Risk-based approach to setting sterile filtration microbial bioburden limits - Focus on biotech-derived products.

Holistic concepts should be applied that reduce risks prior to final bioburden testing and sterile filtration, based on enhanced process and product attribute understanding, which could be key to successful bioburden risk management. Key findings of this paper include.

A Rapid Sterility Method Using Solid Phase Cytometry for Cell-Based Preparations and Culture Media and Buffers.

In this article, we demonstrate a rapid sterility testing method for non-filterable cell-based preparations and its in-process control media/buffers. The selected rapid sterility test (RST) in this work is based on the ScanRDI® system, which detects fluorescently labeled microorganisms with solid-phase cytometry. ScanRDI® has been chosen due to its sensitivity for detecting viable microorganisms down to one microbial cell with a shorter time to detection compared with the compendial sterility test (CST) method. The RST was validated for a CAR-T cell-therapy product with 4 days of time to detection (TTD) and evaluated for in-process control of media/buffers with real-time detection method success according to USP <1223>, Ph. Eur. 5.1.6, and PDA Technical Report No. 33. The validation parameters included limit of detection and equivalence in routine operations, specificity, robustness, ruggedness, and repeatability. For the validation, a combination of pharmacopoeial ATCC strains as well as in-house isolates were used. In addition, the evaluation study of this RST for in-process control of media/buffers was assessed by performing the limit of detection and equivalence with four representative microorganisms. Where applicable, results were statistically evaluated to demonstrate equivalence and no significant difference of the rapid method as compared with the CST method have been detected. All acceptance criteria have been met, and the solid-phase cytometry technology was successfully validated as an alternative sterility test for cell-based preparations and for its in-process control of media/buffer.

Tunable diode laser absorption spectroscopy as method of choice for non-invasive and automated detection of microbial growth in media fills.

Tunable diode laser absorption spectroscopy (TDLAS) was evaluated on its potential to detect bacterial growth of contaminated media fill vials. The target was a replacement/ automation of the traditional visual media fill inspection. TDLAS was used to determine non-invasively O2 and/or CO2 changes in headspaces of such vials being induced by metabolically active microorganisms. Four different vial formats, 34 microorganisms (inoculation volume<10 cells) and two different media (TSB/FTM) were tested. Applying parallel CO2 and O2 headspace measurements all format-organism combinations were detected within <11 days reliably with reproducible results. False negatives were exclusively observed for samples that were intentionally breached with syringes of 0.3mm in diameter. Overall it was shown that TDLAS functionality for a replacement of the visual media fill inspection is given and that investing in further validation and implementation studies is valuable. Nevertheless, some small but vincible challenges remain to have this technology in practical use.

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.

Comparison of Tunable Diode Laser Absorption Spectroscopy and Isothermal Micro-calorimetry for Non-invasive Detection of Microbial Growth in Media Fills.

Two methods were investigated for non-invasive microbial growth-detection in intact glass vials as possible techniques for automated inspection of media-filled units. Tunable diode laser absorption spectroscopy (TDLAS) was used to determine microbially induced changes in O2 and CO2 concentrations within the vial headspaces. Isothermal microcalorimetry (IMC) allowed the detection of metabolic heat production. Bacillus subtilis and Streptococcus salivarius were chosen as test organisms. Parameters as robustness, sensitivity, comparability and time to detection (TtD) were evaluated to assess method adequacy. Both methods robustly detected growth of the tested microorganisms within less than 76 hours using an initial inoculum of <10CFU. TDLA turned out to be less sensitive than TDLA and IMC, as some false negative results were observed. Compared to the visual media-fill examination of spiked samples, the investigated techniques were slightly slower regarding TtD. Although IMC showed shorter TtD than TDLAS the latter is proposed for automating the media-fill inspection, as larger throughput can be achieved. For routine use either TDLA or a combination of TDLA and TDLA should be considered. IMC may be helpful for replacing the sterility assessment of commercial drug products before release.

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.

Bacteria associated with spores of the arbuscular mycorrhizal fungi Glomus geosporum and Glomus constrictum.

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.