The development of compendial rapid sterility tests

Abstract

An Expert Panel was formed under the USP General Chapters - Microbiology Expert Committee to provide recommendations on user requirements specifications (URS) and candidate technologies based on the URS in the area of rapid sterility tests. The Expert Panel provided recommendations for the critical URS for candidate rapid sterility tests, which were: 1) the ability to detect a wide range of microorganisms, i.e., specificity; 2) detection of a low number of microorganisms, i.e., limit of detection; 3) time to result; 4) improved patient safety; 5) sample preparation; and 6) sample quantity, i.e., minimum number of articles tested and quantity per container tested. Based on a review of these user requirements, the Expert Panel recommended that adenosine triphosphate bioluminescence, flow cytometry, isothermal microcalorimetry, nucleic acid amplification, respiration, and solid-phase cytometry advance as candidates for proof-of-concept studies to develop risk-based compendial rapid sterility tests.

Full text

STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies
of the USPC or the USP Council of Experts
The Development of Compendial Rapid Sterility Tests
Members of the USP Modern Microbiological Methods Expert Panela,b,c,d,e
ABSTRACT An Expert Panel was formed under the USP General Chapters—Microbiology Expert
Committee to provide recommendations on user requirements specifications (URS) and candidate
technologies based on the URS in the area of rapid sterility tests. The Expert Panel provided
recommendations for the critical URS for candidate rapid sterility tests, which were: 1) the ability
to detect a wide range of microorganisms, i.e., specificity; 2) detection of a low number of
microorganisms, i.e., limit of detection; 3) time to result; 4) improved patient safety; 5) sample
preparation; and 6) sample quantity, i.e., minimum number of articles tested and quantity per
container tested. Based on a review of these user requirements, the Expert Panel recommended
that adenosine triphosphate bioluminescence, flow cytometry, isothermal microcalorimetry,
nucleic acid amplification, respiration, and solid-phase cytometry advance as candidates for
proof-of-concept studies to develop risk-based compendial rapid sterility tests.
INTRODUCTION
It is widely recognized that the current growth-based sterility tests in Sterility Tests 〈71〉 (1)
with at least a 14-day incubation period are not suitable for short-lived products or those
prepared for immediate use or administered to patients before the completion of the compendial
sterility test. To address the needs of stakeholders making compounded sterile preparations,
radiopharmaceuticals, and cell and gene therapies, the USP Microbiology Expert Committee has
begun work on the development of a new generation of rapid compendial sterility tests.
BACKGROUND
With the primary consideration of improved patient safety, the Expert Panel began by
establishing the user requirements specifications (URS) for rapid sterility tests. The consensus
reached was that not all URS were the same for four main stakeholder groups indicated above.
Therefore, URS were established for: 1) sterile compounding; 2) positron emission tomography
(PET) drugs and other short-lived radiopharmaceuticals; 3) cell therapy; and 4) traditional
pharmaceutical manufacturing.
Once the URS were established, the Expert Panel recommended the most suitable technologies
or analytical platforms as candidates for a compendial rapid sterility test for proof-of-concept
studies. Where analytical platforms were dependent upon instruments and reagents supplied by
vendors, only non-proprietary technologies marketed by two or more instrument manufacturers
were considered as potential candidates. The Expert Panel acknowledges that one or more of
these analytical platforms may be found to have insurmountable technical limitations, which may
prevent them from becoming compendial test methods. Despite being compendial tests, the
rapid sterility tests would need to meet method suitability testing requirements for each
Page 1 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

pharmaceutical and biological product and would be subject to review in their regulatory
submissions.
HISTORY OF USP STERILITY TESTS
It is useful to review the history of how the USP sterility tests evolved (2–4). Table 1 contains a
brief summary of the development of 〈71〉 from 1936–2009.
Table 1. The Evolution of USP Sterility Tests
Compendial
Revision Brief Description of the Sterility Test
USP XI (1936), page
469, Tests for the
Sterility of Liquids 7-day incubation at 37° in a beef extract-peptone-dextrose broth
USP XII (1941),
Sterility Test for
Solids added
Additions: broth for sterility tests under anaerobiosis, inactivating
fluids, and a honey medium for molds and yeast incubated at 22°
–25° for 15 days
USP XVII (1965),
pages 829–832,
Sterility Test
Additions: fluid thioglycollate medium incubated at 30°–32° for 7
days, fluid Sabouraud dextrose medium incubated at 22°–25° for 10
days, and bacteriostasis and fungistasis testing added to
demonstrate the suitability of the method for each specific product
USP XVIII (1970),
pages 851–857,
Sterility Tests 〈71〉
Revisions: fluid thioglycollate medium incubated at 30–35° for 14
days for aseptically filled products and 7 days for terminally
sterilized products; soybean-casein digest medium incubated at 20°
–25° for aseptically filled products and 7 days for terminally
sterilized products; and the incubation period reduced from 14 to 7
days for membrane filtration sterility tests
USP 27 (2004) pages
2157–2162, Sterility
Tests 〈71〉
Harmonization: effective January 1, 2004; however, the compendial
sterility tests contained 11 local non-harmonized requirements; all
incubation times, regardless of product, were 14 days
First Supplement to
USP 32 (2009),
Sterility Tests 〈71〉Revisions: the 11 local non-harmonized requirements were removed
with an official date of August 1, 2009
LIMITATIONS OF THE SELECTED MEDIA
In general, microorganisms that are found in pharmaceutical drug products are present in low
numbers and under stressed conditions due to 1) product formulation (especially the presence of
antimicrobial agents or active ingredients); 2) manufacturing processes; and 3) physicochemical
conditions such as low nutrient levels, pH (deviating from neutral), low-water activities, and
exposure to temperatures above or below ambient temperature. To proliferate in microbiological
growth media, microorganisms need to repair stress-induced damage, activate different
biosynthetic and metabolic pathways, and acclimate to the media before they can enter a
logarithmic growth phase.
Despite the belief that the sterility test media can support the growth of low numbers of
stressed microbial cells, the media selection and incubation conditions of the compendial sterility
test may not be optimal and may, in fact, be seriously compromised in an attempt to isolate the
widest range of microorganisms (2). For example, fluid thioglycollate medium may be considered
suboptimal for 1) strict and facultative anaerobes due to its aerobic incubation, 2) bacterial and
fungal spore germination and growth, and 3) vegetative bacteria and fungi due to its low redox
Page 2 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

potential, medium viscosity, and component toxicity. Soybean–casein digest medium may be
compromised for the isolation of skin-derived bacteria by the low incubation temperature, i.e.,
20°–25°.
The unintended selectivity of the sterility test is illustrated by the common finding that the
majority of sterility failures occur in only one of the two media when the microorganisms are
capable of growth in both media. For example, 55% of the sterility failures had growth in the
soybean-casein digest medium only, 39% grew in the fluid thioglycollate medium, and a mere
9% grew in both media (5) with over 30% of growth occurring between 7 and 14 days of
incubation (6).
SAMPLE SIZE LIMITATIONS
Chapter 〈71〉 defines the quantities of a pharmaceutical drug product per container to be tested
per media and the number of units based on the batch size (see Sterility Tests 〈71〉, Table 2 and
3, respectively). The number of vials tested is a usually 20 or 40 units, depending on the fill
volume of the containers. Considering the statistical power of the sample size with respect to a
typical batch size in excess of 30,000 vials, the test is not capable of detecting a low microbial
contamination rate associated with aseptically filled sterile drug products, i.e., there is only an
18% chance of detecting a 1% contamination rate (4) (see Table 2 below).
Table 2. Probability of Failing the USP Sterility Test with Required Sample Size
Frequency of
Contaminated Units in a
Batch
Probability of Failing the USP Sterility Test with
Required Sample Size (Sterility Tests 〈71〉, Table 2
and 3)
1 in 1000 0.0198 (2%)
5 in 1000 0.0952 (9.5%)
1 in 100 0.1813 (18%)
5 in 100 0.6321 (63.2%)
1 in 10 0.8647 (86.5%)
5 in 10 1.000 (100%)
Furthermore, there are additional challenges with compounded sterile preparations, short-lived
radiopharmaceuticals, and cell therapies compared to most pharmaceutical drug products. The
lot sizes are usually small, the products must be used promptly, and sampling will deplete a
significant portion of each lot, causing an economic loss and reduced availability of the material
for patient treatment.
Currently, the minimum number of articles tested and quantity per container tested per media
are defined in Sterility Tests 〈71〉, Table 2 and 3. This sampling plan is suitable for manufactured
pharmaceuticals, but it depletes the batch, and is therefore unsuitable for products generated by
sterile compounding pharmacies, PET facilities, and cell therapy centers because of their small
batch size and the therapeutic value of the product to the individual patient. A further
consideration is the sample size limitation of these advanced technologies.
Alternative sampling plans have been proposed in compendial chapters. The recommended
approaches to sterility testing of cell therapy products can be found in European Pharmacopoeia
(EP) 2.6.27 for batch sizes less than 40 units.
Page 3 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

The EP provides 2.6.27 Microbiological Examination of Cell-based Preparations to use for cell
therapies when the tests in 2.6.1 Sterility cannot be performed. These limitations may be due to
the nature of the preparation, the process steps during which microbial contamination may be
introduced, the short shelf-life of cell therapy products, the amounts available for testing, and
sampling-related issues. EP positioned this test, not strictly as a sterility test, but as a test to
screen for microbial contamination that may be better suited for certain situations. The chapter
points out that with the use of a single donor or manufacturing-related capacity restraints, the
sample volume available for testing may be limited. Microbial contamination can be missed if the
sample size is not sufficient to ensure suitable sensitivity and specificity of the chosen test
method.
The sample size for cell-based preparations, where the total infusible volume (V) is between 1
mL and 1 L in a single unit, is given in Table 3 below.
Table 3. European Pharmacopoeia 2.6.27 Recommended Sample Sizes
Cell-Based Preparation Volume (mL) Total Test Sample Volume
10 ≤ V < 1000a1% of the total volume
1 ≤ V < 10 100 µL
V < 1 NA
a V is total infusible volume.
In a manner similar to cell-therapy preparations, the sample quantity and sampling plan for
PET radiopharmaceuticals must also accommodate the limited number of vials (usually 1) and the
volume of product produced in a batch (usually less than 15 mL). If the batch consists of a single
container, the sterility test sample size must be at least 1% of the total batch volume. For
example, if a batch consists of 1 vial containing 15 mL, use at least 0.15 mL for purposes of the
sterility test. If the batch consists of more than one container, use a volume from a single
container that represents at least 1% of the total batch volume. If a batch consists of 3 vials
each containing 25 mL, use at least 0.75 mL from 1 vial for purposes of the sterility test.
USER REQUIREMENT SPECIFICATIONS
Based on the work of the USP Expert Panel, 15 major user requirement specifications of
different stakeholders were considered:
• Ability to detect a wide range of microorganisms, i.e., specificity
• Availability of instruments and reagents from multiple vendors
• Availability of Reference Standards
• Data integrity
• Ease of use/simplicity of test and data interpretation
• Low false-positive and false-negative rates
• Limit of detection
• Method suitability
• Improved patient safety
• Regulatory acceptance
• Robustness and reliability of equipment
• Sample preparation
Page 4 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

• Sample quantity, i.e., minimum number of articles tested and quantity per container
tested
• Time to result
• Aseptic test material handling, i.e., open vs. closed systems
DETAILED DESCRIPTIONS OF THE MOST CHALLENGING USER REQUIREMENTS
Challenging user requirements specific to one or more stakeholder groups are:
• Ability to detect a wide range of microorganisms, i.e., specificity
• Limit of detection
• Time to result
• Improved patient safety
• Sample quantity i.e., minimum number of articles tested and quantity per container
tested
• Sample preparation
• Aseptic test material handling, i.e., open vs. closed systems
The user requirements listed above will be discussed in more detail below.
Ability to Detect a Wide Range of Microorganisms
Although all the analytical platforms should have the ability to detect a wide range of bacteria,
yeast, and mold, it is equally important to demonstrate that the rapid sterility test technology
chosen is capable of detecting microorganisms implicated in sterility test failures, infection
outbreaks, and product recalls associated with either compounded sterile preparations,
radiopharmaceuticals, cell therapies, or manufactured pharmaceuticals. This is especially true if
the technology, after risk analysis, is shown to improve patient safety with the administration of
the products unique to that stakeholder group.
For example, a 2014 report from The Pew Charitable Trusts documented over 25 pharmacy
compounding errors, the majority being microbial contamination associated, with 1,049 adverse
events and 89 deaths since 2001. The report identified the bacterium Serratia marcescens as
most frequently implicated in compounded sterile preparation infections (7). In addition, a
prospective, nationwide surveillance study of nosocomial bloodstream infections from U.S.
hospitals over a 7-year period (8) implicated coagulase-negative staphylococci (31%),
Staphylococcus aureus (20%), Enterococcus species (9%), Candida species (5%), Escherichia
coli (3%), Klebsiella species (2%), and Pseudomonas aeruginosa (2%). The absence of strict
anaerobes among microorganisms most responsible for bloodstream infections is notable and is
considered to be due to the high levels of oxygenation of blood.
Limit of Detection
Within the limitations of preparing inocula with one or more colony-forming units (cfu), growth-
based sterility tests can be shown to have at least a theoretical limit of detection (LOD) of 1 cfu
or 3 cfu based on a Poisson distribution. Setting an LOD of a single viable cell with all
technologies is an unrealistic barrier to entry for any sterility test, especially when the signal is
not the colony-forming unit that is amplified by cultural enrichment. The concept of an infectious
dose is well established, especially in food and clinical microbiology (9). Although the absence of
viable microorganisms in the product has generally been accepted as a definition of sterility,
Page 5 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

there is little or no evidence that 1 cfu is an infectious dose (i.e., clinically significant) for
injectable products. To the contrary, well-established evidence from the study of infection rates
due to the administration of platelet concentrates to human cancer patients suggests that the
infectious dose may be 102–103 viable microorganisms, depending on the virulence of the
microorganism. The study of transfusion infection with platelet concentrations provides an
excellent test case to determine the infectious dose as they have an estimated contamination
rate between 0.03% and 0.7%. In a unique study, Jacob et al (2008) determined the bacterial
content of thousands of platelet concentrates immediately prior to infusion (10) and found that a
detection threshold of at least 103 cfu/mL would detect more than 95% of all infection cases and
that a detection threshold of 102 cfu/mL would detect all cases (100%). These general findings
were confirmed in a follow-up publication from the same researchers from Case Western
University (11) and are generally accepted by the transfusion microbiology community (12).
As noted by the authors of a recent study on the use of the 16S rRNA polymerase chain
reaction (PCR) sterility test for stem cells with the demonstrated bacterial sensitivity of 10–100
cfu/mL, a test method with a sensitivity of 100 cfu/mL would be suitable to detect clinically
significant bacterial contamination of blood and cell products (13).
Time to Result
The incubation time for growth-based 〈71〉 sterility tests is at least 14 days; this makes it
unsuitable for PET and cell therapy as these short-life products would be administered before
completion of the test. This time to result is marginally acceptable for sterile compounding, but
generally suitable for pharmaceutical manufacturing. Some PET drugs may be administered
immediately after preparation due to the short half-life of certain PET radionuclides, so a sterility
test needs to be real time for this stakeholder group. The most commonly used PET radionuclide
is fluorine-18, which is normally used within 12 h. For compounded sterile preparations and cell
therapies, sterility tests need to be completed within a maximum of 48 h, especially when the
dose is needed promptly for a waiting patient. Additionally, manufactured pharmaceuticals can be
tested within 5–7 days to shorten the batch release cycle time. See Table 4 for typical expiration
dating.
Page 6 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Table 4. Typical Beyond Use/Expiration Dating of the Stakeholder Products
Stakeholders Representative Products Beyond Use/Expiration Dating
Sterile
compounding
pharmacies
Low Risk: Reconstitution and
transfer of a 1-g vial of
cefazolin into an IV bag
Medium Risk: Distribution
from a 10-g bulk pharmacy
vial of vancomycin among
several final doses
High Risk: Patient-controlled
analgesic from powdered
morphine
Low Risk: 48 h (room temperature);
14 days (2°–8°); 45 days (frozen)
Medium Risk: 30 h (room
temperature); 9 days (2°–8°); 45
days (frozen)
High Risk: 24 h (room temperature);
3 days (2°–8°); 45 days (frozen)
PET facilities
Fluorine-18
fluorodeoxyglucose (half-life
of 110 min)
Cellular therapy products may be
transported for administration in
hours or days without
cryopreservation, or stored in a
cryopreserved state (<−30°)
indefinitely.
Cell therapy
facilities Stem cells
Cellular therapy products may be
transported for administration in
hours or days without
cryopreservation, or stored in a
cryopreserved state (<−30°)
indefinitely.
Pharmaceutical
manufacturers Numerous examples
2–3 years at ambient or refrigeration
temperature
[NOTE—Signals employed by different technologies may be amplified by enrichment culture
with 24–48 h incubation or by concentration, e.g., filtration, selective adsorption and elution, or
centrifugation, to reduce the time to result and lower the limit of detection.]
Improved Patient Safety
It is widely accepted that a rapid sterility test for compounded sterile preparations,
radiopharmaceuticals, and cell therapies will improve patient safety, especially if contaminated
materials can be detected before administration to patients. Furthermore, sterility test methods
that continuously monitor for the presence of viable microorganisms during processing as a
control strategy would be advantageous. Such monitoring after product release, with a reporting
mechanism when a failure is detected, would enable the laboratory to alert the clinician, who
could then intervene as necessary to protect the patient. The ability of a bacterial contaminant to
grow in a product and its virulence when infused into a patient should both be considered.
Other limitations of the compendial sterility test methods that may impact patient safety are as
follows:
• The ability of the sterility test to be affected by antibiotics in the test sample
• The subjectivity of detecting microbial growth in microbiological culture broth
• The lack of detection of culture-negative infectious agents
• The unintended selectivity of culture media and the incubation temperature/conditions
Many compounded sterile preparations are antibiotics. Cell cultures used to produce cell and
gene therapies may include antibiotics to control microbial contamination during aseptic
Page 7 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

manipulations such as cell culture expansion. As the mode of action of antibiotics usually involves
the bacterial cell wall or protein synthesis, residual antibiotics in the sterility test media may
inhibit bacterial growth leading to false-negative test results. Sterility test methods that are not
growth-based generally are not affected by antibiotic residuals.
Microbial growth in broth will appear as turbidity, pellicles, floccular growth, or precipitation.
However, the product may obscure the presence of microbial growth. It is estimated that cell
densities exceeding 106 cfu/mL are needed to make the media turbid for detection by the naked
eye. These assessments are highly subjective and that may result in false-negative test results.
Although rare, culture-negative infections are observed in clinical microbiology, and PCR and
16S rRNA gene sequencing have been used for bacterial detection and identification, e.g.,
Whipple’s disease (14).
The sterility test media may be incapable of detecting a contaminated product. For example, in
2002 and 2003 there were three clusters of three outbreaks of clostridial disease caused by
Clostridium sordelli in cows and sheep in Spain. Ironically, the outbreaks were linked to anti-
clostridial vaccines, all produced by the same manufacturer, that were intrinsically contaminated
with the same strain of C. sordelli (15). The vaccine batches were released to the market using
the harmonized sterility test. The majority of vials (93%) from the implicated batches contained
low counts of C. sordelli when cultured on sulfite-polymyxin-sulfadiazine agar incubated under
anaerobic conditions at 37° for up to 60 days. The fluid thioglycollate medium used in the sterility
test failed to detect the clostridial contamination, presumably due to thioglycollate inhibition and
the shorter incubation time.
Sample Quantity
The minimum number of articles tested and quantity per container tested per media are
defined in Sterility Tests 〈71〉, Table 2 and 3. Whereas this sampling plan is suitable for
manufactured pharmaceuticals, it is unsuitable for products generated by sterile compounding
pharmacies, PET facilities, and cell therapy centers because of their small batch size, high cost,
and therapeutic value to the individual patient. A further consideration is the sample size
limitation of the advanced technology (see Table 6). Alternative sampling plans have been
proposed, as discussed in Sample Size Limitations above.
Sample Preparation
The complexity and number of steps in the sample preparation process add to the analyst’s
hands-on time, as well as the overall reduced recovery of the signal of viable microbial cells.
Furthermore, to obtain a high throughput and short time to results, one needs an easy sample
preparation. However, a complex sample preparation may be acceptable if the method provides
improvements in time to results and LOD or has the potential to be automated. PET drugs and
radiopharmaceuticals have added requirements associated with the safe handling of radioactive
materials and the need for effective shielding to reduce radiation exposure to acceptable levels.
Aseptic Test Material Handling: Open vs. Closed Systems
Advanced technologies with closed systems will mitigate risk of microbial contamination with
live organisms or their artifacts, such as adenosine triphosphate (ATP) or nucleic acid. With open
Page 8 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

systems, a decision may be made at the testing laboratory to conduct the testing in an isolator
system, which adds to the expense and reduces testing throughput.
POTENTIAL TRADE-OFF BETWEEN CONFLICTING USER REQUIREMENTS
There are obvious trade-offs between LOD, sample size, and time to results (see Table 6).
Detecting a contaminated unit prior to administration is paramount in improving patient safety,
therefore the proposed compendial rapid sterility tests must be risk-based with the stakeholder
selecting the technology that best serves the interests of their patients and the beyond-use
dating of their products. For example, patient safety may be served even if the LOD is 10–100
viable microbial cells, if the test can be completed the same day that a low-volume radiotracer is
compounded.
EXPERIENCE WITH BACTERIALLY CONTAMINATED PLATELET CONCENTRATES
The collective experience with the administration of human platelet concentrates is revealing.
This cellular component, which is obtained from whole blood collection or apheresis, is stored on
rocking platforms at ambient temperature for up to 7 days prior to transfusion. These units have
been reported to have bacterial contamination rates of 0.05%–0.2%. Based on the measurement
of the contamination of transfused platelet concentration it was apparent the rates of septic
reactions were about 50 times less and fatality rates were about 250 times lower (see Table 5).
This supports the view that an infectious intravenous dose is not 1 cfu but the order of 10–100
CFU as reported by Jacobs, et al. (10).
Table 5. Rates of Contamination, Septic Reactions, and Deaths with Administration of
Platelet Concentrates
Contamination Rate per
Units Transfused
Rate of Septic Reactions
per Unit Transfused
Fatality Rate per
Units Transfused
0.05% (5000 contaminated
units/million units transfused
annually)
1%–1.3% of the contaminated
units (10–13 septic
reactions/million units)
15%–20% of the septic
reactions (2
deaths/million units)
(From the FDA Draft Guidance for Industry: "Bacterial Risk Control Strategies for Blood
Collection Establishments and Transfusion Services to Enhance the Safety and Availability of
Platelets for Transfusion", March 2016)
SELECTION OF AVAILABLE TECHNOLOGIES WITH POTENTIAL FOR USE AS A RAPID
STERILITY TEST
The Expert Panel selected the following six analytical platforms, listed alphabetically, as
candidates for compendial rapid sterility testing:
• Adenosine triphosphate bioluminescence
• Flow cytometry
• Isothermal microcalorimetry
• Nucleic acid amplification
• Respiration
• Solid phase cytometry
Brief Descriptions of the Six Analytical Platforms
Page 9 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility T...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Each of these candidate advanced analytical platforms is briefly discussed below, and key
references are provided. For an overview, the reader is referred to the 4-volume series of the
Encyclopedia of Rapid Microbiological Methods (16) and a book dedicated to the topic, Rapid
Sterility Testing (17).
Adenosine Triphosphate Bioluminescence
This is a well-established technology that uses luminometers and reagents available from
multiple instrument manufacturers. The energy from living cells is stored as ATP, which can be
measured as light when exposed to luciferase from the American firefly. Each ATP molecule
consumed by luciferase produces 1 photon of light. The result detected by a luminometer is
typically expressed in relative light units (RLU) and is instrument-, reagent-, and organism-
dependent. The ATP content of different microorganisms ranges from 2–4 × 10−18 mol/cfu for
Gram-negative bacteria, 5–8 × 10−18 mol/cfu for Gram-positive bacteria, and 300–800 × 10−18
mol/cfu for fungi (18). Given the high signal-to-noise ratio, the microbiologically relevant
instrument detection limit is on the order of 5000 RLU, equivalent to 103 cfu.
This LOD will detect the presence of microorganisms at 3–4 log lower numbers within an
aliquot of the media than that required for visual detection of growth in the media. For a sterility
test, an enrichment culture, either in liquid media or on a membrane filter on solid media, could
be used with an incubation time of 2–7 days.
Flow Cytometry
Flow cytometry may be used to detect fluorescently labeled viable microbial cells after an
enrichment culture step that takes 24–48 h (19). A labeling reagent consisting of either a
fluorogenic substrate or vital stain is used to differentiate viable cells from dead cells and cellular
debris. Cell viability is indicated by the ability of the intact cell membrane to retain a
fluorochrome generated by non-specific cellular esterase, or by labeling the cell with nucleic acid-
specific vital stain. An argon laser illuminates each cell in the flow stream and the emitted light is
detected by a dual photomultiplier array. The signal is digitized and interpreted by discrimination
software. Instrumentation and reagents may be obtained from multiple vendors. The LOD for this
technology may be, in the best case scenario, 10–100 viable microbial cells in the absence of a
high-particulate background, so an enrichment/concentration step would be necessary unless a
higher LOD than 1 cfu is accepted.
Isothermal Microcalorimetry
Isothermal microcalorimeters monitor enthalpy changes in closed vials (systems) related to
microbial metabolic activity and growth. With current instruments, 104 active microbial cells can
release enough heat to be detected, although enrichment is needed for detection (2–7 days to
result). The system has its origin in the cement and explosive industry. Within the past several
years its use in biology started to receive more attention, and it is being applied in geology (e.g.,
soil testing), parasitology, optimization of fermenting processes, the food industry (e.g.,
monitoring of microbial growth in milk fermentation processes), clinical applications, and
dentistry (20). Recently, the application of isothermal microcalorimetry in pharmaceutical
microbiology has also been evaluated (21).
Nucleic Acid Amplification
Page 10 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Real-time quantitative PCR has the potential to monitor the exponential phase of PCR through
36–48 cycles of amplification using universal primers to estimate the initial quantity of the target
DNA, which is in turn proportional to the number of microbial cells in the test sample. Unlike
DNA, cellular RNA has a rapid metabolic turnover and is a better indicator of viable
microorganisms. For example, E. coli contains 2 molecules of DNA and 20,000 molecules of 16S
rRNA/cell (22). This process is achieved by the conversion of RNA into a complimentary copy of
DNA by the enzyme reverse transcriptase and can be analyzed in real time in either a
quantitative assay (enumeration test) or qualitative assay (sterility test). Alternatively, for DNA-
based PCR, a sample pre-treatment with ethidium monoazide or propidium monoazide may allow
for differentiation between live and dead microbial cells (23, 24).
Realistically, an LOD of 1 viable cell is probably an insurmountable challenge, especially for a
test that relies on a DNA/RNA target and universal primers.
Generally, the LOD ranges from 10–1000 viable cells/mL of sample, and in some reported
cases it ranges from 10–100 viable cells/mL. Recently it was shown that PCR may actually
achieve detection of microorganisms with a limit of 102–103 cfu/mL in a sample containing a high
concentration of up to 106 mammalian cells/mL without the need for pre-incubation in microbial
growth media (25). Adding a growth-based enrichment step for at least 24–48 h and comparing
the PCR results before and after enrichment may provide a practical solution for sterility testing.
Alternatively, concentration methods could be applied to enrich the sample and reduce the
sample volume. Instrumentation and reagents may be sourced from multiple vendors.
The higher LOD of 10–100 viable cells/mL does not mean that PCR methods are unsuitable for
sterility testing. Jacobs, et al. (10) reported the relationship between the bacterial load and
transfusion reactions with platelet concentrates. Based on the data reported they conclude that a
threshold of 1000 cfu/mL would detect more than 95% of all cases of contamination and 90% of
the reactions, whereas a 100 cfu/mL threshold would detect all cases (100%). Data derived from
transfusion medicine are particularly useful (see Table 5), and are used for patients undergoing
bone marrow transplantation or receiving chemotherapy.
Use of non-growth based sterility tests such as PCR increases patient safety for the following
reasons:
• With sterility testing that is close to real-time, the test is completed before the short-lived
product is infused into a patient
• Culture-negative infectious agents are isolated
• The test is unaffected by antibiotics in the test sample
• The test is less sensitive to background noise resulting from animal cell lysis (e.g.,
particles, ATP), as compared to other technologies, because specific microbial genes are
targeted
Respiration
This broad category ranges from classical respirometers to gaseous headspace analyzers to
automated blood culture systems. The use of automated blood culture systems has been
successfully extended to sterility testing of cell therapy products. In 2004, the FDA approved a
supplement to the biologics license held by Genzyme Biosurgery for Carticel, autologous cultured
Page 11 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

chondrocytes, to use the BacT/ALERT™ Microbial Detection System with a 7-day incubation as an
alternative to the compendial sterility test for lot release (26).
Other instruments are available to detect and enumerate respiring microorganisms. For
example, tunable diode laser absorption spectroscopy (TDLAS) can measure O2 depletion or CO2
increase in closed units containing growing microorganisms in culture medium. The system was
developed to monitor gas headspace composition in closed units and also could be used for
automatic media fill inspection (21, 27). TDLAS has gaseous calibration standards, and minor
adaptations are needed if the system is to be used for sterility testing (e.g., calibrating for
higher-volume containers).
Note that all the systems of the respiration platform require microbial growth and metabolic
activity for detection, i.e., the usual time to result of 2–7 days is required. However, the results
can be progressively monitored to detect a sterility test failure earlier in the incubation period,
which is a huge advantage with short-life products.
Solid-Phase Cytometry
Several instrument manufacturers market systems based on solid-phase cytometry. For
instance, the ScanRDI™ microbial analysis system has the most market experience and combines
fluorescent labeling and solid-phase laser scanning cytometry to rapidly enumerate viable
microorganisms in filterable liquids (28). Cells are collected by filtration on 0.45-µm polyester
membranes and treated with background and viability stains. The filters are scanned in a
cytometer by a high-speed, 488-nm argon laser. Fluorescence is detected by multiple
photomultiplier tubes and processed to differentiate between labeled microorganisms and
background noise. The scan is displayed as map that identifies the positions of the fluorescent
events, which are verified using an epifluorescence microscope with an automated motorized
stage to locate the individual events. The system is claimed to identify individual viable
microorganisms in 2–3 h.
In Table 6, the critical operating parameters of representative candidate modern
microbiological methods are provided for informational purposes. These values are estimated,
and may be optimistic in some cases. The list is not all-inclusive and does not constitute an
endorsement of any single technology.
Page 12 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Table 6. Operational Parameters of Candidate Technologies
Representative
Detection System Technology
Limit of
Detection
(cfu/mL)
Time to
Result
Sample
Size
(mL)
Gram stain Classical 104–10530 min 0.1
BacT/ALERT System Respiration 1–10
Overnight to
7 days 5–10
ScanRDI System
Solid-phase
cytometry 1–10 2–3 h 1–500
Milliflex Rapid
System
ATP
bioluminescence 1–10 5–7 days 1–500
FACS analysis Flow cytometry 10–100
6–8 h (pre-
enrichment) 0.1–2
Roche LightCycler
Nucleic acid
amplification 1–100 2–4 h 0.2–2
TAM V
Isothermal
microcalorimetry 1–10 2–7 days 1
Representative Instrumentation Manufacturers of the Candidate Technologies
One requirement for an analytical platform to be considered as a compendial sterility test is
that it is nonproprietary and there are multiple vendors for the technology and associated
reagents. Although it is not all-inclusive, Table 7 provides more details of the justification based
on this requirement.
Page 13 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Table 7. Commercially Available Instrumentation Showing Multiple Vendors
Advanced
Technology Instrument Name Vendor
ATP bioluminescence
Biotrace 2000
Pallchek Rapid System
Milliflex Rapid System
Celsis RapiScan
BioMAYTECTOR
Biotrace International, Bridgend,
UK
Pall Corp., Port Washington, NY
Millipore Corp., Bedford, MA
Charles River Laboratories, Inc.,
Wilmington, MA
Hitachi Plant Technologies,
Tokyo, Japan
Flow cytometry
Bact-Flow
FACSMicroCount
bioMerieux, Hazelwood, MO
Becton, Dickinson & Co. (BD),
Sparks, DE
Isothermal
microcalorimetry
TAM III calorimeter
Biocal 2000 isothermal
calorimeter
48-channel isothermal
microcalorimeter
TA Instruments, Wilmington, DE
Calmetrix, Arlington, MA
SymCell Sverige, Kista, Sweden
Nucleic acid
amplification
Multiple thermocyclers and
amplicor analyzers
Roche Applied Science,
Indianapolis, IN
Applied Biosystems, Foster City,
CA
Cepheid, Sunnyvale, CA
Respiration
Promex 4200 microrespirator
BACTEC System
BioLumix BacT/ALERT 3D
Dual-T System
Pall eBDS System TDLS
PromChem Ltd., Edenbridge, UK
BD Diagnostics, Sparks, DE
BioLumix, Ann Arbor, MI
bioMerieux, Hazelwood, MO
Pall Corp., Port Washington, NY
Lighthouse Instruments,
Charlottesville, VA
Solid-phase
cytometry
ScanRDI System
BioSafe PTS
MuScan System
bioMerieux, Hazelwood, MO
Charles River Laboratories, Inc.,
Wilmington, MA
Innosieve Diagnostics,
Wageningen, The Netherlands
The path forward for the adoption of these analytical platforms as compendial tests for short-
lived products includes 1) writing an informational general chapter on risk-based sterility testing,
2) collaborative development of generic rapid sterility tests and validation of the selected test
methods, and 3) writing and publishing them as official USP tests.
REFERENCES
1. US Pharmacopeia. Sterility Tests 〈71〉. In: USP 40–NF 35. Rockville, MD: US
Pharmacopeial Convention 2017:136.
2. Cundell AM. Review of the media selection and incubation conditions for the compendial
sterility and microbial limit tests. Pharm. Forum 2002;28(6):2034–2041.
3. Cundell AM. The history of the development, applications, and limitations of the USP
sterility test. In: Moldenhauer J, ed. Rapid Sterility Tests. Bethesda, MD: PDA/DHIP;
[2011]:127–170.
Page 14 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

4. Sutton S. The sterility tests. In: Moldenhauer J, ed. Rapid Sterility Tests. Bethesda, MD:
PDA/DHIP; [2011]:7–28.
5. Van Doorne H, Van Kampen B J, et al. Industrial manufacture of parenteral products in
The Netherlands: A survey of eight years of media fills and sterility testing. PDA J. Pharm.
Sci. & Technol. 1998; 52(2):159–164.
6. Bathgate H, Lazzari D, Cameron H, McKay D. The incubation period in sterility testing.
PDA J. Parent. Sci. & Technol. 1993;47(5):254–257.
7. The Pew Charitable Trusts. Report on U.S. Illnesses and Deaths Associated with
Compounded Medications. September 06, 2014.
8. Wisplinghoff H, Bischoff T, Tallent SM , Seiffert H, Wenzel RP, Edmond MB. Nationwide
surveillance study of nosocomial bloodstream infections from U.S. hospitals over a 7-year
period. Clin. Infect. Dis. 2004;39:309–317.
9. Leggett HC, Cornwallis CK, West SA. Mechanisms of pathogenesis, infective dose and
virulence in human parasites. PLOS Pathogens. 2012;8(2):e1002512
10. Jacob MR, Good CE, Lazarus HM, Yomtovian RA. Relationship between bacterial load,
species virulence, and transfusion reaction with transfusion of bacterially contaminated
platelets. Clin. Infect. Dis. 2008;46:1214–1220.
11. Hong H, Xiao W, Lazarus HM, Good CE, Maitta RW, Jabobs MR. Detection of septic
transfusion reactions to platelet transfusions by active and passive surveillance. Blood.
2016;127(4): 496–502.
12. Benjamin R J. Transfusion-related sepsis: a silent epidemic. Blood. 2016;127(4):380
–381.
13. Tokuno O, Hayakawa A, et al. Sterility testing of stem cell products by broad-range
bacterial 16S ribosomal DNA Polymerase Chain Reaction. Lab Med. Winter 2015;46:8–41.
14. Marth T. Tropheryma. ASM Manual of Clinical Microbiology, 10th Edition. 2011;1:1035
–1039.
15. Tellez S, Casimiro R, et al. Unexpected inefficiency of the European Pharmacopoeia
sterility test for detecting contamination in clostridial vaccines. Vaccines. 2006;24:1710
–1715.
16. Miller M J (Editor). Encyclopedia of Rapid Microbiological Methods, Vol. 1–4, PDA/DHIP,
2005–2013.
17. Moldenhauer J (Editor). Rapid Sterility Testing. PDA/DHIP, Bethesda, MD. 2011:488.
18. La Duc M T, Dekas A, Osman S, Moissl C, Newcombe D, Venkateswaran K. Isolation and
characterization of bacteria capable of tolerating the extreme conditions of clean room
environments. Appl. Environ. Microbiol. 2007;71(8):2600–2611.
19. Baumstummler A, Chollet R, Meder H, Rofel C, Venchiarutti A, Ribault S. Detection of
microbial contamination in mammalian cell cultures using a new fluorescence-based
staining method. Lett. Appl. Microbiol. 2010;51:671–677.
20. Braissant O, Bonkat G, Wirz D, Bachmann A. Microbial growth and isothermal
microcalorimetry: Growth models and their application to microcalorimetric data.
Thermochimica Acta. 2013;555:64–71.
21. Bruekner D, Roesti D, Zuber UG, Schmidt R, Kraehenbuehl S, Bonkat G, Braissant O.
Comparison of tunable diode laser absorption spectroscopy and isothermal
microcalorimetry for non-invasive detection of microbial growth in media fills. Scientific
Reports 6. 2016; article 27894; doi: 10.1038/srep27894.
22. Overall macromolecular composition of E. coli. Adapted from F. C. Neidhardt et al.,
“Physiology of the bacterial cell”, Sinauer, 1990, by Katja Tummler and Ron Milo.
Page 15 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml

Accessed 13 January 2017 from the website
http://bionumbers.hms.harvard.edu//bionumber.aspx?id=111490&ver=6.
23. Patel P, Garson JA, Tettmar KI, Ancliff S, McDonald C, Pitt T, Coelho J, Tedder RS.
Development of an ethidium monoazide-enhanced internally controlled universal 16S
rDNA real-time polymerase chain reaction assay for detection of bacterial contamination
in platelet concentrates. Transfusion. 2012;52(7):1423–1432.
24. Nocker A, Cheung C-Y, Camper AK. Comparison of propidium monoazide with ethidium
monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from
dead cells. J. Microbiol. Methods. 2006;67:310–320.
25. Kleinschmidt K, Wilkens E, Glaeser SP, Kaempfer P, Staerk A, Roesti D. Development of a
qualitative real-time PCR for microbiological quality control testing in mammalian cell
culture production. J. Appl. Microbiol. Dec 28, 2016.
26. Kielpinski G, Prinzi S, Duguid J, du Moulin G. Roadmap to approval: use of an automated
sterility test method as a lot release test for Carticel, autologous cultured chondrocytes.
Cytotherapy. 2005;7(6):531–541.
27. Duncan D, Cundell T, Levac L, Veale J, Kuiper S, Rao R. The Application of Noninvasive
Headspace Analysis to Media Fill Inspection. PDA J Pharm. Sci. Technol. May/June
2016;70: 230–247.
28. Smith R, Von Tress M, Ubb C, Vanhaecke E. Evaluation of the ScanRDI as a rapid
alternative to the pharmacopoeial sterility test method: Comparison of limits of detection.
PDA J. Pharm. Sci. & Technol. 2010;64(4):358–363.
a USP Modern Microbiological Methods Expert Panel Members (Listed alphabetically with affiliation): Thierry Bonnevay,
Sanofi Pasteur; Randolph Breton, Infuserve; Claudio Denoya, Particle Measuring Systems Technology; Anthony M. Cundell,
USP Microbiology Expert Committee (Co-chair); John Duguid, Vericel; Matthew Jenkins, UVA Medical Center; Felix Montero
Julian, bioMerieux; James Kenney, FDA/CBER; Amy McDaniel, Pfizer; Michael Miller, Microbiology Consultants, LLC; Gary du
Moulin, Massachusetts College of Pharmacy and Health Sciences; David Newton, USP Compounding Expert Committee; David
Hussong, Chair, USP Microbiology Expert Committee; Kuldip Patel, Duke University Hospital; Steven Richter, Microtest
Laboratories; David Roesti, USP Microbiology Expert Committee; Edward Tidswell, USP Microbiology Expert Committee (Co-
chair); Yongqiang Zhang, BD; Steven Zigler, USP Chemical Medicines Expert Committee.
b Disclaimer:The views presented in this article do not necessarily reflect those of the organizations for which the authors
work. No official support or endorsement by these organizations is intended or should be inferred.
c Disclaimer: Certain commercial equipment, instruments, vendors, or materials are identified in this Stimuli article for
informational purposes. Such identification does not imply approval, endorsement, or certification by USP of a particular
brand or product, nor does it imply that the equipment, instrument, vendor, or material is necessarily the best available for
the purpose or that any other brand or product was judged to be unsatisfactory or inadequate. All product names, logos, and
brands are property of their respective owners
d The conflicts of interest of the named authors of this article are as follows: Yongqiang Zhang is employed by BD
Biosciences; Felix Montero Julian is employed by bioMerieux ; Anthony M. Cundell and Michael J. Miller consult with some of
the technology vendors indicated in this article
e Correspondence should be addressed to: Radhakrishna S.Tirumalai, Ph.D., Principal Scientific Liaison-General Chapters,
US Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD 20852-1790; tel 301-816-8339; e-mail:
rst@usp.org.
Page 16 of 16
43(5) Stimuli to the Revision Process: The Development of Compendial Rapid Sterility...
6/25/2018
http://www.usppf.com/pf/pub/data/v435/GEN_STIMULI_435_s201904.xml