Understanding the Variability in Bioburden Test Results in Biomanufacturing

Peer-Reviewed

Submitted: March 9, 2025

Accepted: March 13, 2025

Abstract

Variability in bioburden testing presents a significant challenge in ensuring accurate microbial enumeration during biopharmaceutical manufacturing. This study provides a quantitative framework for understanding and minimizing errors in colony-forming unit (CFU) counts, integrating regulatory guidance with statistical modeling. Through detailed analysis, the author demonstrates how factors such as microbial distribution, dilution steps, and test volume selection contribute to variability, particularly at low CFU levels where Poisson-distributed behavior dominates. Using models grounded in relative error and confidence-based metrics, the author calculates the percentage error associated with replicate counts and test conditions. A root sum of squares approach is further proposed to estimate total combined error from independent sources that include CFU/plate variability, number of replicates, serial dilution, and sample volume. This work offers a statistically rigorous method to enhance microbial detection reliability, thereby supporting consistent quality control and regulatory compliance in biopharmaceutical environments.

Bioburden testing plays a crucial role in ensuring the microbiological quality of biopharmaceutical products by quantifying microbial contamination at various stages of production. Accurate microbial enumeration is essential for compliance with regulatory standards, product safety, and process control. However, numerous factors introduce variability in colony-forming unit (CFU) counts, including microbial distribution, test volume selection, dilution errors, and plating techniques. Understanding the statistical principles that govern these factors is critical to minimizing error and improving the reliability of bioburden testing.

One of the key challenges in microbial enumeration is establishing an acceptable range for CFU counts per plate to ensure accurate results while avoiding errors caused by overcrowding or underrepresentation. Regulatory guidelines such as United States Pharmacopeia (USP) <1227>, Validation of Microbial Recovery from Pharmaceutical Articles, and American Society for Testing and Materials (ASTM) standards provide specific CFU ranges to maintain consistency in bioburden assessments. Additionally, sources of variability such as pipetting errors and microbial recovery rates can significantly impact CFU enumeration. Addressing these challenges requires the application of statistical models to quantify errors and establish robust bioburden testing methodologies that enhance data integrity.

References

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About the author

Naveenganesh Muralidharan, nmural@agcbio.com, is senior manager, Manufacturing Science and Technology (MSAT), at AGC Biologics.

Article details

Pharmaceutical Technology®
Vol. 49, No. 4
May 2025
Pages: 20-23

Citation

When referring to this article, please cite it as Muralidharan, N. Understanding the Variability in Bioburden Test Results in Biomanufacturing. Pharmaceutical Technology 2025 49 (4).