The Future of Microbial Contamination Testing: Rapid and Real-Time

How Pharmaceutical Companies Are Ensuring Water Safety and Fighting Bioburden with On-Line Monitoring

دقائق

12 Dec, 2024

Areen Kalantari, Thornton

In the realm of pharmaceutical manufacturing, the purity of water is paramount. Microbial contamination, or bioburden, poses a significant threat to both consumer health and the reputation of pharma companies. Traditional microbial testing methods are often time-consuming, error-prone, and labor-intensive. The emergence of real-time microbial analyzers offers a promising solution to these challenges, revolutionizing the way pharmaceutical manufacturers approach bioburden control.

water testing pharmaceutical manufacturing

Microbial contamination, also known as bioburden in the context of pharmaceutical waters, refers to the number and types of viable microorganisms present in a water sample, including bacteria and fungi. Such contamination is a significant concern for pharmaceutical companies, as microbial contamination can compromise the safety and efficacy of medicinal products. Pharmaceutical waters must meet stringent purity standards set by regulatory governing bodies, as they serve as a critical component in the manufacturing of medications. Consequently, rigorous microbial testing is essential to ensure water purity and protect both patient health and the integrity of pharmaceutical manufacturing processes.

The Dangers of Bioburden in Pharmaceutical Waters

The risks associated with microbial contamination in pharmaceutical waters cannot be overstated. Contaminated water poses a direct threat to consumer health, particularly for individuals with compromised immune systems or those undergoing critical treatments. Ingesting or being exposed to water contaminated with pathogenic microorganisms can lead to severe health complications, such as bloodstream infections, gastrointestinal illnesses, and other life-threatening conditions.

For many drug companies, the implications of water contamination can be equally devastating in terms of financial and reputational damage. The pharma industry estimates that the cost of a single recall can range from hundreds of thousands to millions of dollars, depending on the severity and scope of the issue. Such an incident can tarnish a company’s reputation through the erosion of consumer trust and long-lasting negative brand association.

In an age where information spreads rapidly through social media and online platforms, negative publicity can lead to substantial declines in sales, stock prices, and investor confidence.

For pharma brands, the stakes are incredibly high: navigating the balance between maintaining operational efficiency and ensuring regulatory compliance with the highest standards of water quality is imperative for sustainable success.

Traditional Testing Methods for Monitoring Microbial Limits

The history of microbial testing can be traced back to the late 19th century, during the burgeoning field of microbiology. Pioneering scientists like Louis Pasteur, Julius Petri, and Robert Koch laid the groundwork for understanding the role of microorganisms in health and disease. By the early 20th century, as the pharmaceutical industry began to grow and the production of medicines became more complex, the need for reliable methods to detect microbial contamination in products, including water, became apparent.

The first bioburden enumeration tests primarily relied on direct observation and culturing techniques. It wasn't until the mid-20th century that standardized methods for quantifying microorganisms in water were developed, leading to the establishment of the first pharmacopeial guidelines. In the United States, the United States Pharmacopeia (USP) began to outline acceptable microbial limits and testing methods for pharmaceutical products, emphasizing the importance of quality control in water used for manufacturing.

Plate Counting

Direct plate counting involves collecting water samples, culturing them in a laboratory, and counting the colonies that form over several days. While these techniques have been standard practice, they come with significant limitations:

Time-consuming: Plate counting is a labor-intensive process that typically requires 5-7 days to produce results.

Subjective: Numerating colonies can be subjective and prone to human error due to manual counting and variations in incubation conditions.

Limited sensitivity and specificity: Plate counting may not be sensitive enough to detect low levels of contamination. Moreover, plate counting provides only a snapshot of contamination levels at a specific point in time, rather than a continuous view of water quality.

Membrane Filtration

Membrane filtration became popular in the mid-20th century, particularly for testing larger volumes of water. In this technique, water samples are passed through a filter with pores small enough to trap microorganisms (typically 0.45 μm in diameter). The filter is then placed on a culture medium and incubated, similar to the plate-counting method. Therefore, membrane filtration faces similar drawbacks:

Lengthy process: Membrane filtration can be time-consuming, especially if large volumes of water are being tested.

Error-prone: The process requires meticulous handling and can be sensitive to the physical and chemical properties of the water sample, which might inhibit microbial growth. Additionally, any microorganisms that are present but not culturable will go undetected, leading to potential gaps in monitoring and possible overgrowth of dangerous colony-forming units.

While traditional methods for microbial load testing have served as critical tools for many years, they come with significant limitations that can jeopardize water quality and, consequently, patient safety. Both techniques mentioned specifically rely on counting colony-forming units (CFUs) which requires time for the microbes to grow into colonies that can be physically counted. Thus, these techniques are resource-intensive, requiring significant time and labor for sample collection, processing, and analysis, and provide only a snapshot of microbial levels at a specific moment. They therefore don't provide continuous monitoring of water quality. In an industry where contamination events can occur rapidly and have immediate consequences, this delay in obtaining results can be particularly problematic.

Furthermore, not all microorganisms can be easily cultivated in laboratory settings, meaning that some pathogens may remain undetected. This selectivity may lead to an underestimation of the total microbial load, as some microorganisms present in the sample may not be able to grow on the chosen media.

Enter continuous, real-time microbial analyzers, which present a modern solution to the challenges posed by traditional methods. These advanced systems provide immediate detection of microbial contamination, enabling proactive intervention.

Real-time microbial detection, as used in analyzers like our 7000RMS, relies on two established optical measurement techniques: Laser Induced Fluorescence (LIF) and Mie Scattering. These techniques work together to detect and quantify microorganisms in high-purity waters like those used in pharmaceutical production.

Here’s how the process works:

Laser Induced Fluorescence (LIF)  takes center stage by harnessing the natural fluorescence of microbial metabolites, such as NADH and riboflavin. When the 405 nm laser in the analyzer illuminates the water flowing through it, these metabolites absorb the light and enter an excited state. As they return to their original state, they release energy in the form of fluorescent light, which is then captured by a photodiode in the 7000RMS.

Mie Scattering complements this process by measuring how light interacts with the microorganisms themselves. As the laser light passes through the water, any microorganisms present scatter the light. A detector records how the light is scattered, revealing critical information about the size and quantity of the particles. The 7000RMS synthesizes data from both the fluorescence and scattering detectors. When both signals align under specific criteria, the analyzer identifies a single microorganism as an auto fluorescent unit (AFU). The results are displayed live on a user-friendly touchscreen interface, providing clear and immediate insights into water quality.

At a basic level, AFU counting is like instantly snapping a vibrant and detailed photo while CFU counting is like waiting for a basic and hollow sketch to be rendered. This real-time capability is vital for pharmaceutical industries where swift detection of contamination is crucial.

Additionally, AFU allows for a direct-count method of numerating individual cells, unlike CFU counting, which only estimates the population of an entire colony. Subsequent AFU measurements can be taken directly within the process environment, reducing the risk of contamination associated with traditional sampling methods. This in-situ monitoring provides continuous insights into water quality, offering a dynamic view of microbial activity

Benefits of Continuous Water Testing

Speed	Continuous	Direct Measurements	Enhanced Sensitivity	Immediate Actions
With results every two seconds, real-time monitoring offers rapid detection of contamination. While plate counting can take 5-7 days, the 7000RMS captures 216,000+ measurements in the same time period.	Unlike static plate counts, which provide a snapshot at a single moment, the 7000RMS continuously analyzes water flow, enabling proactive trend identification and early interventions.	Advanced optical measurement technology counts individual microorganisms and does not rely on growth, eliminating the variances that come from colony formation in plate methods.	The 7000RMS is capable of detecting microorganisms as small as 0.3 µm. The analyzer also reveals viable but non-culturable (VBNC) bacteria —microorganisms that traditional methods might miss.	The trending data from the 7000RMS helps determine when water system sanitization is necessary and facilitates quick responses to contamination. This proactive approach allows for risk reduction of releasing contaminated water while optimizing sanitization frequency.

How On-Line Water Monitoring Supports Pharma Compliance

In the pharma industry, microbial monitoring of Purified Water, Ultrapure Water, and Water for Injection is not merely a good quality control practice; it’s a regulatory requirement. Standards established by the United States Pharmacopeia (USP) dictate the conditions under which microbial contamination must be tested.

For instance, USP <61> outlines requirements for microbial limits, specifying acceptable levels of microorganisms in non-sterile products and emphasizing the importance of water quality. USP <62> details methods for detecting specific pathogens, including E. coli, Salmonella, and Pseudomonas aeruginosa, in sterilized water. Moreover, USP <1231> on Water for Pharmaceutical Purposes from the USP has consistently endorsed on-line, continuous monitoring of pharmaceutical waters. This approach ensures that historical in-process data is collected, allowing for effective control of water systems, and maintaining the production of water that meets acceptable quality standards.

The 7000RMS aligns with USP <1223>, which encourages the validation of alternative methods that demonstrate advantages in accuracy and sensitivity. It also adheres to guidelines published by the FDA and EMA for alternative microbiological measurement methods, ensuring compliance with regulatory standards while enhancing the monitoring process.

The future of testing bioburden levels in pharmaceutical water lies in the adoption of real-time microbial analyzers. These advanced technologies offer a paradigm shift in how pharma companies can approach microbiological contamination control, enabling a more proactive, data-driven approach.

By adopting real-time microbial analyzers, pharma companies can not only ensure the safety and quality of their products but also demonstrate their commitment to regulatory compliance.

The advancements in microbial testing exemplified by the 7000RMS ensure that pharmaceutical manufacturers can deliver safe and effective products, safeguarding both patient health and their own operational integrity in an increasingly demanding regulatory landscape.

As the industry continues to evolve, embracing real-time monitoring will be paramount for maintaining the highest standards of water quality, reducing risks to consumer health, and protecting brand reputation.

Real-Time, On-Line Microbial Monitoring for Pharmaceutical Waters

METTLER TOLEDO Thornton’s 7000RMS™ is an on-line analyzer for real-time measurement of microbial contamination (bioburden) in pharmaceutical water. Laser-based technology enables immediate detection and quantification of microorganisms directly from the water sample, overcoming limitations of time-consuming growth-based methods.

Areen Kalantari

Global Pharmaceutical Segment Specialist

Areen Kalantari holds a B.S. in Biological/Chemical Engineering and a Master of Business Administration (MBA) in International Business. He has experience in both biotechnology applications and pharmaceutical waters. Areen has previously worked as a pH, dissolved oxygen, and CO2 product manager, working with global pharmaceutical and biotech customers to implement effective SOPs and to train personnel for their cell culture and fermentation processes. Areen is a voting member of the American Society of Mechanical Engineers – Bioprocess Equipment (ASME-BPE) standards committee. He contributes to the Process Instrumentation subgroup to help implement standards for the bioprocessing and pharmaceutical industries, focusing on conductivity, pH, TOC, and various other process technologies. In his current role, he supports the global market for pharmaceutical water systems and USP regulatory aspects of WFI and PW systems regarding required tests to be conducted.