554612

research-article2014
JLAXXX10.1177/2211068214554612Journal of Laboratory AutomationPeris-Vicente et al.

Review Article
Journal of Laboratory Automation

Validation of Rapid Microbiological Methods

2015, Vol. 20(3) 259­–264
© 2014 Society for Laboratory
Automation and Screening
DOI: 10.1177/2211068214554612
jala.sagepub.com

Juan Peris-Vicente1, Samuel Carda-Broch1, and

Josep Esteve-Romero1

Abstract
Classical microbiological methods currently have unacceptably long cycle times. Rapid microbiological methods have
been available on the market for decades and have been applied by the clinical and food industries. However, their
implementation in the pharmaceutical industry has been hampered by stringent regulations on validation and comparison
with classical methods. To encourage the implementation of these methodologies, they must be validated to assess that the
results are straightforward. A comparison with traditional methods should be also performed. In this review, information
about the validation of rapid microbiological methods reported in the literature is provided as well as an explanation of
the difficulty of validation of these methods. A comparison with traditional methods is also discussed. This information is
useful for industries and laboratories that can potentially implement these methods.

Keywords
analysis, guideline, laboratory, microorganism, validation

Introduction growth. These limitations have motivated sponsors and

Gene therapy products and human somatic cell therapy are
emerging and present multiple challenges to safety, purity,
and potency. Gene therapy products include vectors (such
as nucleic acid, virus, or genetically modified microorgan-
isms) that are directly administered to patients and cells that
are transduced with a vector ex vivo prior to administration
to the patient. Certain genetically modified microorganisms
(bacteria and yeast), cellular therapy products, and cells
transduced with a gene therapy vector present similar chal-
lenges to sterility assurance and can be considered cell-
based products. These products cannot be cryopreserved or
otherwise stored without affecting viability and potency.
Most cell-based products are manufactured using aseptic
manipulations because they can undergo sterile filtration or
sterilization.1 However, the incubation times needed for
these methods is usually too long.2 Thus, rapid and effective
testing is needed because many cell-based products have a
potential short dating period, which often necessitates
administration of the final product to a patient before steril-
ity test results. Because of the challenges associated with
cell-based products, there is a significant need to develop,
validate, and implement sterility test methods.3
Traditional methods, described by Title 21 of the Code
of Federal Regulations, 610.12 (21 CFR 610.12),4 are used
for the isolation of microorganisms. However, the present
limitations related to cell-based products include low pro-
duction volumes, limited manufacturing time, short dating
periods, requirement for large sample volumes, and the
need for manual and visual examination of cultures to detect
manufacturers to develop rapid microbiological methods
(RMMs) based on techniques that reportedly yield accurate
and reliable test results in less time and often with less oper-
ator intervention. Therefore, the use of RMMs for cell-
based products might provide rapid results that could be
applied for product release.5
RMMs are methods designed to provide performance
equivalent to the sterility traditional methods while provid-
ing results in significantly less time. In general, RMMs
involve technologies that can be growth-based, viability-
based, or surrogate-based cellular markers for a microor-
ganism (e.g., nucleic acid based, fatty acid based). RMMs
are usually automated, and many have been used in clinical
laboratories to detect viable microorganisms in patient
specimens. These methods show improved sensitivity in
detecting changes in the sample matrix (e.g., by-products of
microbial metabolism), under conditions that favor the
growth of microorganisms. The RMMs can be applied in
the manufacturing of cell-based products to test the compo-
nents (raw material, excipients), for in-process testing, for
drug substance testing, and to test the drug product in its
final container.6
1
QFA, ESTCE, Universitat Jaume I, 12071, Castelló, Spain
Received May 13, 2014.
Corresponding Author:
Juan Peris-Vicente, Universitat Jaume I, QFA, ESTCE, UJI, Campus del
Riu Sec, Av/Sos Banyat s/n, Castelló, 12071, Spain.
Email: vicentej@uji.es
260
To apply these methods, the operator must prove that
RMMs provide the same assurance of effectiveness of the bio-
logical product as traditional methods do. A validation guide
should be followed to indicate the protocol is needed to assess
the usefulness of the method. Several guides have been sug-
gested and can be applied to validation of RMMs, such as the
Guides for Industry: Validation of Growth-Based Rapid
Microbiological Methods for Sterility Testing of Cellular and
Gene Therapy Products, described by the Food and Drug
Administration (FDA)3; Guidance for Industry PAT—A
Framework for Innovative Pharmaceutical Development,
Manufacturing, and Quality Assurance, September 20046; and
Presidential Task Force on Best Practices in Microbiological
Methodology performed by AOAC International, 2006.7 These
guides require detailed information on all of the validation
parameters: accuracy, precision, ruggedness, specificity, limit
of quantification (LOQ), limit of detection (LOD), linearity,
range, equivalence, and quality by design. An analytical meth-
odology is accepted if the validation parameters are under
those guideline requirements.
Although RMMs are now routinely used in limited set-
tings, such as clinical testing, they have not been compre-
hensively validated for use in a variety of manufacturing
settings. Cell-based products present especially challenging
manufacturing issues, as these products often introduce
additional product or process variables that would likely
affect the test outcome. These variables should be consid-
ered in a validation study to demonstrate that the method is
suitable for its intended product and application.
The aim of this work is to discuss the two main guides
proposed to validate RMMs: the Guidance for Industry:
Validation of Growth-Based RMMs for Sterility Testing and
Gene Therapy Products and the Guidance for Industry
PAT—A Framework for Innovative Pharmaceutical
Development, Manufacturing, and Quality Assurance focus-
ing on the description of the applied validation protocol. The
ability to recover and detect organisms from the product and
demonstrate their viability by multiplication in liquid media
must be evaluated. The guidance has been developed for
RMMs with qualitative results (e.g., detection of microor-
ganisms). Reliance on validated sterility testing methods is a
critical element in assuring the safety of a product. Validation
of most methods for final product testing, including those
assessing sterility, is required under 21 CFR 211.165(e) and
21 CFR 11.194(a).4 Validation of critical methods proves that
the methods are suitable for their intended purpose, to mea-
sure the quantity of microorganisms in the sample, and pro-
vides assurance about the accuracy and reproducibility.
Description of the Guides
Guidance for Industry:Validation of Growth-Based Rapid
Microbiological Methods for Sterility Testing and Gene
Therapy Products has been developed by the Center for
Journal of Laboratory Automation 20(3)
Biologics Evaluation and Research/FDA, from the U.S.
Department of Health and Human Services. FDA guidance
exposes a recommendation to test the suitability of the vali-
dation of the RMMs, but it has not law level.3
This guide proposes recommendations for manufactur-
ers and users for validation and sterility testing of therapy
and gene therapy products, as well as growth-based RMMs.
However, the guideline is not directly applicable to human
cells, tissues, or cellular and tissue products.3
The guidance applies to the following products.
1. Somatic cell therapy products: Autologous or allo-
geneic cells that have been propagated, expanded,
selected, pharmacologically treated, or otherwise
altered in biological characteristics ex vivo, to be
administered to humans and applicable to the pre-
vention, treatment, cure, diagnosis, or mitigation of
disease or injuries.8
2. Gene therapy products: All products that mediate
their effects by transcription and/or translation of
transferred genetic material and/or by integrating
into the host genome and that are administered as
nucleic acids, viruses, or genetically engineered
microorganisms. The products may be used to mod-
ify cells in vivo or transferred to cells ex vivo prior
to administration to the recipient.9
Guidance for Industry PAT—A Framework for Innovative
Pharmaceutical Development, Manufacturing, and Quality
Assurance was developed by the U.S. Department of Health
and Human Services, FDA, in September 2004. The guide
is intended to describe a regulatory framework (process
analytical technology [PAT]) that will encourage industry
and research laboratories to develop and implement innova-
tive pharmaceutical development, manufacturing, and qual-
ity assurance. Working with existing regulations, the agency
has developed an innovative approach for helping the phar-
maceutical industry address anticipated technical and regu-
latory issues and questions.6
The Presidential Task Force on Best Practices in
Microbiological Methodology was performed by AOAC
International in September 2006.7 This guide is devoted to
the validation microbiological methods for the industry and
refers to laboratories working in the field of food safety,
quality assurance, clinical diagnostics, veterinary diagnos-
tics, and engineering.7
Validation Procedure
The final objective of validation of an analytical method is
to demonstrate that it is suitable for its intended purpose.
Thus, RMMs must be compared with traditional methods to
prove that they are equivalent or even that they provide less
uncertainty.10
Peris-Vicente et al.
Contamination Studies
During the production of cell-based products and microor-
ganisms, several mistakes, such as incorrectly controlled
process streams and failures in aseptic processing tech-
niques, can cause the introduction of microorganisms. Thus,
a serious assessment of the production must be performed
to identify and, eventually, avoid potential routes of micro-
bial contamination8.
The main risk of contamination comes from the follow-
ing: source material, level manipulation, processing path,
cell culture, incubation duration, and cell culture vessel.11
Validation Parameters
The validation parameters that must be tested during valida-
tion are described below.10,12
•• Limit of detection (LOD): The lowest number of
microorganisms detectable in the sample matrix.
LOD is essential to define what is considered to be
contaminated. The LOD is determined by inoculat-
ing samples with serial dilutions of viable contami-
nants, with colony-forming units (CFUs) confirmed
at inoculation by plate counts. Challenge testing is
performed with each challenge microorganism using
an amount between 10 and 100 CFU. Any growth is
recovered and identified to confirm identity.
•• Incubation time robustness: Time range required for
detection of microorganisms at a determinate level.
•• Accuracy: Closeness between measured value and
true value of microorganism amount.
•• Precision: Agreement among individual test results
for multiple samplings from a homogeneous sample.
Repeatability refers to using the test procedure
within a laboratory over a short time period.
•• Linearity: The linearity is the ability (within a given
range) to obtain a detection signal that is directly
proportional to the CFU of a microorganism in a
sample, with suitable accuracy and precision. The
evaluation of linearity is assessed by the determina-
tion coefficient (r2), which should be greater than
0.95 to consider that linearity is adequate.
•• Limit of quantification (LOQ): The lowest CFU of
microorganisms quantifiable in cell culture with ade-
quate accuracy and precision.
•• Calibration range: Minimal and maximal amount of
CFU, which could be reliably quantified.
•• Recovery: Ratio between the measured CFU and the
CFU really incubated in the cell culture. The accep-
tance criterion is more than 70%.
•• Viability recovery: Ratio between the CFU count
after reincubation and traditional method count. The
acceptance criterion is more than 70%.
261
•• Specificity: The ability of the test method to detect a
panel of organisms within the sample matrix estab-
lished in the validation protocol. Specificity is demon-
strated by challenging the RMMs to detect variations in
microbial growth characteristics and concentrations.
Challenge testing is performed with organisms, includ-
ing those recovered in the manufacturing environment
and from sterility test positives, as well as common
technical and operator contaminants. Known quantities
of organisms inoculated into product samples need to
be detected within the detection limit (see LOD), recov-
ered, and identity confirmed.
•• Ruggedness: Degree of reproducibility of results
obtained by analysis of the same sample under a variety
of normal test conditions, such as different analysts, dif-
ferent instruments, and different reagent lots.
Ruggedness is the lack of influence on the test results of
operational and environmental variables of the micro-
biological method and should be assessed by being pre-
pared by different analysts, as specified in the
procedures. Positive and negative controls should be
included and should test true to their characteristics.
Ruggedness is evaluated by tabulating the mean time to
detection for each inoculate and determining the differ-
ence between the means for each analyst.
•• Robustness: Capacity of a method to remain unaf-
fected by slight, but deliberate, variations in method
parameters.
Selection of Microorganisms for Validation
The RMMs must be tested using the microorganism more rel-
evant to the product and manufacturing method. The guide
suggests the inclusion of microorganisms belonging to several
categories: gram-negative bacteria, gram-positive bacteria,
aerobic bacteria, anaerobic bacteria, yeast, fungi, isolates
detected in starting materials, isolates detected by in-process
testing or during preliminary product testing, isolates detected
by environmental monitoring of your manufacturing facility,
isolates from production areas that represent low-nutrient and
high-stress environments, and microorganisms from commer-
cial sources that have continually been exposed to high-nutri-
ent growth media and slow-growing bacteria.3
The same microorganism can show strong differences in
growth-rate kinetics, depending on the source, significantly
affecting the needed incubation time and the temperature to
detect growth. Thus, the validation should include the study
of growth under aerobic and anaerobic conditions, espe-
cially for products manufactured in closed systems.3
Quality by Design of Method Validation
The composition of the test sample should be representative of
the product samples that RMMs would test. The parameters to
262
consider are cell concentration, media, and additives, as well as
preservation and antimicrobial agents, with a bacteriostatic or
fungistatic effect. Several matrix compositions should be
tested to validate the method for multiple sample types of the
same cellular product.
The application of RMMs must be validated for in-pro-
cess and final product in a cell culture production. The cell
culture medium can contain antibiotics in different stages of
the in-process, which might contain a preservation agent as
well as a higher cell concentration. Thus, the different steps
of cell production must be separately validated because of
their different compositions.
To validate the performance of a commercially available
testing system, studies using characteristic organisms under
prescribed assay conditions should be performed, and the
results should be compared with those provided by the
supplier.
In the validation study design, the potential for the materi-
als being tested to generate false-positive or false-negative
results should be evaluated using the appropriate controls.
This will depend on the product matrix, additives and preser-
vatives, and unique characteristics of the product. For exam-
ple, if a freshly isolated cellular product is tested for sterility
using a detection method based on CO2 production, then the
same freshly isolated cellular product should serve as a con-
trol to determine whether uncontaminated cells generate lev-
els of CO2 that would produce a false-positive result. The use
of additional positive and negative controls containing prod-
uct components, but not cells, is also recommended.3
Comparison between RMMs and Traditional
Methods
To compare RMMs and traditional methods, the same amount
of potential microbial contaminants as microorganisms indi-
cated in the previous section are inoculated in cell cultures
(measured in CFU/volume). The ability of both methods to
identify the microorganisms is tested, together with recovery,
detection limit, and assay performance. Several dilutions of
microorganisms are tested (10–99 CFU/sample), in order to
evaluate the usefulness of RMMs at the main working con-
centration levels of microorganisms.3
Revalidation Method
Any changes in product manufacturing, including formula-
tion, or changes in the RMMs that can potentially inhibit or
enhance detection of viable microorganisms need to revali-
date the RMMs. Changes in cell types, harvesting proce-
dures, culture media, additives, critical processing steps, or
postprocessing handling can potentially affect the detection
of viable microorganisms. Initial validation can be designed
to minimize the effect of some changes by designing assay
conditions that encompass known or proposed changes
Journal of Laboratory Automation 20(3)
(e.g., worst case, matrix approach). Revalidation of the
RMM should be performed whenever there are changes in
the process that could potentially inhibit (e.g., the addition
of antimicrobials) or enhance detection of viable microor-
ganisms. Verification of critical parameters of the test
method postprocess, or product changes using the microor-
ganisms most difficult to detect, can serve as an indication
of the need to revalidate the method. Validation of the steril-
ity test should be performed on all new products.3
Advantages of RMMs
The implementation of RMMs also provides interesting
economic features, because of the significant reductions in
time to result over conventional methods. Normally, the
production cycle is stopped or a product is kept until
checking the absence of contamination. If the time to
result is shortened, the product would be more rapidly
released. Moreover, RMMs allow continuous sampling
and data collection and provide time-sensitive outputs and
real-time data analysis. Therefore, more screenings and
repeat tests could be performed in all stages of production,
improving the surveillance process.13 Real-time control
and early warning of contamination are essential before
introducing raw materials and other product components
to the process, validating manufacturing, or distributing
finished products.14
RMMs involves an easy-to-handle protocol, with a neg-
ligible production of waste, minimal operator variability,
reduced staff training requirement, and possibility of auto-
mation. They also facilitate research through generating
earlier results. Moreover, the shorter analysis time reduces
the probability of contamination, improving the robustness.
A shorter time to result facilitates replication of an analysis
if a contamination or a strange result occurs. Thus, RMMs
can improve manufacturing consistency by permitting
faster implementation of corrective actions and fostering
opportunities to improve the safety of the process. Moreover,
RMMs also show improved sensitivity, efficiency, selectiv-
ity, accuracy, high throughput, and low false-positive rate,
leading to higher efficiency.13 These features of RMMs pro-
vide an opportunity to assess the significance of viable but
nonculturable or stressed microorganisms.15,16
Application of Validation of RMMs
The Milliflex Quantum method is an RMM based in fluo-
rescence detection for the quantification of microorgan-
isms. The culture media are prefilled tryptic soy agar plates,
R2A plates, and Sabouraud dextrose agarplates. The authors
have validated this methodology for the analysis of contami-
nants in water. The tested microorganisms were the following
American Type Culture Collection (ATCC) strains (TM):
Candida albicans (10231D-5), Aspergillus brasiliensis
Peris-Vicente et al.
(16404D-2), Staphylococcus epidermidis (12228D-5),
Ralstonia pickettii (27511), Brevundimonas diminuta (19146),
Staphylococcus aureus (700699D-5), Pseudomonas aerugi-
nosa (47085D-5), Bacillus subtilis (23857D-5), and Escherichia
coli (10798D-5). The experimental procedure was described
Meder et al.12 This method was compared with a compendial
method.
•• Incubation time robustness was tested at 10 CFU and
was found to be between 8 and 28 h, depending on
the microorganism.
•• Ruggedness was evaluated using different media
lots, membrane lots, and reagent lots and treating the
results by analysis of variance. No significant differ-
ences were detected in fluorescence or viability
recovery.
•• Fluorescence recovery was 98% to 102%, and vali-
dation recovery was 97% to 102%. Thus, no signifi-
cant differences were found between the Milliflex
Quantum and traditional method.
•• Accuracy was 90% to 112%.
•• Linearity was found at r2 > 0.96 between 4 to 10
CFU and 100 CFU.
•• The LOD was 1 CFU.
•• Specificity: the challenged microorganisms were
correctly identified.
The validated RMM and the compendial method were
applied to the detection of microorganisms in purified water
sampled in pharmaceutical plants. Milliflex Quantum pro-
vides results in 24 to 40 h, whereas the compendial method
takes 5 to 7 d. The obtained results were similar in both
cases.12
RMMs offer evident improvements in comparison with
traditional methods, and they especially provided results in
a shorter time. This review outlines the basic principles of
validation and verification in an implementation process for
microbiological methods. The main validation parameters
have been described. Thus, the implementation of these
methodologies is encouraged. According to the literature,
RMMs provide significant improvements with the same
results as traditional methods. This encourages their imple-
mentation in laboratories carrying out microbiological stud-
ies for clinical or food safety purposes.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
263
work has been supported by a project from the University Jaume I
(project P1.1B2012-36).
References
1. Food and Drug Administration. Guidance for Industry:
Sterile Drug Products Produced by Aseptic Processing—
Current Good Manufacturing Practice (September 2004).
http://www.fda.gov/cber/guidelines.html (accessed May 14,
2014).
2. Verdonk, G. P. H. T.; Willemse, M. J.; Hoefs, S. G. G.;
et al. The Most Probable Limit of Detection (MPL) for Rapid
Microbiological Methods. J. Microbiol. Meth. 2010, 82,
193–197.
3. Food and Drug Administration. Guidance for Industry: Validation
of Growth-Based Rapid Microbiological Methods for Sterility
Testing of Cellular and Gene Therapy Products (February 2008).
http://www.fda.gov/downloads/BiologicsBloodVaccines/
GuidanceComplianceRegulatoryInformation/Guidances/
CellularandGeneTherapy/ucm078696.pdf (accessed May 14,
2014).
4. United States Pharmacopeia 28 and National Formulary 23
(USP 28 NF 23), Sterility Tests, General Chapter 71, 2005;
pp 1–13.
5. Miller, M. J. Case Study of a New Growth-Based Rapid
Microbiological Method (RMM) That Detects the Presence
of Specific Organisms and Provides an Estimation of Viable
Cell Count. Am. Pharmaceut. Rev. 2012, 15(2).
6. Food and Drug Administration. Guidance for Industry
PAT—A Framework for Innovative Pharmaceutical
Development, Manufacturing, and Quality Assurance
(September 2004). http://www.fda.gov/downloads/Drugs/
Guidances/ucm070305.pdf (accessed May 14, 2014).
7. Food and Drug Administration; AOAC International. Final
Report and Executive Summaries, Presidential Task Force
on Best Practices in Microbiological Methodology (August
2006). http://www.fda.gov/Food/FoodScienceResearch/Labora
toryMethods/ucm124900.htm (accessed May 14, 2014).
8. Food and Drug Administration. Application of Current
Statutory Authorities to Human Somatic Cell Therapy Products
and Gene Therapy Products (58 FR 53248) (October 1993).
http://www.fda.gov/downloads/BiologicsBloodVaccines/
SafetyAvailability/UCM148113.pdf (accessed May 14,
2014).
9. Food and Drug Administration. Guidance for Industry:
Gene Therapy Clinical Trials—Observing Participants for
Delayed Adverse Events (November 2006). http://www
.fda.gov/BiologicsBloodVaccines/GuidanceCompliance
RegulatoryInformation/Guidances/CellularandGeneTherapy/
ucm072957.htm (accessed May 14, 2014).
10. ICH Expert Working Group. The International Conference
on Harmonisation (ICH) Guidance; Q2(R1), Validation of
Analytical Procedures: Text and Methodology (November
2005). http://www.ich.org/fileadmin/Public_Web_Site/ICH_
Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__
Guideline.pdf (accessed May 14, 2014).
11. ICH Expert Working Group. The International Conference
on Harmonisation (ICH) Guidance; Q9, Quality Risk
Management (June 2006). http://www.ich.org/fileadmin/
264
Public_Web_Site/ICH_Products/Guidelines/Quality/Q9/
Step4/Q9_Guideline.pdf (accessed May 14, 2014).
12. Meder, H.; Baumstummler, A.; Chollet, R.; et al. Fluorescence-
Based Rapid Detection of Microbiological Contaminants in
Water Samples. Sci. World J. 2012, 234858.
13. Miller, M. J., , ed. Encyclopedia of Rapid Microbiological
Methods; DHI Publishing, LLC: River Grove, IL; 2005.
Journal of Laboratory Automation 20(3)
14. Cundell, A. M. Opportunities for Rapid Microbial Methods.
Eur. Pharm. Rev. 2006, 1, 64–70.
15. Newby, P. The Significance and Detection of VBNC
Microorganisms. Eur. Pharm. Rev. 2007, 3, 87–92.
16. Sandle, T. A Review of Cleanroom Microflora: Types,
Trends, and Patterns. PDA J. Pharm. Sci. Technol. 2011, 65,
392–403.