A review published in Discover Pharmaceutical Sciences highlights significant limitations in the traditional growth-based microbiological methods currently used to ensure the sterility of pharmaceutical products. The study, authored by Nagi M. Marsit, Sakina Saadawi, and Khairi Alennabi, examines why conventional compendial guidelines are often insufficient for detecting the absolute absence of microorganisms in sterile manufacturing. The authors argue that while these methods remain the mainstay of the industry, they face critical challenges that can compromise patient safety and lead to costly product recalls.
Nonsterile parenteral products pose serious public health risks, including systemic bloodstream infections, bacteremia, and septic shock. Contaminated ophthalmic solutions can cause endophthalmitis and vision loss. Beyond health consequences, failures in sterility assurance result in significant financial damage to manufacturers and healthcare providers. Data from published reports indicate that a majority of recalled drugs are due to a lack of sterility, with a specific analysis of US Food and Drug Agency recalls between 2017 and 2019 revealing that 83.7% of drugs were recalled for this reason.
Traditional bioburden estimation and sterility testing rely heavily on culturing samples in growth media to detect microorganisms. These growth-based techniques are dominant in compendia because they are easy to apply and have reasonable costs. However, the review identifies several arguable limitations, including inefficiency in detecting all microbial contamination and the inability to discriminate between viable and non-viable microorganisms. A significant challenge is the potential for false-positive and false-negative results, which can lead to erroneous judgments about microbial limits in the manufacturing process.
One major limitation is the sampling procedure itself. The standards for bioburden monitoring often use sample sizes that are not adequate to provide sufficient statistical confidence in sterility assurance. For example, the pharmacopeia determines a sample size of only 20 items for a batch of over 500 filled containers. This small sample size, combined with the non-uniform distribution of microbial contamination, can lead to false-negative results. Contaminated samples may not be detected until after distribution, when microorganisms adapt to the product formulation and proliferate during storage.
The review also addresses the issue of viable but not culturable (VBNC) bacteria. These bacteria can exist in adverse conditions, such as the presence of antimicrobial preservatives or stress from disinfection. In this state, they undergo metabolic changes that prevent them from forming colonies on culture media, making them undetectable by normal growth-based methods. Many VBNC bacteria are endotoxin-producing Gram-negative pathogenic species, including Burkholderia cepacia and Escherichia coli. These organisms pose a health risk when resuscitated, as their virulence and ability to produce endotoxins remain unchanged despite their dormant state.
Culture media limitations further complicate detection. The review notes that current compendial media, such as fluid thioglycolate medium (FTM) and tryptic soy broth (TSB), may be suboptimal for certain microorganisms. For instance, FTM aerobic incubation may be unfavorable for strict anaerobes and fungal spores. Additionally, the composition of culture media, including nutrients and selective constituents, can sometimes interfere with microbial growth. Research has shown that autoclaving media containing agar and phosphate can yield oxidizing agents like hydrogen peroxide, which can initially inhibit microbial growth.
Incubation conditions also present variability that affects test outcomes. There is disagreement among regulatory bodies regarding the optimal temperature and duration for incubation. The European Pharmacopoeia recommends separate incubation at 20–25 °C for TSB and 30–35 °C for FTM for at least 14 days. In contrast, the United States Pharmacopeia recommends two subsequent incubations of the same samples to enhance the recovery of human contaminants. This divergence in standards reflects the difficulty in maximizing microbial recovery across different types of potential contamination.
Visual assessment of microbial growth is another source of error. Counting colony-forming units (CFUs) on agar plates is a manual process that is tedious and subject to inter-individual variations. A study of over 13,000 agar plates found 50% variations in counting accuracy among operators, with errors often stemming from the difficulty of counting small colonies or distinguishing artifacts from microbial growth. To address this, the US FDA has recommended double plate counting or secondary analyst confirmation to improve data integrity and reduce the impact of human error on bioburden results.
The traditional sterility test itself has inherent limitations that prevent it from guaranteeing absolute sterility. The long incubation period, typically 14 days, is necessary to support the growth of slow-growing bacteria like Cutibacterium acnes. However, extending the test period further may be unsuccessful if microbes have converted to the VBNC state. Furthermore, broth turbidity becomes visible only when growth density reaches approximately 10^6 CFU/mL, creating uncertainty for less intensive growth. This delay in results can mandate the quarantine of product batches, imposing further delays on market release.
Rapid microbiological methods (RMMs) offer a potential solution to these challenges. These automated systems can detect and quantify microbial contaminants in a fraction of the time required by traditional methods, with some providing results in as little as a few minutes. RMMs include both growth-dependent and growth-independent technologies, such as nucleic acid-based methods, mass spectrometry, and flow cytometry. These methods can detect negligible counts of microbes and verify the absence of contaminants, including VBNC, with higher sensitivity than compendial methods.
Despite their advantages, the adoption of RMMs in large-scale pharmaceutical manufacturing has been hesitant. The review cites high expenses, the need for specialized technical expertise, and regulatory restrictions as key barriers. Validation of these methods is complex, as there is no single method applicable to all types of pharmaceutical products. Each product requires extensive testing to meet method suitability requirements, and regulatory review is necessary to ensure that results are accurate and reproducible.
The authors conclude that while growth-based methods cannot unequivocally determine the absolute absence of viable microorganisms, an integrated strategy is needed to improve sterility assurance. This strategy should include the implementation of RMMs, process automation to reduce human intervention, and a shift towards risk management. By combining traditional methods with advanced detection technologies, manufacturers can better identify and quantify microbiological risks, ultimately enhancing the safety and quality of sterile pharmaceutical products.
The review details specific regulatory frameworks that currently govern these testing procedures, highlighting the practical constraints manufacturers face. The United States Pharmacopeia chapter <71> outlines the traditional sterility test, which utilizes two distinct approaches: direct and indirect inoculation. In the direct method, batch representative samples are aseptically transferred to liquid cultures of tryptic soy broth and fluid thioglycolate medium for incubation. Conversely, the indirect method involves rinsing or solubilizing the sample in a recovery medium, such as physiological saline, before filtering it through a sterile membrane with a pore size not greater than 0.22 micrometers. The resulting suspension is then placed on tryptic soy agar plates. Results from these tests are typically obtained after a 14-day incubation period, with visual inspections scheduled at specific intervals, such as days 3, 5, 7, and 14, to monitor for turbidity or colony growth.
Environmental monitoring programs play a critical role in assessing the impact of the manufacturing environment on product quality. These programs employ various microbiological tests to monitor the bioburden of air, surfaces, operators, and equipment. Common techniques include settle plates, air sampling, finger plates, swabs, and contact plates. Sampling for bioburden testing is performed at various stages, from raw materials to the finished product, and is particularly rigorous for products manufactured with aseptic processing that are not terminally sterilized. Regulatory bodies such as the FDA and the European Medicines Agency provide specific guidance on sampling, requiring that pharmaceutical samples be taken aseptically in sterile containers at the end of the process. The United States Pharmacopeia <1116> details important sampling sites, which are typically located adjacent to areas with potentially high bioburden, such as those in proximity to exposed product, closures, and containers, or in hard-to-clean places and traffic areas responsible for microbial dispersal.
The review also examines the specific types of microbial contaminants that have historically led to product recalls, providing context for the risks associated with sterility failures. The FDA reported that recalls of non-sterile drugs released into the US market between 2012 and 2019 were primarily due to Gram-negative bacteria. The Burkholderia cepacia complex was the predominant identified species, responsible for 102 recalls, followed by Ralstonia pickettii with 45 recalls and Salmonella species with 28 recalls. The Burkholderia genus is frequently identified as a water contaminant in pharmaceutical formulations. In addition to bacterial contamination, 52 incidents were attributed to yeast and mold contamination in non-sterile drugs. Fungal contamination, while less common than bacterial contamination, is involved in approximately 20% of all recalls according to FDA data. A notable example of the severity of fungal contamination is the 2012–2013 outbreak associated with Exserohilum rostratum, a melanized fungus, which resulted in 753 patients suffering from serious complications, including fungal meningitis and spinal infections, with an 8% mortality rate.
Bioburden estimation techniques involve specific culture media designed to detect different classes of microorganisms. Tryptic soy broth is commonly used for the detection of aerobes, facultative anaerobes, and fungi, while fluid thioglycolate medium is utilized for detecting both aerobic and anaerobic bacteria. Solid agar media, such as tryptic soy agar and plate count agar, are typically used for facultative aerobic bacteria. For the cultivation of yeast and molds, media such as Sabouraud dextrose agar, tryptic soy agar, and potato dextrose agar are generally employed. The choice between liquid broth bottles and solid agar plates depends on the specific bioburden estimation standard methods being applied. For sterile products, the total aerobic microbial count and total combined yeast and mold count are used to assess the colony-forming unit count of bacteria and fungi in liquid product samples. Non-liquid samples are extracted using a validated method, often involving rinsing in physiological saline, and are then aseptically filtered through 0.45 micrometer membrane filters to retain microorganisms for subsequent incubation and analysis.
The review emphasizes that the presence of bioburden and sterility assurance are crucial issues in the sterile manufacturing of pharmaceuticals, with failure to control unexpected bioburden levels creating serious health risks. These risks range from severe complications to life-threatening conditions. For instance, contaminated ophthalmic solutions can cause endophthalmitis and risks for vision loss, while non-sterile parenteral products can introduce systemic bloodstream infections, resulting in bacteremia or septicemia. If endotoxin contamination is involved, septic shock may occur. Strict adherence to regulatory requirements for aseptic processing techniques, good manufacturing practices, and sterilization is fundamental to ensuring the sterility of such products. However, despite these standards, microbial contamination remains an existing problem in the sterile manufacturing of pharmaceutical products. The consequences of such failures extend beyond health risks to significant financial burdens, as the recall of released products imposes costs on both manufacturers and healthcare providers. Data from published reports reveal that most recalled drugs were due to a lack of sterility, underscoring the critical need for effective contamination control programs.
Subject of Research: Challenges of growth-based microbiological methods in sterility assurance of pharmaceutical product manufacturing
Article Title: Challenges of growth-based microbiological methods in sterility assurance of pharmaceutical product manufacturing
Article References: Marsit, N. M., Saadawi, S., & Alennabi, K. (2025). Challenges of growth-based microbiological methods in sterility assurance of pharmaceutical product manufacturing. Discover Pharmaceutical Sciences, 1(1), Article 13. https://doi.org/10.1007/s44395-025-00020-6
Image Credits: AI Generated
DOI: 10.1007/s44395-025-00020-6
Keywords: Challenges, growth-based, microbiological, methods, sterility, assurance, pharmaceutical, product, manufacturing, scientific research
News Source: Ophelia Keating. (November 10, 2026). Growth-Based Tests Fail to Guarantee Pharmaceutical Sterility. Scienmag.
