The pharmaceutical industry is increasingly seeking modern microbial methods (alternative microbiological methods) to comply with evolving regulatory standards like Annex 1 for contamination control and to improve efficiency. A critical aspect of adopting alternative methods is demonstrating their correlation to traditional plate counts, often involving a variety of microorganisms, such as those outlined in USP <61>, USP <62>, E.P. 2.6.12, EP 2.6.13 and JP 4.05.
This research presents a comprehensive comparison study to evaluate the performance of the Sievers Soleil Rapid Bioburden Analyzer against the compendial method involving 11 microorganisms and a combination of common water isolates, which are tested across multiple laboratory sites, analysts, and instruments. This research was originally presented as a poster at the PDA Pharmaceutical Microbiology Conference.
The Sievers Soleil microorganism verification study aligns with parameters mentioned in USP <1223> “VALIDATION OF ALTERNATIVE MICROBIOLOGICAL METHODS”. Findings demonstrate that the Sievers Soleil Rapid Bioburden Analyzer successfully detects and quantifies Gram-positive, Gram-negative bacteria, yeasts, and molds, showing strong correlation with traditional plate counts in ultrapure water (UPW). The study confirmed performance across key metrics including accuracy, linearity, precision, range, robustness, and ruggedness, with a Limit of Detection (LOD) of 5 CFU/100 mL. These results demonstrate Sievers Soleil is a reliable, rapid, and sensitive microbial enumeration method for pharmaceutical water testing.
Background: Implementing Modern Microbial Methods (MMMs) for Annex 1 Compliance
The publication of Annex 1 sparked increased interest among pharmaceutical companies in implementing Modern Microbial Methods (MMMs). MMMs represent a significant advancement toward obtaining bioburden results to enable proactive decision-making, instead of reactive.
Alternative rapid micro methods offer a promising alternative to traditional plating methods by enhancing efficiency and reliability in microbial testing. However, comprehensive correlation studies and adherence to pharmacopoeial guidelines, such as USP <1223> criteria, remain essential to ensure their efficacy and reliability for widespread adoption.
When selecting an MMM as a Process Analytical Tool (PAT), it is crucial to ensure correlation with traditional methods.
Global pharmacopoeias recommend comparing results from alternative methods to compendial methods, typically using common microorganisms. The Japanese Pharmacopoeia specifically suggests using microorganisms in a starved state to simulate real-world contamination events.
In response to these guidelines and industry needs, a comprehensive comparison study was conducted between the Sievers Soleil Rapid Bioburden Analyzer and traditional bioburden test plating methods. This extensive study involved:
- 11 individual microorganisms and a mixed culture
- Two laboratory sites
- Six analysts
- Six instruments
The evaluation followed USP <1223> (Validation of Alternative Microbiological Methods) guidelines, assessing:
- Range
- Linearity
- Robustness
- Precision
- Reproducibility
- Ruggedness
This research presents the methodology, results, and conclusions of the correlation study, demonstrating the efficacy and reliability of the Sievers Soleil Rapid Bioburden Analyzer as an alternative to traditional plating methods in pharmaceutical microbial testing.
Study Design: Comparison of Traditional Plating Methods to Rapid Bioburden Analysis for Bioburden Detection
As part of the daily start up, Negative Controls and System Suitability Standards were run and had to pass the acceptance criteria before testing could begin.
Working stock solutions were created for the following organisms:
- A. brasiliensis
- B. cepacia
- B. diminuta
- B. subtilis
- C. albicans
- E. coli
- P. aeruginosa
- R. pickettii
- S. aureus
- S. enterica
- S. maltophilia
- Mixture of B. diminuta, R. pickettii, S. maltophilia, & B. cepacia
Sample Preparation:
- Concentrations targeted at 0.05, 0.1, 1, 10, and 100 CFU/mL
- Sample volumes were made in 250mL bottles then aliquoted into 100mL samples—one run on Soleil, one for plating
- Serial dilutions were performed to achieve desired concentrations, and solutions were added to buffered Water For Cell Culture (WFCC) to maintain cell integrity.
Traditional Plating Method:
- Agar plates were prepared using: T
- ryptic Soy Agar (TSA) for bacteria
- Sabouraud Dextrose Agar (SDA) for fungi (as directed per USP <61> and USP <62>)
- Sample filtration
- Each solution filtered through a manifold onto a sterile filter
- Filter aseptically transferred to the appropriate agar plate Incubation
- Plates incubated in a cell incubator Minimum incubation period: 3 days
Note: For the 100 CFU/mL samples, flood plates were used.
Results and Conclusions: Demonstration of Detection and Quantification Per Criteria Outlined in USP <1223>
The average % recovery is detailed in the chart below. Average linearity of all organisms was 0.983 and average coefficient of variation (CV%) was 28% for Soleil and compendial plates.
The comprehensive correlation study between the Sievers Soleil Rapid Bioburden Analyzer and traditional plating methods met the criteria outlined in USP <1223>.
- Detection and Quantitation: Successfully detected and quantified Gram-positive bacteria, Gram-negative bacteria, Yeasts, and Mold
- Performance metrics: Demonstrated acceptable Accuracy, Linearity, Precision, Range, Robustness, and Ruggedness
- Sensitivity: Limit of Detection (LoD): 0.05 CFU/mL; Limit of Quantitation (LoQ): ≤1.0 CFU/mL
In conclusion, the Sievers Soleil demonstrated correlation to traditional plate counts in CFU/mL. The above criteria demonstrates that the Soleil is a reliable, efficient, sensitive alternative to compendial plating methods.
Modern Microbial Methods with Sievers Soleil
The Sievers Soleil Rapid Bioburden Analyzer serves as a promising complementary tool to compendial testing, delivering near real-time, actionable data with demonstrated correlation to traditional plate counts in accordance with USP <1223> guidelines. As a Process Analytical Technology (PAT) solution that empowers manufacturers to implement more robust, risk-based contamination control strategies, Soleil can enhance overall process control by enabling rapid detection of microbial excursions and triggering same-day corrective actions.
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Sievers Soleil Rapid Microbial Method Verification Testing for USP <1223>
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