One of the most important aspects of brand protection in Pharmaceutical, Medical Device, Personal Care and Cosmetics, Nutraceuticals and Biotechnology industries is environmental monitoring and the documented control of microorganisms

Routine environmental monitoring, accurate identification and tracking of microorganisms is required to recognize potential routes of contamination and demonstrate acceptable sterility levels throughout a given manufacturing process.

Regulatory Recommendations

According to the FDA’s Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice:

“The goal of microbiological monitoring is to reproducibly detect microorganisms for purposes of monitoring the state of environmental control. Consistent methods will yield a database that allows for sound data comparisons and interpretations.”

Many identification methods have been used over the past decade, with varied levels of success, to characterize recovered microorganisms and yield a quality database.

Genotypic Methods

AccuGENX-ID™

The use of ribosomal DNA sequences for the purposes of microbial taxonomic classification has been in practice for many decades. At Accugenix with the AccuGENX-ID™ service, all or part of the 16S rRNA gene is amplified and sequenced for bacterial identification, BacSeq. For fungi, FunITS, the ITS2 region of the rRNA gene region is primarily analyzed. We can also use the D2 region. The data are then analyzed and aligned in order of increasing genetic distance to relevant sequences in our validated library.  The results are clearly displayed on our cGMP compliant reports.

Thorough identification of the microbial population in the manufacturing environment allows for rapid and definitive resolution of sterility failures and other excursions. Additionally, this information can be used to create tracking and trending reports on a regular or routine basis, providing detailed analysis regarding the state of environmental control within the manufacturing area for our customers.

Strain Typing

Strain typing or characterization of microorganisms in industrial settings, especially in aseptic manufacturing industries, is an important part of a comprehensive environmental monitoring program. Of equal importance to the probiotics and nutraceutical industries is the confirmation of specific bacterial strains used for production. While standard genotypic identification targets, such as 16S, D2 and ITS rDNA sequencing, along with the novel proteotypic identification method, increase the ability to accurately and consistently ID and track and trend microorganisms at the species-level, some common microbes cannot be resolved by this approach alone. Furthermore, in the case of a major excursion or sterility failure where characterization to the strain level can be critical, strain typing is the best resource.

Increased discrimination at the strain level can be achieved by looking at highly variable single or multiple loci (regions) in the genome, usually housekeeping genes. By combining standard genotypic identification methods with multi-locus sequence typing (MLST) or single-locus sequence typing (SLST) it is possible to resolve some of the most difficult organisms to trend and track in the pharmaceutical, medical device and nutraceutical industries.

The RiboPrinter^® is an additional microbial characterization system that compares bacteria for strain typing. Ribotyping is an automated process which extracts and cuts DNA with restriction enzymes, then visualizes the banding pattern using a chemiluminescent probe. The resulting pattern or fingerprint is compared to others in a database and assigned to a RiboGroup for characterization. This fingerprint can be used for tracking at the subspecies or strain level.

Proteotypic Method

AccuPRO-ID^®

AccuPRO-ID^®, the first practical proteotypic identification method for bacteria, utilizes matrix-assisted laser desorption/ ionization – time of flight (MALDI-TOF) mass spectrometry. The analysis of a small amount of sample results in a protein spectrum based on ribosomal proteins. The spectra are then compared to a validated database for identification and generation of a cGMP compliant report. Accugenix optimized MALDI-TOF for use in bacterial identification and backs the method with our 16S rDNA sequencing technology and its substantial library when an initial ID cannot be made by MALDI-TOF.

Phenotypic Methods

VITEK^® 2 Compact

This clinical-based phenotypic method requires a healthy organism with specific growth conditions prior to processing, and usually relies on visual analysis of biochemical reactions catalyzed by an organism. The results are dependent on growth parameters and effected by the variability in the isolate’s cellular and colonial morphology, staining characteristics and phenotypic properties and require ancillary testing, such as a Gram stain. Interpretation of these results can be very subjective leading to higher inaccuracies in identifications.

FAME (fatty acid methyl esters)

This phenotypic method provides microbial identification based on cellular fatty acids that are extracted and methylated. The resultant methyl esters are separated by gas chromatography, and their patterns are compared against a database. Fatty Acid identification is dependent on growth conditions, media and temperature used to grow the organism, which is similar to other phenotypic methods and leads to a higher rate of inaccurate identifications.

Biolog GEN III Microbial ID System

This phenotypic method is based on the carbon source utilization of microorganisms. The results are based on quantifying color reactions in microplate wells, which test the organism’s ability to utilize specific substrates.  The color pattern (biochemical characterization) is compared to established databases. Like all phenotypic systems, data from biochemical reactions can vary due to health of the organism and growth conditions making the results unreliable.

Regulatory Recommendations

DNA sequencing rapidly provides data for identification that are substantially more accurate, robust and reproducible than relying solely on visual phenotypic characteristics since the sequence-based result is not dependent on growth conditions or ancillary testing. This is so well understood and accepted that the FDA recommended the use of genetic methods in their 2004 update to the guidance document, “FDA Guidance for Industry.  Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice.”

“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 failures (e.g., sterility test; media fill contamination).” … “Sterility test isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is also found in the laboratory and production environments, personnel, or product bio-burden. Advanced identification methods (e.g., nucleic-acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and sterility positives, both identifications should be performed using the same methodology.”