Suitability of

ScanRDI as a
®

Rapid Sterility
Testing Method
 Table of Contents

3 Eagle ScanRDI® Sterility Test Protocol

Ross Caputo, PhD

4 Evaluation of the ScanRDI® as a Rapid Alternative to the Pharmacopeial

Sterility Test Method: Comparison of the Limits of Detection
Smith, R., Von Tress, M., Tubb, C., & Vanhaecke, E.

6 Sterility Testing: Compendial and Alternative Rapid Methods

David Hussong, PhD

9 ScanRDI® System Performance Data as a Sterility Test Method for

Pharmacy Compounded Preparations: A Ten Year Survey
Ross Caputo, PhD, and David Hussong, PhD

15 USP Stimuli to the Revision Process: The Development of Compendial

Rapid Sterility Tests
Members of the USP Modern Microbiological Methods Expert Panel

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© 2018 Eagle Analytical Services, Inc.
 Eagle ScanRDI® Sterility Test Protocol
Ross Caputo, PhD, President at Eagle

Would your sterile compounding process pass an FDA inspection?

In order to fully comply with USP <797>, a compounding pharmacy creating sterile preparations must
develop its own formal quality assurance (QA) program. The characteristics of a QA program require
the consideration of environmental testing and preparation verification results, with the recommended
testing method outlined by USP <71>.

Because the compendial method of testing takes 14 to 18 days to complete, it is extremely limiting in
the compounding industry. A sterile compound with a standard 30 day beyond-use date (BUD) will
lose approximately half of its shelf life due to sterility testing. This delay may discourage compounders
from developing or adhering to a QA program, leaving them at risk of failing an FDA inspection.

However, there is a faster way to comply with USP <797> and ensure your patients are receiving
quality compounds. The ScanRDI Sterility Test Protocol is an FDA-accepted alternative to the
official compendial sterility test method. ScanRDI rapidly detects viable microbial cells down to
one microorganisms without the need for growth or cell multiplication. This removes the extended
incubation period included in the USP <71> test method, so results are ready in just one day.

According to USP General Notices, alternate testing methods may be used if they can be shown to
provide equivalent or better results. ScanRDI uses the same sampling protocols as USP <71>, detects
all of the standard USP organisms, and has been shown to provide consistent and reliable results.1

Additionally, ScanRDI fulfills the method suitability requirement as outlined in USP <1223>. A
suitability test is performed on every unique drug formula sample submitted for ScanRDI testing. If the
compound does not pass this suitability test, Eagle will inform the compounding pharmacist that it was
incompatible with the ScanRDI method.

1
Smith, R., Von Tress, M., Tubb, C., & Vanhaecke, E. (2010). Evaluation of the ScanRDI as a rapid alternative to the pharmacopoeial sterility
test method: Comparison of limits of detection. PDA Journal of Pharmaceutical Science and Technology, 64(4), 356-363.

PAGE 3
© 2018 Eagle Analytical Services, Inc.
 Evaluation of the ScanRDI® as a Rapid Alternative to the
Pharmacopeial Sterility Test Method: Comparison of the
Limits of Detection
Smith, R., Von Tress, M., Tubb, C., & Vanhaecke, E. (2010). Evaluation of the ScanRDI as
a rapid alternative to the pharmacopoeial sterility test method: Comparison of limits of
detection. PDA Journal of Pharmaceutical Science and Technology, 64(4), 356-363.

TABLE II
Paired T-Tests of Non-Inferiority of the ScanRDI® and Compendial Sterility Test
Difference Lower
between Standard Confidence
Organism Mean MPNa Deviation n zn P Limitb
Clostridium sporogenes 0.05 0.29 24 3.518 0.0009 -0.050
Propionibacterium acnes 0.58 0.40 15 7.128 <0.0001 0.398
Escherichia coli 0.20 0.30 25 5.812 <0.0001 0.092
Pseudomonas aeruginosa 0.95 0.37 13 10.972 <0.0001 0.762
Staphylococcus aureus 0.39 0.29 14 7.067 <0.0001 0.251
Bacillus subtilis 0.43 0.23 12 8.904 <0.0001 0.308
Aspergillus niger 0.67 0.41 16 8.115 <0.0001 0.490
Candida albicans 0.64 0.46 13 6.278 <0.0001 0.412
a
Mean log10 MPN for ScanRDI minus mean log10 MPN for compendial sterility method.
b
The acceptance criteria were satisfied if the lower one-sided 95.25% confidence limit on the paired difference was less
than -0.1549, which is log10(0.70).

TABLE IV
Comparison of the Limits of Detection (cells/mL-1)

ScanRDI Compendial
Organism Method Method
Clostridium sporogenes 0.000070 0.002992
Propionibacterium acnes 0.000153 0.002805
Escherichia coli 0.000805 0.003846
Pseudomonas aeruginosa 0.001172 0.017179
Staphylococcus aureus 0.000489 0.002871
Bacillus subtilis 0.000192 0.001832
Aspergillus niger 0.000690 0.003199
Candida albicans 0.000178 0.005370

PAGE 4
FIGURE 1
Difference between mean concentrations of bacteria detected by ScanRDI and the compendial methods for each of
eight bacteria. Data points falling within the shaded region would indicate that the ScanRDI method was inferior to the
compendial sterility test method for detecting contaminants. Error bars represent the two 1-sided 95% confidence intervals.

FIGURE 2
Percent of culture tubes showing the presence of microorganisms at three dilution levels. Open symbols depict data
collected using the ScanRDI method, while closed symbols depict data collected using the reference compenidal sterility
test method. Values alongside the names of organisms in the legend indicate the total number of tubes tested for a given
organism. Horizontal lines (dashed or dotted) depict the overall mean for all organisms at a given dilution.

PAGE 5
 Sterility Testing: Compendial and Alternative
Rapid Methods
David Hussong, PhD, Chief Technical Officer at Eagle

Introduction

The compendial USP <71> sterility tests are growth-based and require at least a 14-day incubation
to yield results. This time requirement is not suitable for short-lived products or those prepared for
immediate use that are frequently infused into patients before the completion of the compendial
test. Additionally, this reduces the useable time period while adding significant cost to the
release testing. To address the needs of stakeholders preparing compounded sterile preparations,
radiopharmaceuticals, and cell and gene therapies, the USP Microbiology Expert Committee assessed
and has proposed alternative test methods for addition to the compendia, including new-generation
rapid sterility tests. The complete proposal will be published in Pharmacopeial Forum (September,
2017; 43(5)). This paper summarizes some findings related to the status of rapid sterility test methods.

Background

With primary consideration of improved patient safety, USP has established user requirement
specifications (URS) for rapid sterility tests. They determined that the URS are different for various
therapies and diagnostics, and the means of producing those therapies by the four major users:
1. Sterile compounding
2. Positron emission tomography (and other short-lived radiopharmaceuticals)
3. Cell therapy
4. Traditional pharmaceutical manufacturing

Findings

The USP has identified several technologies that are suitable for use as alternative, rapid sterility
tests. These offer acceptable results to meet the user requirement specifications pending suitability
assessment, just as the compendial <71> methods are subject to suitability testing with each product
or product class. Additionally, there are various vendors for each technology. There are some platforms
that have been in use for longer periods, and for which more data exists.

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© 2018 Eagle Analytical Services, Inc.
The following table provides a list of these technologies and suppliers:

Advanced Technology Instrument Name Vendor

ATP bioluminescence Biotrace 2000 Biotrace International, Brigend,

UK
Pallcheck Rapid System Pall Corp., Port Washington, NY
Milliflex Rapid System Millipore Corp., Bedford, MA
Celsis RapiScan Charles River Laboratories, Inc.,
Wilmington, MA
BioMAYTECTOR Hitachi Plant Technologies, Tokyo,
Japan

Flow cytometry Bact-Flow bioMerieux, Hazelwood, MO

FACSMicroCount Becton, Dickerson & Co., Sparks,
DE

Isothermal microcalorimetry TAM III Calorimeter TA Instruments, Wilmington, DE

Biocal 2000 Isothermal Calimetrix, Arlington, MA
Calorimeter
48-challen isothermal SymCell Sverige, Kista, Sweden
microcalorimeter

Nucleic acid amplification Multiple thermocyclers and Roche Applied Science,

amplicor analyzers Indianopolis, IN
Applied Biosystems, Foster City,
CA
Cepheid, Sunnyvale, CA

Respiration Promex Microrespirator 4200 PromChem Ltd., Edenbridge, UK

BACTEC System BD Diagnostics, Sparks, DE
BioLumix BioLumix, Ann Arbor, MI
BacT/ALERT 3D Dual-T System bioMerieux, Hazelwood, MO
eBDS Pall System Pall Corp., Port Washington, NY
TDLS Lighthouse Instruments,
Charlottesville, VA

Solid phase cytometry ScanRDI® System bioMerieux, Hazelwood, MO

BioSafe PTS Charles River Laboratories, Inc.,
Wilmington, MA
MuScan System Innosieve Diagnostics,
Wageningen, The Netherlands

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© 2018 Eagle Analytical Services, Inc.
Among the technologies is ScanRDI, a long-used solid-phase technology that has an established
record for detection of filterable organisms in solutions or soluble products.1 Cells are collected by
filtration on 0.45-micron 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, processed to differentiate between labeled microorganisms and
background noise. The scan is displayed as a map that identifies the position of the fluorescent events
that 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 hours.
Technologies using similar principles were accepted for clinical use beginning in 19872 and also offered
the advantage of detecting senescent microorganisms not recoverable by cultivation.3,4

Discussion

Rapid microbiology methods have been accepted for FDA approved products. Carticel, a cell therapy
product, uses the BacT/Alert system that detects headspace gases generated during several days of
incubation.5 Similarly, the National Institutes of Health has used the Bactec system for clinical supply
(ING) products.6 Bactect also requires up to 14-days incubation but reliable product release results
generally occur within 7-days. Although unpublished, there are approved drug applications using the
ScanRDI for sterility tests for product release. This assay can yield results in one day. None of the rapid
methods appear suitable for all products’ needs, just as the compendial methods need to be adjusted
for proper results. However, the alternative methods provide reliable results in less time. As noted in
USP General Notices, section 6.30, Alternative and Harmonized Methods and Procedures, “Alternative
methods and/or procedures may be used if they provide advantages in terms of accuracy, sensitivity,
precision, selectivity, or adaptability to automation or computerized data reduction, or in other special
circumstances.”

References
1. Smith, R., Von Tress, M., Tubb, C., & Vanhaecke, E. (2010). Evaluation of the ScanRDI as a rapid alternative to the pharmacopoeial
sterility test method: Comparison of limits of detection. PDA Journal of Pharmaceutical Science and Technology, 64(4), 356-363.
2. Kronvall, G., and Myhre, E. (1977). Differential staining of bacteria in clinical specimens using acridine orange buffered at low pH. Acta
Pathologica et Microbiologica Scandinavica. Section B, Microbiology, 85(4), 249-254.
3. Lauer, B. A., Reller, L. B., & Mirrett, S. (1981). Comparison of acridine orange and Gram stains for detection of microorganisms in
cerebrospinal fluids and other clinical specimens. Journal of Clinical Microbiology, 14(2), 201-205.
4. Daley, R. J., & Hobbie, J. E. (1975). Direct counts of aquatic bacteria by a modified epifluorescence technique. Limnology &
Oceanography, 20(5), 875-882.
5. Kielpinski, G., Prinzi, S., Duguid, J., & du Moulin, G. (2005). Roadmap to approval: Use of an automated sterility test method as a lot
release test for Carticel, autologous cultured chondrocytes. Cytotherapy, 7(6), 531-541.
6. Khuu, H. M., Patel, N., Carter, C. S., Murray, P. R., & Read, E. J. (2006). Sterility testing of cell therapy products: Parallel comparison of
automated methods with a CFR-compliant method. Transfusion, 46(12), 2071-2082.

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© 2018 Eagle Analytical Services, Inc.
 ScanRDI® System Performance Data as a Sterility Test
Method for Pharmacy Compounded Preparations:
A Ten Year Survey
Ross Caputo, PhD, President at Eagle
David Hussong, PhD, Chief Technical Officer at Eagle

Abstract

Since 2007, Eagle has utilized the ScanRDI system to determine the sterility of pharmacy compounded
products. During this time period nearly 40,000 tests have been performed, encompassing nearly 1500
different drug compound categories for nearly 500 different pharmacy locations. This is believed to
be the most complex data set ever accumulated. Of the products tested 0.96% (383) were found to be
non-sterile, 96% sterile and 3.4% were found to be incompatible (no test) with the process. The data
indicated that there was no statistical difference between pharmacy locations for the same product
preparations compounded nor was there a difference in data between trained Eagle scientists. During
this same time period traditional sterility testing outlined in USP 71 was also performed. During this
time period over 45000 tests were performed for approximately 1000 different pharmacy locations
over 1500 drug compound categories. The sterility positive rate was .63% (288) This evaluation
suggests that the Eagle ScanRDI method functions as an effective, efficient, alternative to the USP
Sterility test method.

Introduction

Proposed USP <797> revisions will dramatically reduce current beyond use dates (BUDs) for sterile
compounded preparations.1 Finished product testing required to assign the maximum allowable BUD
requires a final preparation sterility test to be performed. The United States Pharmacopeia (USP) test
for sterility, USP <71>, requires a minimum of 14 days for completion and can take up to 18 days,
which could severely affect the availability of compounded preparations. For example, in the absence
of a sterility test, sterile compounded preparations are permitted a BUD of 4 to 6 days when stored
at room temperature. Once a sterility test has been completed, however, the permitted BUD extends
to 42 days. Even if these dates are ultimately extended, it is apparent that it is critical to minimize the
testing period so as to ensure the delivery/dispensing of prescribed products to the patient within the
required time of not only dispensing but patient use.

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© 2018 Eagle Analytical Services, Inc.
Since 2004, Eagle has used and perfected the ScanRDI analyzer for the sterility testing of
compounded product preparations. This system is the subject of an FDA Type V Drug Master File
(DMF 14621) which contains data to support the technology for the detection of bacteria, spores,
yeasts, and molds.2 This master file also contains the validation data for the process of water
microbial analysis, which supports previously published data.3 Using this process, a sterility test can
be performed in one day. Eagle now presents data compiled from using the ScanRDI system over
a decade for sterility testing, analysis, and research onunique patient-specific sterile compounds
prepared in multiple pharmacy locations. The data show that ScanRDI performance supports the
utilization of this method for the sterility testing of compounded preparations.

Method

All samples were prepared and managing in conformance with Eagle’s standard operating
procedures. This was, in the beginning, a research procedural system focused on the research
associate documenting what was performed. As procedures were developed, they were codified
but as with all dynamic operations a functional change control system exists for tracking
effective dates for all procedures. The ScanRDI units were installed by the manufacturer who
was also tasked with the performance of Installation Qualification, Operational Qualification, and
Performance Qualification (IQ/OQ/PQ), to include final reports. All recommended maintenance
is performed, and has always been, on contract by the manufacturer. All reagents and supporting
material are supplied by the manufacturer. All personnel involved in the performance of tests are
trained directly by the manufacturer. Only degreed scientists have been trained to use this system.

As a summary, the ScanRDI system can be described as follows. ScanRDI uses a combination
of fluorescent labeling and solid phase laser cytometry to identify viable microorganisms from
filterable samples. The system sensitivity can detect a single cell within 3 hours. Proprietary stains
which consist of non –fluorescent membrane permeant substrates are used which are cleaved by
non-specific esterases and retained in viable cells. This accumulated measurable chromophore is
then detected during the laser scanning step in the ScanRDI analyzer. Within 3 minutes, results are
displayed without operator interpretation. Some products are incompatible with this system due to
auto fluorescence of the preparation. The unit will automatically indicate incompatibility, and the
results would not be reported as a sterility failure. A scan map display showing the precise location
of each organism for visual confirmation is a system microscope option that is used by Eagle to
confirm all results. The reader is referred to the manufacturer for a more detailed description of the
operation.4

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© 2018 Eagle Analytical Services, Inc.
 Results
Sales

ScanRDI Breakup of Tests — ScanRDI and USP <71>

39,689
46.58%
Of the 85,211 sterility tests conducted over the
time period studied:
• 39,689 (46.58%) were ScanRDI rapid
sterility tests
• 45,522 (53.42%) were USP <71> 14-day
sterility tests

USP <71>
46,522
53.42%
2nd Qtr 1st Qtr

Sales

ScanRDI
383
Fails/Positives — ScanRDI and USP <71>
57.08%

ScanRDI USP <71>

Pass (Negative) 37,955 45,234

Percent of Total 95.63% 99.37%

Fail (Positive) 383 288

Percent of Total 0.97% 0.63%

Incompatible 1,351 --

Percent of Total 3.40% --

USP <71>
288 Total 39,689 45,522
42.92%
2nd Qtr 1st Qtr

ScanRDI Method USP <71> Method

PAGE 11
© 2018 Eagle Analytical Services, Inc.
 Results

Number of Tests — ScanRDI and USP <71> Over Time

13,000 12,662

12,000

11,000 10,817

10,000 9,731
Distinct Count of Sample and Method

9,532

9,000

8,000 7,956

7,765

7,000

6,000

5,000
4,582
4,582

4,000

3,000
2,525
2,282 2,317 2,202
1,865 2,347
2,000
1,764

1,000 1,319 1,264

921

0
2007 2008 2009 2010 2011 2012 2013 2014 2015

Year of Analysis Date

ScanRDI Method USP <71> Method

PAGE 12
© 2018 Eagle Analytical Services, Inc.
 Results

Number of Tests — ScanRDI and USP <71> Over Time

10,000

9,025 9,116
9,000

8,000

7,585
7,000
Distinct Count of Sample ID

6,000

5,000
4,500
4,000

3,000
2,118
2,000 2,239
1,267 1,205
900
1,000 667
397
36 6 47 76 78 68 175
0
16 6 12 8 30 40 39 19 5

-1,000

100% 96.06% 97.72% 95.33% 96.19% 97.66%

95.64%
92.74%
95.40% 97.68%
Percent of Total Distinct Count of Sample ID

80%

60%

40%

20%

6.85%
2.73% 3.72% 3.45% 3.32% 4.16%
1.63% 1.48% 2.25%
0%
1.21% 0.65% 0.95% 0.36% 1.28% 0.87% 0.40% 0.20% 0.06%

-20%
2007 2008 2009 2010 2011 2012 2013 2014 2015

Year of Analysis Date

Pass / Negative Fail / Positive Inconclusive / Incompatible

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© 2018 Eagle Analytical Services, Inc.
 Results

Top 10 Categories for Samples Failed

BEVACIZUMAB 33 10

HCG 13 15

VITAMIN 12 16 7

VITAMIN B COMPOUNDS 9 10

BETAMETHASEONE COMPOUNDS 1 13
Categories

ASCORBIC ACID 6 7

SCM 10 3

TRIMIX 4 6

HOMEOPATHIC PREPARATIONS 1 8

SODIUM COMPOUNDS 7 2

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

Distinct Count of Sample and Method

ScanRDI Method USP <71> Method

Conclusion

As described by Smith, “…the ScanRDI method is appropriate for use as a rapid alternative to the
growth-based sterility test method.”5 Furthermore, given short beyond-use dating requirements,
Eagle is not aware of any other option than the ScanRDI method at this time which has been
evaluated as extensively with such a large data base of this specific use to evaluate end product
sterility of compounded preparations.

References
1. United States Pharmacopeial Convention (2018). <797> pharmaceutical compounding – Sterile preparations [In-process revision:
General chapters]. Pharmacopeial Forum, 44(5). Retrieved from http://www.usppf.com/pf/pub/
2. bioMérieux (n.d.). FDA Drug Master File #14621.
3. Jones, D. L., Brailsford, M. A., & Drocourt, J.-L. (1999) Solid-phase, laser-scanning cytometry: A new two-hour method for the
enumeration of microorganisms in pharmaceutical water. Pharmacopeial Forum, 25, 7626-7645.
4. bioMérieux (n.d.). ScanRDI®. Retrieved from http://www.biomerieux-industry.com/biopharma/scanrdi
5. Smith, R., Von Tress, M., Tubb, C., & Vanhaecke, E. (2010). Evaluation of the ScanRDI as a rapid alternative to the pharmacopoeial
sterility test method: Comparison of limits of detection. PDA Journal of Pharmaceutical Science and Technology, 64(4), 356-363.

PAGE 14
© 2018 Eagle Analytical Services, Inc.
 USP STIMULI TO THE REVISION PROCESS
Stimuli articles do not necessarily reflect the policies of the USP or the USP Council of Experts.

The Development of Compendial Rapid Sterility Tests

Members of the USP Modern Microbiological Methods Expert Panel

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.

PAGE 15
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 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.2,3,4 Table 1 contains a brief
summary of the development of <71> from 1936–2009.

Table 1:

Compendial Revision Brief Description of the Sterility Test

USP XI (1936), page 469, Tests for 7-day incubation in 37° in a beef extract-peptone-dextrose broth
the Sterility of Liquids

USP XII (1941), Sterility Test for Additions: broth for sterility tests under anaerobiosis, inactivating
Solids added fluids, and a honey medium for molds and yeast incubated at 22° -
22° for 15 days

USP XVII (1965), pages 829-832, Additions: fluid thioglycollate medium incubated at 30°-32° for 7
Sterility Test days, fluid Sabouraud dextrose medium incubated at 22° - 25° for 10
days, and bacteriostasis and fungistatis testing added to demonstrate
the suitability of the method for each specific product

USP XVIII (1970), pages 851-857, Revisions: fluid thioglycollate medium incubated at 30°-35° for 14
Sterility Tests 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, Harmonization: effective January 1; however, the compendial sterility
Sterility Tests <71> tests contained 11 local non-harmonized requirements; all incubation
times, regardless of product, were 14 days

First Supplement to the USP 32 Revisions: the 11 local non-harmonized requirements were removed
(2009), Sterility Tests<71> with an official date of August 1, 2009

PAGE 16
 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 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, 33% grew in the fluid thioglycollate medium, and a mere 9% grew in both media5 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 rate4 (see Table 2).

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.

PAGE 17
 Table 2: Probability of Failing the USP Sterility Test with Required Sample Size
Probability of Failing the USP Sterility Test with
Frequency of Contaminated Units in a Batch
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%)

Currently, the minimum number of articles tested and quantity per container tested per media
are defined in Sterility Tests <71>, Tables 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.

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.

Table 3: European Pharmacopoeia 2.6.27 Recommended Sample Sizes

Cell-Based Preparation Volume (mL) Total Test Sample Volume

10 < V < 1000 1% of the total volume

1 < V < 10 100 µL
V<1 NA

PAGE 18
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
• 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

PAGE 19
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 infections7. In addition, a prospective, nationwide
surveillance study of nosocomial bloodstream infections from U.S. hospitals over a 7-year period8
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, 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 infusion10 and found that
a detection threshold of at least 103 cfu/Ml would detect more than 95% of all infection cases and

PAGE 20
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 University11 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

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.

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.

PAGE 21
 Table 4: Typical Beyond Use/Expiration Dating of Stakeholder Products

Stakeholders Representative Products Beyond Use/Expiration Dating

Sterile Low Risk: reconstitution and transfer of Low Risk: 48 h (room temperature); 14 days
compounding a 1-g vial of cefazolin into an IV bag (2°-8°); 45 days (frozen)
pharmacies Medium Risk: Distribution from a 10-g Medium Risk: 30h (room temperature); 9
bulk pharmacy vial of vancomycin days (2°-8°); 45 days (frozen)
among several final doses High Risk: 24h (room temperature); 3 days
High Risk: Patient-controlled analgesic (2°-8°); 45 days (frozen)
from powdered morphine
PET facilities Fluorine-18 fluorodeoxyglucose (half- Cellular therapy products may be
life of 110 min) transported for administration in hours or
days without cryopreservation, or stored in a
cryopreserved state (<-30°) indefinitely.
Cell therapy Stem cells Cellular therapy products may be
facilities transported for administration in hours or
days without cryopreservation, or stored in a
cryopreserved state (<-30°) indefinitely.
Pharmaceutical Numerous examples 2-3 years at ambient or refrigeration
manufacturers temperature

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 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

PAGE 22
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 level.

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 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 throughout.

PAGE 23
 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.10

Table 5: Rates of Contamination, Septic Reactions, and Deaths with Administration of Platelet Concentrates
Contamination Rate per Units Rate of Septic Reactions per Unit
Fatality Rate per Units Transfused
Transfused Transfused
0.05% (5000 contaminated units/ 1%-1.3% of the contaminated 15% - 20% of the septic reactions
million units transfused annually) units (2 deaths/million units)
(10-13 septic reactions/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

PAGE 24
Brief Descriptions of the Six Analytical Platforms

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, edited by Michael J. Miller, and a book dedicated to the topic, Rapid Sterility
Testing, edited by Jeanne Moldenhauer.

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.16 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.17 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.

PAGE 25
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.18 Recently, the application of isothermal
microcalorimetry in pharmaceutical microbiology has also been evaluated.19

Nucleic Acid Amplification

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.20 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.21,22

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.23

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

PAGE 26
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 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.24

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.19,25 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.26 Cells are collected by filtration on 0.45-µm polyester membranes and treated

PAGE 27
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.

Table 6: Operational Parameters of Candidate Technologies

Representative Limit of Detection
Technology Time to Result Sample Size (mL)
Detection System (cfu/mL)
Gram stain Classical 104 - 105 30 min 0.1
BacT/ALERT System Respiration 1-10 Overnight - 7 days 5-10
Solid-phase
ScanRDI System 1-10 2-3 h 1-500
cytometry
Milliflex Rapid ATP
1-10 5-7 days 1-500
System bioluminescence
6-8 h
FACS analysis Flow cytometry 10-100 0.1-2
(pre-enrichment)
Nucleic acid
Roche LightCycler 1-100 2-4 h 0.2-2
amplification
Isothermal
TAM V 1-10 2-7 days 1
microclorimetry

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 28
 Table 7: Commercially Available Instrumentation Showing Multiple Vendors

Advanced Technology Instrument Name Vendor

ATP bioluminescence Biotrace 2000 Biotrace International, Brigend,

UK
Pallcheck Rapid System Pall Corp., Port Washington, NY
Milliflex Rapid System Millipore Corp., Bedford, MA
Celsis RapiScan Charles River Laboratories, Inc.,
Wilmington, MA
BioMAYTECTOR Hitachi Plant Technologies, Tokyo,
Japan

Flow cytometry Bact-Flow bioMerieux, Hazelwood, MO

FACSMicroCount Becton, Dickerson & Co., Sparks,
DE

Isothermal microcalorimetry TAM III Calorimeter TA Instruments, Wilmington, DE

Biocal 2000 Isothermal Calimetrix, Arlington, MA
Calorimeter
48-challen isothermal SymCell Sverige, Kista, Sweden
microcalorimeter

Nucleic acid amplification Multiple thermocyclers and Roche Applied Science,

amplicor analyzers Indianopolis, IN
Applied Biosystems, Foster City,
CA
Cepheid, Sunnyvale, CA

Respiration Promex Microrespirator 4200 PromChem Ltd., Edenbridge, UK

BACTEC System BD Diagnostics, Sparks, DE
BioLumix BioLumix, Ann Arbor, MI
BacT/ALERT 3D Dual-T System bioMerieux, Hazelwood, MO
eBDS Pall System Pall Corp., Port Washington, NY
TDLS Lighthouse Instruments,
Charlottesville, VA

Solid phase cytometry ScanRDI® System bioMerieux, Hazelwood, MO

BioSafe PTS Charles River Laboratories, Inc.,
Wilmington, MA
MuScan System Innosieve Diagnostics,
Wageningen, The Netherlands

PAGE 29
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

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40th ed. & NF 35th ed.). Rockville, MD: United States Pharmacopeial Convention, Inc.
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4. Sutton, S. (2011). Sterility tests. Rapid Sterility Tests, 7, 7-28.
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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: [email protected].

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