuvanted vaccines, liposome-based drug delivery solutions, and similar surfactant or emulsion-based product fluids increase the likelihood of such penetration events (4). Here we demonstrate that some 0.2 µm rated sterilizing-grade filters from different filter manufacturers may perform less effectively than expected when their membranes are challenged with one of these bacterial penetration risk-related solutions. Some filters provided very little sterility assurance (titer reductions < 6 logs) and others provided substantial sterility assurance (titer reduction > 8 log). From this, it is clear that the product formulation most likely to lead to filter penetration must be identified early in the design process to facilitate process design and minimize qualification costs. In this way, the solution can be matched with the appropriate sterilizing-grade filter and the appropriate process conditions. LAY ABSTRACT: Bacterial penetration of intact sterilizing-grade filters during filter qualification, although rare, is not a new phenomenon in the biopharmaceutical industry. Because these incidences are identified in the filter validation process, there is no risk to the drug product end-user, but these failures do incur additional expense to the pharmaceutical manufacturer and could prevent the manufacture of very important drug formulations. Formulations such as some adjuvanted vaccines, liposome-based drug delivery solutions, and similar surfactant or emulsion-based product fluids have been documented to lead to an increased risk of penetration events. Here we demonstrate that some sterilizing-grade filters may perform differently from expected when their membranes are challenged with one of these solutions related to bacterial penetration risk. Some filters provided very little sterility assurance and others provided substantial sterility assurance. From this, it is clear that the pharmaceutical products and product formulation most likely to lead to filter penetration must be identified early in the design process to facilitate process design and minimize qualification costs. Moreover, that solution should be matched with the appropriate sterilizing-grade filter and process conditions to ensure expected sterility.

Nutritional effects of culture media on mycoplasma cell size and removal by filtration.

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.

Nutritional Effects on the Growth, Cell Size, and Resistance to Stress of Acholeplasma laidlawii.

Due to their lack of a cell wall, mycoplasmas are useful models for the study of biological membranes. However, they are much less appreciated by cell culture laboratories due to their unfortunate tendency to contaminate cell culture lines (1-5). One of the primary protections of a cell line is through filtration of cell culture media. It is necessary to use 0.1 µm-rated filters because mycoplasmas can often penetrate the more commonly used 0.2 (or 0.22) µm-rated filters. In the battle against mycoplasma contamination, it is helpful to delineate factors that may affect mycoplasma replication, cell size, and resistance to contamination control. The addition of cholesterol and unsaturated long chain fatty acids to growth media enhance replication of Acholeplasma laidlawii (6-10). The absence of glucose, presence of unsaturated fatty acids, increased incubation period, and exposure to stress promote the production of small mycoplasma cells (6-12). Exposure to a high salt concentration, hydrogen peroxide, and UV light, which are commonly used for contamination control, can enhance the production of small resistant cells (13-15).

Re-identification of the halotolerant, biosurfactant-producing Bacillus licheniformis strain JF-2 as Bacillus mojavensis strain JF-2.

Bacillus strain JF-2 (ATCC 39307) is a halotolerant, biosurfactant-producing bacterium that was initially described as a member of the species Bacillus licheniformis based on a limited set of phenotypic characteristics. Here, genetic and phenotypic analyses were employed to determine the relationship of Bacillus strain JF-2 to other Bacillus strains. The restriction patterns with AluI and analysis of gyrA and 16S rRNA gene sequences grouped Bacillus strain JF-2 with B. mojavensis(T) and not with B. licheniformis(T). DNA-DNA similarity showed JF-2 was 75% similar to B. mojavensis(T) and only 11% similar to B. licheniformis(T). Both strain JF-2 and B. mojavensis(T) required DNA for anaerobic growth, but B. licheniformis(T) did not. B. mojavensis(T) and strain JF-2 did not grow anaerobically in thioglycollate medium or aerobically with propionate while B. licheniformis(T) grew under these conditions. DNA-DNA similarity, gene sequence data and phenotypic characteristics all support the assignment of JF-2 as a member of the species B. mojavensis.

Anaerobic growth of Bacillus mojavensis and Bacillus subtilis requires deoxyribonucleosides or DNA.

Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191(T) and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions. Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth. The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions. The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B. mojavensis JF-2 for the four deoxyribonucleosides. However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth. The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B. mojavensis JF-2 in the presence of hydroxyurea. B. mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source. These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.

Bioaerosol Concentration at an Outdoor Composting Center.

Compost centers are one of many environments that produce airborne microorganisms. The objective of this study was to compare the bacterial, fungal, and acti-nomycete concentrations at the Norman, OK, compost center to background concentration of these same microorganisms. For this comparison, a modified Andersen Microbial Sampler was used. Sampling was performed at three sites at the outdoor compost center and at two background sites. The concentration of each microorganism was measured as total colony forming units per cubic meter (CFU/m3). The predominantly downwind compost center site had a 10-fold increase in all the microorganisms in comparison with the other sites (p < 0.05). The median concentrations (95% confidence interval) of total viable bacteria, Gram-negative bacteria, fungi, and actinomycetes at this site were 5059 (CI95= 4952-9600) CFU/m3, 2023 (CI95= 2586-6806) CFU/m3, 972 (CI95= 964-1943) CFU/m3, and 2159 (CI95= 1755-4190) CFU/m3, respectively.