Compliance and Microbial Identification

by Christine E. Farrance, Ph.D.

Recently, the dietary supplement industry has come under heavy regulatory scrutiny to ensure manufacturers are undertaking appropriate cGMP (current good manufacturing practice) quality control (QC) measures for product safety. To be in compliance, dietary supplements, herbal products and topicals should be tested to determine the microbiological quality and identity of the raw materials and of the final formulations. Manufacturers must confirm that production processes are sufficiently controlled to prevent adulteration of the product and risks to consumers.

Establishing an environmental monitoring (EM) program is one of the most important manufacturing production and process control systems. The program should provide reliable and comprehensive information on the status of environmental quality and the state of facility control by identifying microorganisms. Accurate classification of unknown isolates is an essential first step in understanding the impact organisms have on the manufacturing environment. The identification of microorganisms can be done by different processes, and these methods have different levels of accuracy and reproducibility, with DNA sequencing being recognized as the gold standard for microbial identification. Accurate and reproducible identification methods will yield data that allow for comprehensive and reliable tracking and trending during routine monitoring of the state of production environment control.

The role of a testing program is to verify compliance to specifications set by the manufacturer and confirm the manufacturing processes are rigorously controlled to prevent adulteration of the product. The program establishes a baseline profile of the microbial ecology of a manufacturing environment. It  then acts as a surveillance system that measures against that baseline and documents consistent QC.. The objectives should be to monitor the effectiveness of cleaning and sanitation through surface monitoring, of gowning and personnel behaviors, and to monitor air quality and critical utilities such as water and compressed gasses, focusing on critical control points in the production process. EM programs should specifically detect and reliably identify organisms including routine flora, organisms of concern and indicators of objectionable organisms. The data gathered in a well-designed and executed EM program provide critical information for tracking and trending and allow alerts from normal operating conditions to be addressed before a critical excursion occurs, which can disrupt production and cause supply chain issues. Inaccurate and inconsistent microbial identification can lead to a false sense of control and potentially inappropriate remediation actions. A better understanding of the manufacturing process from the raw materials to the finished product with respect to microbiological quality is gained through an EM program comprising accurate microbial identification and proactive microbial tracking and trending.

When bacterial or fungal isolates are recovered from a production facility, it is extremely important to be able to accurately identify the organism to the species and possibly strain level in order to track the potential origin of the contamination to avoid delays in product release or to complete investigations. Accuracy of identification depends on the method used to generate and interpret the data as well as the library database used as the reference. In order to correctly identify unknown isolates from EM programs, the libraries must contain species relevant to the manufacturing environment. If the library lacks depth of coverage, the interpretation of the data may be inaccurate and unreliable.

Three broad categories of identification methods are currently used: genotypic, proteotypic and phenotypic. FDA states, genotypic methods have been shown to be more accurate and precise than traditional biochemical and phenotypic techniques. These methods are especially valuable for investigations into [critical] failures." Genotypic identification methods involve sequencing different regions of the microorganisms ribosomal RNA genes (16S, ITS2), resulting in an identification to the species or occasionally the subspecies level. Sequencing and analysis of more highly variable genes, such as protein coding genes, is an effective way to provide identification to the strain level for isolates of the same species. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an effective proteotypic technology, which uses a protein spectral fingerprint, consisting primarily of ribosomal proteins, to identify bacteria to the species level. Finally, phenotypic technologies use either biochemical reactions or cell-surface component compositions to identify a microorganism.

Each technology has advantages and disadvantages. DNA sequencing provides the most consistent and unambiguous data set, followed by MALDI-TOF, but both technologies require a substantial capital investment for in-house testing. However, the high level of accuracy can be obtained without the fiscal commitment by outsourcing. The key is to balance the cost effectiveness of the technologies while maintaining a high level of accurate and reproducible microbial identifications.

Consistent identification methods yield a reliable historical database that allows for comprehensive comparisons and interpretations. Systematic and accurate identification of the microbial population in the raw materials, final product and manufacturing environment facilitates  rapid and definitive resolution of alerts and other excursions. If a major excursion occurs that results in a product on hold or production stoppage, a thorough characterization of the microbial population through strain or sequence typing is the preferred method for sourcing the contaminant. Data from EM sample sites should reflect operational considerations, and should be proactively used to create tracking and trending reports on a frequent and routine basis, providing detailed analysis regarding the state of environmental control within the manufacturing area. Any significant change in microbial flora should be considered in a review of the ongoing monitoring data and used in investigations of excursions to affect the mitigation process and root cause determination.

Assuring compliance to the regulatory guidelines for microbial monitoring during the production of dietary supplements, nutraceuticals, cosmetics, personal care products and probiotics requires accurate identification of the organisms in the microbial environment of the production facility and in the components or the product. By using dependable methods and reference databases to obtain identifications, brand owners can be certain that tracking the organisms in the environment, or the organisms that are in the product, will be accurate, consistent and aid in documenting control of the production environment and leading to overall brand protection and consumer confidence.

Christine E. Farrance ([email protected]), Ph.D., senior applications scientist, Accugenix Inc, received her doctorate in cellular and molecular biology from the University of Pennsylvania, a bachelor's in biochemistry from Cornell University and has more than 17 years of experience conducting both applied and basic research using molecular, microbial, genetic and biochemical techniques. At Accugenix, she is involved in coordinating research studies, developing the technical content for marketing materials, designing and implementing technical training for customers and leading efforts in creating publications and presentations.