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Charles River Laboratories
Jon Kallay, Senior Technology and Market Development Manager
Why Strain Typing Should Be In Your Microbial Control Toolbox
Jon Kallay
Pharmaceutical manufacturers share a common goal of formulating a safe and effective product. To achieve this, a robust contamination control strategy is a necessity. The importance of this wholistic approach is evident by the recent regulatory emphasis in Annex 1 and PDA’s Technical Report 88 “Microbial Data Deviation Investigations in the Pharmaceutical Industry.” However, despite manufacturers’ best efforts, microbial contamination events are all too frequent and demand a thorough investigation to identify root cause.
Poor root cause investigations are commonly cited in FDA Warning Letters. An example of this from a recent letter states:“For all OOS results found by the retrospective review to have an inconclusive or no root cause identified in the laboratory, include a thorough review of production (e.g., batch manufacturing records, adequacy of the manufacturing steps, suitability of equipment/facilities, variability of raw materials, process capability, deviation history, complaint history, batch failure history). Provide a summary of potential manufacturing root causes for each investigation, and any manufacturing operation improvements. A comprehensive review and remediation plan for your OOS result investigation systems. The CAPA should include but not be limited to addressing the following:
• Quality unit oversight of laboratory investigations
• Identification of adverse laboratory control trends
• Resolution of causes of laboratory variation
• Initiation of thorough investigations of potential manufacturing causes whenever a laboratory cause cannot be conclusively identified
•Adequate scoping of each investigation and its CAPA
• Revised OOS investigation procedures with these and other remediations”
Given the scope of what’s expected for a contamination investigation, it is worth investing in prevention and an environmental monitoring program.
When contamination investigations are required, it is important for microbiologists to use all available tools to quickly find the source of the contamination. Technical Report 88 includes some well-thought-out carefully considered questions to assist in the investigation. One of the key starting points is a secure microbial identification to the species-level. This can help direct the investigation by reviewing the environmental monitoring data to see if the organism has been recovered. If so, strain typing is a valuable tool in identifying and confirming the source.
"When contamination investigations are required, it is important for microbiologists to use all available tools to quickly find the source of the contamination"
Strain typing a is a molecular method that compares DNA sequences of conserved housekeeping genes to look for nucleotide differences. If two organisms are the same strain, it can indicate that they come from the same source. Thus, strain typing can aid in investigations, and this is supported by regulatory and industry guidance. “Any isolates should be identified at least to species and, if available, strain-typed to aid the follow-up investigations with isolates recovered from the manufacturing or material bioburden… If the same species of microorganism was recovered from EM or supporting activities during batch manufacturing or testing, these colonies should be identified to the strain-type level” (TR 88). Another PDA publication, Microbial IDs- Keys to a Successful Program, also states that “having strain typing as part of your identification scheme is very valuable” (pg 77) and “strain typing provides the ability of tracking the contaminant to its root cause” (pg 146). USP <1113> also explains the utility and benefit of strain typing.
If the microbial contaminant is the same as a laboratory QC strain, it’s important to first eliminate laboratory error. This can also be done through strain typing. For example, Staphylococcus aureus can be typed through the spaA gene and Pseudomonas aeruginosa can be typed through the ascA, guaA, mutL, and nuoD genes. The genes are sequenced, and then phylogenetic trees are generated and interpreted. In the example below, the laboratory QC strain Pseudomonas aeruginosa ATCC# 9027 was different from the product contaminant and thus the contamination was not due to lab error.
Figure 1. The genetic differences in the ascA, guaA, mutL, and nuoD genes indicate that the laboratory QC strain is different from the product contaminant.
After laboratory error is ruled out, a review of environmental monitoring data is required. Some additional sampling may also be required. If the same species is recovered, strain typing can identify the source. In the case of a media fill failure, the microbial contaminant was identified as Micrococcus luteus.The species-level identification provided direction to focus the investigation on operators since Micrococcus is a common skin flora. A review of EM trends found recent hits on personnel monitoring as well as some surface sampling. By strain typing the gsp, prfA, and sahH genes, the isolates can be compared. In this case, the strain from Operator A was identical to the media fill strain, indicating that Operator A was likely the source. Operator B contaminated the Pass Through, and all the other isolates were different strains.
Figure 2. Micrococcus luteus isolates that were strain typed using the gsp, prfA, and sahH genes. The phylogenetic trees represent the relatedness of the isolates to each other and are interpreted to make conclusions.
Strain typing can provide beneficial information to quickly locate the root cause of microbial contamination. The primary drawback is that since strain typing is a comparison of different isolates, multiple isolates must first be found and identified. If the same species is not discovered through environmental monitoring, then no definitive root cause can be determined.
While strain typing can be an intensive and technical undertaking, it is become more widely available through contract labs and in core laboratories. The technology has advanced rapidly with results becoming faster and less expensive. The adoption of whole genome sequencing for strain typing is not just a valuable tool for public outbreaks, but also for pharmaceutical manufacturers in investigations. Given the regulatory and business pressure for thorough root cause analysis, microbiologists need to utilize every tool available to quickly remediate contamination events.
The articles from these contributors are based on their personal expertise and viewpoints, and do not necessarily reflect the opinions of their employers or affiliated organizations.
