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
Linked Data in Production: Moving Beyond Ontologies
Managing Objectionable Events in cGMP Cleanrooms: A Polyphasic Approach.
1. E N V I R O N M E N TA L M O N I T O R I N G
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 ac- manufacturers to take more rational and manageable
curate and comprehensive knowledge of the entire process approaches to environmental montioring.
flow is a critical part of a thorough cGMP microbial We recommend a polyphasic approach, one that
control program. The goal is to have precise data highlight- makes use of key characteristics or properties of a
ing where incursion points may exist, and control those particular unknown organism in tandem with its
points to limit or prevent migration into the process flow. genetic information. Such an approach provides a more
Historically, the identification of cleanroom environmental definitive taxonomic identification; it also avoids the
monitoring (EM) isolates has been limited to the genus or
group level for most organisms, with some ability to pro- Genetic ID
vide species level identification only if absolutely required. Comparisons to genetically similar microorganisms
Recently, rapid technologies have become
Genetic Distance Genus Species
available, especially DNA-sequencing based systems,
to more quickly identify microorganisms. These 0.000 Shigella sonnei
techniques can help manufacturers to understand and 0.000 Shigella flexneri
investigate potential physical and temporal sources 0.000 Escherichia coli
of contamination. However, while such genetic-based
0.000 Shigella boydii
systems may be rapid, they are still inherently limited
by their inability to discriminate between species and 0.000 Escherichia sp.
some genera in some critical categories. Additionally, 0.007 Enterobacter hormaechei
an identification match is only possible if the organism 0.0019 Shigella dysenteriae
and its reference are adequately differentiated and in
0.0022 Enterobacter sp.
the manufacturers’ database. Thus, with this increase
in technology comes the likelihood of identifying 0.0031 Cronobacter sakazakii
a far greater number of potentially “objectionable” 0.0081 Citrobacter freundii
environmental organisms (e.g., coliforms, pathogens)
0.0090 Enterobacter dissolvens
than ever before, as well as challenges in knowing what to
do with the additional data. 0.0100 Kiebsiella oxytoca
It’s important, therefore, that new tools and the data Microbial ID Family: Enterobacteriaceae
they produce not take precedence over the practical Conclusion
application of good microbiological practices. If Figure 1. Genetic-based identification reported the unknown isolate as
anything, these technologies increase the necessity for “Family: Enterobacteriaceae.”
2. E N V I R O N M E N TA L M O N I T O R I N G
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.
Figure 2. Polyphasic Microbial Identification (PMID) identified the isolate as Escherichia coli. The following organisms were used
for Quality Control purposes in the
Identities = 416/418 (99%), Gaps = 1/418 (0%) various analytical tests performed:
E. coli ATCC 8739, Shigella sonnei
E. coli O157 23
ATCC 25931, Klebsiella pneumoniae
ACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACAC 82
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
E. coli K-12 213094 ACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACAC 213035
ATCC 10031, and Klebsiella oxytoca
E. coli O157 83 GCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGT 142
E. coli K-12 213034
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGT 212975
ATCC 43863. These were obtained
E. coli O157 143 AGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGG 202
as lyophilized cultures, reconstituted
E. coli K-12 212974
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGG 212915 and sub-cultured as recommended
E. coli O157 203 ATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCCGA 262 for isolated colonies. The study
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
E. coli K-12 212914 ATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCCGA 212855 was further supplemented with
E. coli O157 263 TGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
322 ATCC-derived strains, Enterobacter
E. coli K-12 212854 TGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACC 212795 cloacae ATCC 23355, Pseudomonas
E. coli O157 323 GTTTCCAGTAGTTATCCCNNCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGC
|||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||
382
aeruginosa ATCC 9027, E. coli ATCC
E. coli K-12 212794 GTTTCCAGTAGTTATCCCC-CTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGC 212736
25922 and E. coli O157:H7 ATCC
E. coli O157 383 CACTCGTCAGCAAAGAAGCAAGCTTCTTCCTGTTACCGTTCGACTTGCATGTGTTAGG 440
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 35150, from MEI’s QC collection as
E. coli K-12 212735 CACTCGTCAGCAAAGAAGCAAGCTTCTTCCTGTTACCGTTCGACTTGCATGTGTTAGG 212678
well as two additional E. coli strains
Figure 3. Alignment comparison of partial 16S rRNA genetic sequence from platform strain E. coli from lab and environmental sources.
K-12 with genetic sequence from pathogenic strain E. coli O157:H7. Note that >99 percent homol- Additionally, Althea submitted
ogy at this sequence length is considered as indistinguishable. an isolate of the Master Cell Bank
platform strain used in processing.
pitfalls of potentially misidentifying combining a genetic-based microbial Representative cultures of each
organisms due to the limitations ID assay (16S rRNA sequencing) with microorganism were subjected to
of various commercial phenotypic a broad spectrum for phenotypic and 16S rRNA sequencing, colonial,
and genotypic microbial ID systems biochemical analyses, to accurately morphological, biochemical (Vitek
and their databases. This article identify a potentially objectionable GNI, bioMérieux) observations and
will detail such an approach taken environmental organism submitted DNA fingerprinting using pulsed
recently during work at facilities of by Althea. The organism of con- field gel electrophoresis (PFGE
Althea Technologies in San Diego. cern was recovered during routine analysis—with restriction enzymes
environmental monitoring, while XbaI, AvrII and SpeI). Analysis of
AN ORGANISM OF CONCERN aliquoting in a tissue culture hood. toxigenic and pathogenic potential
In the current study, Molecular The procedure involved a biofermen- was conducted using a proprietary
Epidemiology Inc. (MEI) used a tation Master Cell Bank (MCB), with polymerase chain reaction (PCR)
polyphasic identification approach, Escherichia coli as the platform. method (MEI, 2010) to determine the
44 MAY 2012
3. E N V I R O N M E N TA L M O N I T O R I N G
Figure 4. PCR results for various targeted
marker genes: upper image shows recovery
of genomic DNA (lane 8, negative, is buffer
control); middle image shows GAPDH target
specific to members of Enterobacteriaceae
(note lane 5, negative, is Pseudomonas aeru-
ginosa); lower image shows recovery of target
toxin and attachment genes in control strain E.
coli O157:H7 (lane 7); lane 2 is study strain.
presence or absence of toxin markers
and pathogenicity factors in the
reference and subject strains.
CONFIRMING IDENTIFICATION
An initial isolate recovered from a
cleanroom sanitary fill operation
(aliquoting of MCB) was received for
identification by 16S rRNA genetic Figures 5a, 5b, 5c. A comparison of genetic sub-typing patterns of entire genomes by pulsed field
sequencing. The isolate was reported gel electrophoresis showing distinctly different patterns between EM isolate (Althea Tech.) and
as a member of the Family Entero- other E. coli, pathogenic forms of E. coli and other selected pathogens and non-pathogens in Fam-
bacteriaceae (Figure 1)—consistent ily Enterobacteriaceae. Note the indistinguishable patterns of EM isolate and Platform E. coli under
with the industry and federally conditions of three different restriction enzymatic digests: XbaI, AvrII, and SpeI.
recognized guidelines (CLSI, 2008)
for genetic sequencing techniques culture of E. coli O157:H7 ATCC (PCR) analyses for specifically
for identification. Further taxonomic 35150. Furthermore, sequence targeted toxigenic and pathogenic
classification was required, and the alignment data (Figure 3) indicate markers demonstrated the absence
application of a polyphasic micro- the indistinguishable similarity of these markers in the submitted
biological ID (PMID) method clearly in the 16S rRNA gene sequence organism (Figure 4, lane 2) when
differentiated and reported the associated with the pathogenic compared to the control organisms
isolate as E. coli (Figure 2). species. Therefore, reliance on (Figure 4, lane 7).
Further comparisons with related genetic-based ID or even a microbial Subsequent to the PMID of the EM
strains with pathogenic potential ID conclusion would not rule out the isolate as E. coli, DNA Fingerprinting
differentiated and confirmed this possibility of a frank pathogen. comparison of the entire genome
identification (data not shown). Of To confirm that the EM isolate by PFGE analysis (employing three
concern was the equally identical had not acquired any pathogenic distinct restriction enzyme digests)
taxonomic ID presented by the QC potential, polymerase chain reaction demonstrated the differences in
4. E N V I R O N M E N TA L M O N I T O R I N G
Coliform in
Classified Area
the facility is verified to be operating
with suitable environmental control
Microbial Identification
regarding the detection of coliforms.
Polyphasic Microbial ID Genetic ID EM AND EXCURSION RESPONSE
A company should have an estab-
Species Level ID: E. coli
Family Level Output: lished SOP which clearly identifies
Enterobacteriaceae
the actions to be taken, including
Strain Characterization Toxigenicity and
clear directions if and when regula-
Species/Strains of Note
by PFGE Pathogenicity analysis by PCR tory notification (FDA, USDA, CDC,
etc.) would be required. In many cas-
Match to Platform Strain
No toxin or pathogen
Shigella sonnei
E. coli O157:H7 (and other es, the detection of an enteric organ-
markers detected pathogenic serotypes)
ism or a frank pathogen may include
No Risk Undefined Risk
the determination of non-pathoge-
nicity, or absence of virulence factors
and potential for toxigenicity, which
Figure 6. MEI’s proprietary Polyphasic System Approach
CAPA
allows the manufacturer to better
(PSA) towards the investigation of a potential “objection- manage associated risk. The key is to
able” organism. understand the process and potential
incursion points, and to complement
genetic profiles from other similarly demonstrated to be unequivocally them with Corrective Action/Preven-
named control and environmental non-pathogenic and non-toxigenic tive Action, based on the potential
strains (Figures 5a, 5b, 5c). by PCR analysis of pathogenic and toxigenicity or pathogenicity of
The isolate under investigation toxigenic potential. The entire pro- the organism. This, coupled with a
is identified in the dendrograms cess using a thorough science-based science-based Risk Assessment, al-
and its genomic pattern is clearly evaluation is illustrated in Figure 6. lows for a more rigorous evaluation
distinguishable from the other As such, this isolate was not of potential EM excursions.
strains and yet indistinguishable an “objectionable” organism. The
from the Master Cell Bank platform manipulation of the fermentation References
strain. This, having already strain—sampling, pipetting with 1. Clinical Laboratory Standards Institute.
demonstrated the strain’s lack of expected minute aspirations, etc.— Interpretive Criteria for Identification
associated pathogenicity, confirms its can lead to isolated incidents of of Bacteria and Fungi by DNA Target
clonal relationship with the MCB. detection of the bioprocess strain Sequencing: Approved Guideline. CLSI
in any well-controlled facility. document MM18-A. Wayne, PA, 2008.
BIOPROCESS STRAIN: A SCI- The results presented here show 2. Molecular Epidemiology, Inc. dba IEH
ENCE-BASED EVALUATION the benefits of polyphasic ID Laboratories & Consulting Group. IEH
In our study, the detection of an E. and the limitations of genetic or E. coli O157, Stx-producing E. coli, (STEC)
coli recovered during environmen- phenotypic (rapid) microbial ID with Intiman and Salmonella Test System.
tal/production monitoring in the methods when used individually. AOAC-RI PTM 100701, AOAC Research
tissue culture hood was cause for Incorrect, incomplete or inadequate Institute, Gaithersburg, MD, USA, 2010.
thorough and appropriate investiga- identification and characterization
tion. Through the use of a detailed of strains of E. coli cannot exclude About the Authors
polyphasic analysis approach, the potential pathogenic forms such as Jaspreet S. Sidhu, Ph.D. (VP, Business Devel-
isolate was verified to be the same specific pathogenic serotypes (e.g., opment and Pharmaceutical Microbiology),
taxonomic genus and species as the O157; O104), Enterohemorrhagic Connor Tyler (Scientist), Greg Ma (Director
bioprocess strain. Further testing via strains (EHEC) as well as E. coli of General Microbiology), and Mansour Sa-
DNA fingerprinting by pulsed field subtypes STEC, ETEC, and EPEC, madpour, Ph.D. (President, CEO) represent
gel electrophoresis demonstrated respectively. By detailed polyphasic Molecular Epidemiology, Inc. in Lake Forest
that this was indeed the exact same analysis and demonstrating that any Park, Washington. E.J. Brandreth is VP of
strain as used in the fermentation isolate of E. coli is linked directly to Quality and Regulatory Affairs for Althea
process. In addition, the strain was the upstream production process, Technologies, San Diego.
46 MAY 2012