Validation of Milliflex® Quantum for Bioburden Testing of Pharmaceutical Products.

This article reports the validation strategy used to demonstrate that the Milliflex® Quantum yielded non-inferior results to the traditional bioburden method. It was validated according to USP <1223>, European Pharmacopoeia 5.1.6, and Parenteral Drug Association Technical Report No. 33 and comprised the validation parameters robustness, ruggedness, repeatability, specificity, limit of detection and quantification, accuracy, precision, linearity, range, and equivalence in routine operation. For the validation, a combination of pharmacopeial ATCC strains as well as a broad selection of in-house isolates were used. In-house isolates were used in stressed state. Results were statistically evaluated regarding the pharmacopeial acceptance criterion of ≥70% recovery compared to the traditional method. Post-hoc test power calculations verified the appropriateness of the used sample size to detect such a difference. Furthermore, equivalence tests verified non-inferiority of the rapid method as compared to the traditional method. In conclusion, the rapid bioburden on basis of the Milliflex® Quantum was successfully validated as alternative method to the traditional bioburden test.LAY ABSTRACT: Pharmaceutical drug products must fulfill specified quality criteria regarding their microbial content in order to ensure patient safety. Drugs that are delivered into the body via injection, infusion, or implantation must be sterile (i.e., devoid of living microorganisms). Bioburden testing measures the levels of microbes present in the bulk solution of a drug before sterilization, and thus it provides important information for manufacturing a safe product. In general, bioburden testing has to be performed using the methods described in the pharmacopoeias (membrane filtration or plate count). These methods are well established and validated regarding their effectiveness; however, the incubation time required to visually identify microbial colonies is long. Thus, alternative methods that detect microbial contamination faster will improve control over the manufacturing process and speed up product release. Before alternative methods may be used, they must undergo a side-by-side comparison with pharmacopeial methods. In this comparison, referred to as validation, it must be shown in a statistically verified manner that the effectiveness of the alternative method is at least equivalent to that of the pharmacopeial methods. Here we describe the successful validation of an alternative bioburden testing method based on fluorescent staining of growing microorganisms applying the Milliflex® Quantum system by MilliporeSigma.

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

Contact plates, dipslides, and swabs are used for the microbiological monitoring of surfaces in controlled environments such as pharmaceutical clean rooms. In the present study, three different swab types using two different methods (direct streaking on agar versus elution followed by membrane filtration) were evaluated. In a first study, representative surfaces in pharmaceutical clean rooms were artificially inoculated using three different environmental strains (in vitro study). In a second study, a naturally inoculated floor was swabbed with the same three swab types, again using the two different recovery methods (in situ study). With the in vitro study, clear differences were found between the three swab types as well as between the two recovery methods. In addition, recovery rate of the swab type was dependent on the recovery method (interactive effect). One swab type showed a higher recovery rate with direct streaking on agar, while the other swab type showed better results using the elution/membrane filtration method. This difference can be explained by the fact that both swabs were each developed for their specific application. The type of surface also had a highly significant effect on the recovery rates. Recovery on stainless steel was better than for the other surfaces, while lexan had the lowest recovery rate. From the three different strains applied in the in vitro study, Micrococcus luteus had significantly higher recovery results compared to the other two strains (Bacillus thuringiensis, Aspergillus brasiliensis). The differences in recovery between the swab type and recovery method were less pronounced in the in situ study. In particular, the recovery of the swab type depending on the recovery method was not found. In conclusion, if swabs are to be used for environmental monitoring, their suitability should first be evaluated. This can be approached with artificially inoculated surfaces. However, naturally inoculated surfaces might be more realistic and might better reflect what is found in pharmaceutical clean rooms. LAY ABSTRACT: 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.

Comparison of Different Calculation Approaches for Defining Microbiological Control Levels Based on Historical Data.

UNLABELLED: In the present work we compared different calculation approaches for their ability to accurately define microbiological control levels based on historical data. To that end, real microbiological data were used for simulation experiments. The results of our study confirmed that assuming a normal distribution is not appropriate for that purpose. In addition, assumption of a Poisson distribution generally underestimated the control level, and the predictive power for future values was highly insufficient. The non-parametric Excel percentile strongly predicted future values in our simulation experiments (although not as good as some of the parametric models). With the limited amount of data used in the simulations, the calculated control levels for the upper percentiles were on average higher and more variable compared to the parametric models. This was due to the fact that the largest observed value was generally defined as the control level. Accordingly, the Excel percentile is less robust towards outliers and requires more data to accurately define control levels as compared to parametric models. The negative binomial as well as the zero-inflated negative binomial distribution, both parametric models, had good predictive power for future values. Nonetheless, on basis of our simulation experiments, we saw no evidence to generally prefer the zero-inflated model over the non-inflated one. Finally, with our data, the gamma distribution on average had at least as good predictive power as the negative binomial distribution and zero-inflated negative binomial distribution for percentiles ≥98%, indicating that it may represent a viable option for calculating microbiological control levels at high percentiles. Presumably, this was based on the fact that the gamma distribution fitted the upper end of the distribution better than other models. Since in general microbiological control levels would be based on the upper percentiles, microbiologists may exclusively rely on the gamma distribution for calculation of their control levels. As the gamma distribution can conveniently be calculated in standard office calculation software, it may represent a superior alternative to the widely used percentile functions or other distribution models. LAY ABSTRACT: During the manufacturing of pharmaceutical drug products, the counts of microorganisms are monitored in the cleanroom environment, water, the product's raw materials, and the final product. This enables manufacturers to ensure that high numbers of microorganisms that may impair the product's microbiological quality are detected before the product is released to the patient. Microbiological control levels must be set to determine at which number a count is considered too high. Exceeding such levels may require an investigation to determine the root cause explaining why such high numbers of microorganisms occurred, and a set of actions should be performed with the aim of eliminating this root cause. In order to really differentiate higher-than-usual counts, microbiological control levels should be based on historical data. In the present work we analyzed different calculation approaches towards that purpose. We used real microbiological data and performed simulation experiments to determine which statistical method could calculate the most realistic control levels that would provide the best prediction for future routine testing. Better predictions would ensure that only significant contaminations lead to an excursion of the microbiological control level, which would avoid wasting resources by investigating non-issues or normal/controlled conditions.

Comparison of different incubation conditions for microbiological environmental monitoring.

Environmental monitoring represents an integral part of the microbiological quality control system of a pharmaceutical manufacturing operation. However, guidance documents differ regarding recommendation of a procedure, particularly regarding incubation time, incubation temperature, or nutrient media. Because of these discrepancies, many manufacturers decide for a particular environmental monitoring sample incubation strategy and support this decision with validation data. Such validations are typically laboratory-based in vitro studies, meaning that these are based on comparing incubation conditions and nutrient media through use of cultured microorganisms. An informal survey of the results of these in vitro studies performed at Novartis or European manufacturing sites of different pharmaceutical companies highlighted that no consensus regarding the optimal incubation conditions for microbial recovery existed. To address this question differently, we collected a significant amount of samples directly from air, inanimate surfaces, and personnel in pharmaceutical production and packaging rooms during manufacturing operation (in situ study). Samples were incubated under different conditions suggested in regulatory guidelines, and recovery of total aerobic microorganisms as well as moulds was assessed. We found the highest recovery of total aerobic count from areas with personnel flow using a general microbiological growth medium incubated at 30-35 °C. The highest recovery of moulds was obtained with mycological medium incubated at 20-25 °C. Single-plate strategies (two-temperature incubation or an intermediate incubation temperature of 25-30 °C) also yielded reasonable recovery of total aerobic count and moulds. However, recovery of moulds was found to be highly inefficient at 30-35 °C compared to lower incubation temperatures. This deficiency could not be rectified by subsequent incubation at 20-25 °C. A laboratory-based in vitro study performed in parallel was inconclusive. We consider our results potentially conferrable to other pharmaceutical manufacturing sites in moderate climate zones and believe that these should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples. LAY ABSTRACT: Microbiological environmental monitoring confirms that pharmaceutical cleanrooms are in an appropriate hygienic condition for manufacturing of drug products. Guidance documents from different health authorities or expert groups differ regarding recommendation of the applied incubation time, incubation temperature, or nutrient media. Therefore, many pharmaceutical manufacturers perform studies that aim to identify the optimal incubation setup for environmental monitoring samples. An informal survey of the results of such studies, which had been performed at Novartis or European manufacturing sites of different pharmaceutical companies, highlighted no consensus regarding the optimal incubation conditions for microbial recovery. All these studies had been conducted in the laboratory using selections of cultured microbial strains. We tried to solve this disagreement by collecting a significant amount of real environmental monitoring samples directly from the environment in pharmaceutical production and packaging rooms during manufacturing operation. These samples were then incubated under different conditions suggested in the regulatory guidelines. We believe that the results of our study are more meaningful than laboratory-based experiments because we used environmental samples with microorganisms directly isolated from the manufacturing area. Therefore, we believe that our results should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples.

Identification of micro-organisms after milliflex rapid detection--a possibility to identify nonsterile findings in the milliflex rapid sterility test.

The Milliflex Rapid System is used as a rapid microbiological method based on adenosine triphosphate (ATP) bioluminescence in the pharmaceutical industry to quantify the amount of micro-organisms present in water and in bioburden samples. The system can also be used for qualitative analyses, for example, to perform a rapid sterility test. This rapid sterility test has been successfully validated and implemented at Novartis and Sandoz. As the reagents used for the ATP bioluminescence reaction, which are directly sprayed on a micro-colony, disrupt the walls/membranes of the present cells to release ATP and therefore no intact cells for subsequent identification were believed to be present, the identification was supposed to be impossible until now. During development and validation of a rapid sterility test with the Milliflex Rapid System, a possibility to identify contaminants was found. A method based on regrowth of the Milliflex Rapid-treated microbial cells and consecutive genotypic identification reproduced feasible and robust results. The data presented here show that sufficient recovery of the micro-colonies detected with the Milliflex Rapid System was reached with the test strains, except with Penicillium spec. The chosen micro-organisms represent the full spectrum of environmental isolates and ATCC strains, and it was shown that they are not destroyed after application of the reagents for the ATP bioluminescence reaction. Overall, 22 stressed microbial strains were examined during the study. LAY ABSTRACT: After Milliflex Rapid System detection, it was supposed that a subsequent identification of the contaminant is not possible. In this paper it is shown how contaminants can be identified in the rapid sterility test application.

Growth-promoting Properties of Different Solid Nutrient Media Evaluated with Stressed and Unstressed Micro-organisms: Prestudy for the Validation of a Rapid Sterility Test.

Currently, sterility testing in the pharmaceutical industry-a mandatory release test for all sterile drug products-takes an incubation time of at least 14 days and is based on liquid media according to the pharmacopoeias. The search is on for a rapid sterility test to reduce this rather long time frame. For this we have chosen the Millipore Milliflex Rapid Microbiology Detection System, which is based on solid nutrient media. As a prerequisite for the validation of this rapid sterility test, a solid nutrient medium promoting the growth of stressed and unstressed micro-organisms replacing tryptic soy broth and fluid thioglycollate medium from the traditional sterility test had to be found. For this a wide variety of appropriate nutrient media were evaluated. After a prestudy with 10 different nutrient agar media, tryptic soy agar, Center for Disease Control (CDC) anaerobic blood agar, Schaedler blood agar, and Difco brewer anaerobic agar were tested in detail using a range of 22 micro-organisms (7 ATCC strains and 15 production site-specific strains). These strains were inoculated in their unstressed and in a stressed state. Stress was evoked by heat treatment and nutrient starvation in the case of the sporulating bacteria. This stress effect-resulting in deceleration in growth-was experimentally confirmed based on growth curve analysis. It was statistically evaluated which media and which incubation temperatures are best suitable. The resulting data showed that Schaedler blood agar has the best growth-promoting properties among the agars tested and is going to be used in the rapid sterility test with the incubation temperatures 20-25 °C for aerobes, 30-35 °C for aerobes, and also 30-35 °C for anaerobic micro-organisms.