Managing Objectionable Events in cGMP Cleanrooms: A Polyphasic Approach

New technologies are finding far more organisms than before, creating the need for a rational, comprehensive approach to environmental monitoring.

By J.S. Sidhu, C.T. Tyler, G. Ma, and M. Samadpour, Molecular Epidemiology, Inc., and E.J. Brandreth, Althea Technologies

In biopharmaceutical manufacturing, an accurate and comprehensive knowledge of the entire process flow is a critical part of a thorough cGMP microbial control program. The goal is to have precise data highlighting where incursion points may exist, and control those points to limit or prevent migration into the process flow. Historically, the identification of cleanroom environmental monitoring (EM) isolates has been limited to the genus or group level for most organisms, with some ability to provide species level identification only if absolutely required.

Recently, rapid technologies have become available, especially DNA-sequencing based systems, to more quickly identify microorganisms. These techniques can help manufacturers to understand and investigate potential physical and temporal sources of contamination. However, while such genetic-based systems may be rapid, they are still inherently limited by their inability to discriminate between species and some genera in some critical categories. Additionally, an identification match is only possible if the organism and its reference are adequately differentiated and in the manufacturers’ database. Thus, with this increase in technology comes the likelihood of identifying a far greater number of potentially “objectionable” environmental organisms (e.g., coliforms, pathogens) than ever before, as well as challenges in knowing what to do with the additional data.

It’s important, therefore, that new tools and the data they produce not take precedence over the practical application of good microbiological practices. If anything, these technologies increase the necessity for manufacturers to take more rational and manageable approaches to environmental monitoring.

We recommend a polyphasic approach, one that makes use of key characteristics or properties of a particular unknown organism in tandem with its genetic information. Such an approach provides a more definitive taxonomic identification; it also avoids the pitfalls of potentially misidentifying organisms due to the limitations of various commercial phenotypic and genotypic microbial ID systems and their databases. This article will detail such an approach taken recently during work at facilities of Althea Technologies in San Diego.

An Organism of Concern

In the current study, Molecular Epidemiology Inc. (MEI) used a polyphasic identification approach, combining a genetic-based microbial ID assay (16S rRNA sequencing) with a broad spectrum for phenotypic and biochemical analyses, to accurately identify a potentially objectionable environmental organism submitted by Althea. The organism of concern was recovered during routine environmental monitoring, while aliquoting in a tissue culture hood. The procedure involved a biofermentation Master Cell Bank (MCB), with Escherichia coli as the platform.

Since the polyphasic determination presented the EM isolate as belonging to the same genus and species as the MCB and was a potentially objectionable organism (coliform), further analysis of its potential for pathogenicity was required. The process included targeted PCR analysis of toxins as well as attachment factors associated with recognized pathogenic forms, such as Enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC), Enterohemorrhagic E. coli (EHEC), and Enteropathogenic E. coli (EPEC), or closely related species such as Shigella spp.

The following organisms were used for Quality Control purposes in the various analytical tests performed: E. coli ATCC 8739, Shigella sonnei ATCC 25931, Klebsiella pneumoniae ATCC 10031, and Klebsiella oxytoca ATCC 43863. These were obtained as lyophilized cultures, reconstituted and sub-cultured as recommended for isolated colonies. The study was further supplemented with ATCC-derived strains, Enterobacter cloacae ATCC 23355, Pseudomonas aeruginosa ATCC 9027, E. coli ATCC 25922 and E. coli O157:H7 ATCC 35150, from MEI’s QC collection as well as two additional E. coli strains from lab and environmental sources.

Additionally, Althea submitted an isolate of the Master Cell Bank platform strain used in processing. Representative cultures of each microorganism were subjected to 16S rRNA sequencing, colonial, morphological, biochemical (Vitek GNI, bioMérieux) observations and DNA fingerprinting using pulsed field gel electrophoresis (PFGE analysis—with restriction enzymes XbaI, AvrII and SpeI). Analysis of toxigenic and pathogenic potential was conducted using a proprietary polymerase chain reaction (PCR) method (MEI, 2010) to determine the presence or absence of toxin markers and pathogenicity factors in the reference and subject strains.

Confirming Identification
Microbiology
An initial isolate recovered from a cleanroom sanitary fill operation (aliquoting of MCB) was received for identification by 16S rRNA genetic sequencing. The isolate was reported as a member of the Family Enterobacteriaceae (Figure 1)—consistent with the industry and federally recognized guidelines (CLSI, 2008) for genetic sequencing techniques for identification. Further taxonomic classification was required, and the application of a polyphasic microbiological ID (PMID) method clearly differentiated and reported the isolate as E. coli (Figure 2).

Further comparisons with related strains with pathogenic potential differentiated and confirmed this identification (data not shown). Of concern was the equally identical taxonomic ID presented by the QC culture of E. coli O157:H7 ATCC 35150. Furthermore, sequence alignment data (Figure 3) indicate the indistinguishable similarity in the 16S rRNA gene sequence associated with the pathogenic species. Therefore, reliance on genetic-based ID or even a microbial ID conclusion would not rule out the possibility of a frank pathogen.

Microbiology

Microbiology
To confirm that the EM isolate had not acquired any pathogenic potential, polymerase chain reaction (PCR) analyses for specifically targeted toxigenic and pathogenic markers demonstrated the absence of these markers in the submitted organism (Figure 4, lane 2) when compared to the control organisms (Figure 4, lane 7).

Microbiology
Subsequent to the PMID of the EM isolate as E. coli, DNA Fingerprinting comparison of the entire genome by PFGE analysis (employing three distinct restriction enzyme digests) demonstrated the differences in genetic profiles from other similarly named control and environmental strains (Figures 5a, 5b, 5c).

The isolate under investigation is identified in the dendrograms and its genomic pattern is clearly distinguishable from the other strains and yet indistinguishable from the Master Cell Bank platform strain. This, having already demonstrated the strain’s lack of associated pathogenicity, confirms its clonal relationship with the MCB.

Bioprocess Strain: A Science-Based Evaluation

In our study, the detection of an E. coli recovered during environmental/production monitoring in the tissue culture hood was cause for thorough and appropriate investigation. Through the use of a detailed polyphasic analysis approach, the isolate was verified to be the same taxonomic genus and species as the bioprocess strain. Further testing via DNA fingerprinting by pulsed field gel electrophoresis demonstrated that this was indeed the exact same strain as used in the fermentation process. In addition, the strain was demonstrated to be unequivocally non-pathogenic and non-toxigenic by PCR analysis of pathogenic and toxigenic potential. The entire process using a thorough science-based evaluation is illustrated in Figure 6.

MicrobiologyAs such, this isolate was not an “objectionable” organism. The manipulation of the fermentation strain—sampling, pipetting with expected minute aspirations, etc.—can lead to isolated incidents of detection of the bioprocess strain in any well-controlled facility. The results presented here show the benefits of polyphasic ID and the limitations of genetic or phenotypic (rapid) microbial ID methods when used individually. Incorrect, incomplete or inadequate identification and characterization of strains of E. coli cannot exclude potential pathogenic forms such as specific pathogenic serotypes (e.g., O157; O104), Enterohemorrhagic strains (EHEC) as well as E. coli subtypes STEC, ETEC, and EPEC, respectively. By detailed polyphasic analysis and demonstrating that any isolate of E. coli is linked directly to the upstream production process, the facility is verified to be operating with suitable environmental control regarding the detection of coliforms.

EM and Excursion Response

A company should have an established SOP which clearly identifies the actions to be taken, including clear directions if and when regulatory notification (FDA, USDA, CDC, etc.) would be required. In many cases, the detection of an enteric organism or a frank pathogen may include the determination of non-pathogenicity, or absence of virulence factors and potential for toxigenicity, which allows the manufacturer to better manage associated risk.

Microbiology

The key is to understand the process and potential incursion points, and to complement them with Corrective Action/Preventive Action, based on the potential toxigenicity or pathogenicity of the organism. This, coupled with a science-based Risk Assessment, allows for a more rigorous evaluation of potential EM excursions.


References
1. Clinical Laboratory Standards Institute. Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing: Approved Guideline. CLSI document MM18-A. Wayne, PA, 2008.
2. Molecular Epidemiology, Inc. dba IEH Laboratories & Consulting Group. IEH E. coli O157, Stx-producing E. coli, (STEC) with Intiman and Salmonella Test System. AOAC-RI PTM 100701, AOAC Research Institute, Gaithersburg, MD, USA, 2010.

About the Authors
Jaspreet S. Sidhu, Ph.D. (VP, Business Development and Pharmaceutical Microbiology), Connor Tyler (Scientist), Greg Ma (Director of General Microbiology), and Mansour Samadpour, Ph.D. (President, CEO) represent Molecular Epidemiology, Inc. in Lake Forest Park, Washington. E.J. Brandreth is VP of Quality and Regulatory Affairs for Althea Technologies, San Diego.

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