Quality control for potential microbial contamination in pharmaceutical manufacturing is of growing importance. While traditional methods for microbial detection require visual acuity under optimum conditions, rapid methods are being developed that are more objective and less impacted by subjectivity or variability. The transformation to Rapid Microbial Methods (RMM) has been initiated to overcome some of the impediments associated with traditional methods, for example, time to results is now days, compared to weeks.

Vicki A. Barbur Ph.D., Senior Director at Battelle discusses real-time microbiology, the critical factors to determining the rapid method of choice, adoption in the pharmaceutical industry, and accessibility to new technologies. –KB
 
Contract Pharma: What are the driving forces for Real-Time microbiology?

Vicki A. Barbur: Quality control for potential microbial contamination associated events in pharmaceutical manufacturing is receiving increased attention in the current risk-averse environment, although these industries are routinely slow to embrace change. Traditional methods for microbial detection depend on the growth of microorganisms in cultured media. During the growth period, frequent checking by eye is necessary to assess activity. Visual acuity, which varies person to person, and can be even more severely impacted under less than optimum lighting conditions, means microbes are detected only when growth reaches a high number of colony-forming units.

Rapid methods are by design more objective and are less impacted by observer subjectivity or variability. The transformation to Rapid Microbial Methods (RMM) has been initiated by the development of new approaches that eliminate many of the downsides associated with classical methods, for example, time to results is now days, often hours, compared with the weeks of the past. Additionally, it is well recognized that traditional methods of microbial detection tend to be labor-intensive, whereas now it has been proven that RMM can be sensitive, precise and quick. Many companies are considering RMM to improve their bottom line as they implement lean manufacturing principles, thus obtaining quicker product release is key to eliminating several forms of waste, namely, inventory and hold times. Industry is now actively looking at serious return-on-investment calculations to justify the implementation of RMM.
 
CP: What are critical factors to determining the rapid method of choice?

VB: The various RMM in use have different strengths and limits of detection. Selection still depends on the application. For example, an RMM may align more closely with an individual company’s process flow or work more effectively with a specific type of sample or product type. Selection requires the user typically to determine what performance criteria are important for their manufacturing or laboratory process. Some of the RMM attributes that a user would want to assess are performance criteria, including specificity, accuracy, limits of detection, limits of quantification, linearity, ruggedness and robustness. In addition, the user will consider the cost to implement, validate and operate on a routine basis (e.g., any consumables). Tradeoffs exist when evaluating strengths and weaknesses. For instance, an end user may require that any positive results identify the organisms. In this case, they would require the test method to be nondestructive. The tradeoff may be a longer time to result, yet the method would meet the requirement of being nondestructive.

Detection levels vary depending on the test method. Most systems have some interference from background noise. For return on investment, it makes sense to select a system that can screen the broadest range of a company’s product line for bacteria, yeast and mold. Different systems present different challenges. An RMM that detects carbon dioxide will have difficulty with anaerobic bacteria or buffered products. A system that looks for color changes will not be able to test, for example, pigmented products. Solid or highly viscous samples may pose challenges for systems that rely on filtering.

The detection limit varies with different rapid methods. In general, enumerative and DNA-based methods have a lower limit of detection. Growth-based methods offer a greater sensitivity yet take more time and are less accurate. Nongrowth-based rapid methods may have greater accuracy than growth-based rapid methods. Organisms present in the sample may be viable yet nonculturable because of injury or media conditions, and therefore they cannot be detected by growth-based rapid methods.

CP: What is the adoption curve in the pharmaceutical industry?

VB: Regulatory agencies world-wide are enhancing awareness of RMM as submissions and approvals become more common. Global regulatory agencies are committed to accepting process changes or improvements if the proposed change is equivalent to or better than the existing process. The European, Japanese and U.S. Pharmacopeias state clearly that an alternative method may be used. For products regulated by the U.S. Food and Drug Administration, the comparability protocol streamlines the submission and approval process. Submitted for review and approval before initiating the validation process, the comparability protocol simply outlines the studies that will be performed and how the study results will be interpreted. Overall the goal is to demonstrate that the proposed protocol change produces results that are equivalent to or better than the existing method.

CP: What accessibility is there to new technologies?

VB: The pharmaceutical industry has been slow to adopt RMM because no one method generally meets all the industry expectations with respect to detection limit, notably enumerative capability and routinely low cost. We advocate for a new RMM utilizing Raman spectroscopy that provides a resource effective bio-identification system (REBS) and eliminates the need for microbial growth. Raman scattering occurs usually from a laser light in the visible, near infrared or near ultraviolet range. When the laser interacts with a molecule, the energy of the laser photons is shifted up or down. This shift in energy provides information about the vibrations and rotations of the molecule. Because each molecule has its own unique Raman spectrum, or fingerprint, Raman spectroscopic techniques can therefore be used to identify microorganisms.

With REBS, typically a test sample is first retained on a supported film, and the surface is examined for microscopic particulates. A spectral signature is provided for each particle, which is then statistically correlated with spectral signatures in a library composed of Raman signatures of known microorganisms. If there is a match, a microbial identification is provided. The technology can target a single cell for microbial identification, and, under the right conditions, enumeration, within minutes. Additionally, REBS can be used to detect viruses too, whenever the need arises. The capabilities offered by this kind of system allow for the analysis of air and liquid samples, and almost anything that can be passed through the filtration membrane/support film in a static or dynamic mode.

Optical spectroscopic-based RMM represent the next generation in real-time or close to real-time detection and identification opportunities for the pharmaceutical and biopharmaceutical industries. These types of technologies offer a unique advantage over conventional, growth-based methods for monitoring the state of microbiological control in manufacturing environments. They represent significant progress toward the acceptance of microbiology Process Analytical Technology (PAT) solutions that may eventually support replacement of offline or laboratory-based assays and deliver real-time release of products.