AAPS PharmSciTech, Vol. 12, No. 1, March 2011 ( # 2011)
`DOI: 10.1208/s12249-010-9575-9
`
`Review Article
`Theme: Sterile Products: Advances and Challenges in Formulation, Manufacturing, Devices and Regulatory Aspects
`Guest Editors: Lavinia Lewis, Jim Agalloco, Bill Lambert, Russell Madsen, and Mark Staples
`
`Comparison of Compendial Antimicrobial Effectiveness Tests: A Review
`
`Cheryl L. Moser1 and Brian K. Meyer1,2
`
`Received 9 August 2010; accepted 16 December 2010; published online 8 January 2011
`Abstract. The antimicrobial effectiveness or preservative effectiveness test is described in the tripartite
`compendia for sterile parenteral multi-dose formulated products. The execution of the test is essentially
`harmonized with respect to inoculum preparation and test execution but not the acceptance criteria. This
`article describes how a single test can be performed that procedurally satisfies all of the compendia and
`their acceptance criteria.
`
`KEY WORDS: antimicrobial effectiveness; pharmacopeia; preservative effectiveness.
`
`INTRODUCTION
`
`Antimicrobial preservatives are added to sterile multi-
`dose parenteral products for the purpose of inhibiting and/or
`killing the growth of microorganisms that may have been
`inadvertently introduced during multiple withdrawals of the
`product from its container. Common antimicrobial preserva-
`tives that are utilized in parenteral products include phenol,
`m-cresol, benzyl alcohol, cholorobutanol, phenoxyethanol,
`methyl, and proplyparaben, and in some cases, thimerosal
`(1–3). The ability of these antimicrobial preservatives to
`inhibit or kill microorganisms in parenteral formulations is
`evaluated using antimicrobial effectiveness tests (AETs).
`The antimicrobial effectiveness test, also known as the
`preservative effectiveness test, is a compendial test performed
`during formulation development and stability testing of a
`parenteral drug product intended as a multi-dose product.
`The test procedures and acceptance criteria are described in
`the three major compendia. The procedures are the United
`States Pharmacopeia (USP) <51> Antimicrobial Effectiveness
`Testing, the European Pharmacopeia (Ph. Eur. or EP) 5.1.3
`Efficacy of Antimicrobial Preservation, and the Japanese
`Pharmacopeia (JP) 19, Preservative Effectiveness Tests
`(4–6). These compendial chapters are essentially harmonized
`with respect to how the test is performed; however, there are
`minor differences with respect
`to selection of challenge
`organisms,
`test intervals, and the acceptance criteria. In
`addition to the compendia,
`there are other references
`describing how to evaluate the efficacy of antimicrobial
`preservatives (7). The article presented here will review each
`of the preservative effectiveness tests in the three Pharmaco-
`
`1 Merck & Co., 770 Sumneytown Pike( PO Box 4( West Point,
`Pennsylvania 19486, USA.
`2 To whom correspondence should be addressed. (e-mail:
`brian_meyer@merck.com)
`
`peia and demonstrate how they can be applied to sterile
`multi-dose parenteral drug products.
`
`THE DRUG PRODUCT
`
`During the development of a multi-dose parenteral
`product, formulation scientists and microbiologists must make
`a decision as to which preservative and what concentration
`will be utilized in the drug formulation. Interactions of the
`preservative with the drug product must be considered as well
`as with the container and closure (2,3). The preservative must
`also remain effective, not just “present” or measureable, in
`the formulated product throughout its shelf life at the labeled
`storage conditions. Historical data from other marketed
`products can be useful in choosing the appropriate preserva-
`tive and concentration for the product (2,3). It is also possible
`that the drug substance and pharmaceutical ingredients or
`excipients may possess some intrinsic antimicrobial activity
`that will add to or enhance the antimicrobial effectiveness of
`the formulated drug product, and help to minimize the
`amount of preservative that needs to be added. Furthermore,
`the physicochemical attributes of
`the product such as
`extremes in pH or osmotonicity may have antimicrobial
`properties.
`A major consideration for selecting an antimicrobial
`preservative for a parenteral formulation is the “use period”
`or storage conditions and time after the initial product
`withdrawal. Some multi-use parenteral formulations, due to
`chemical or microbial stability, must be used within a 24-h
`period whereas others may remain stored for up to 1 week at
`2–8 C following the initial use (1). The Ph. Eur. requires
`testing of antimicrobial activity at 6 and 24 h after the
`microbial challenge. This activity ensures that any micro-
`organisms inadvertently added to the product are killed prior
`to repeat administration. However,
`the USP tests are
`designed to evaluate antimicrobial activity after 7 days.
`
`1530-9932/11/0100-0222/0 # 2011 American Association of Pharmaceutical Scientists
`
`222
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`LUPIN EX 1056
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`Page 1 of 5
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`

`
`Comparison of Compendial Antimicrobial Effectiveness Tests
`
`223
`
`Table I. Strains from Major Culture Collection Recommended for the Test (4–6)
`
`Organism
`
`ATCC (USP/EP/JP)
`
`NCIMB (EP)
`
`CIP (EP)
`
`NCTC (EP)
`
`NCPF (EP)
`
`IP (EP)
`
`IMI (EP)
`
`NBRC (JP)
`
`S. aureus
`P. aeruginosa
`E. coli
`C. albicans
`A. brasiliensis
`
`6538
`9027
`8739
`10231
`16404
`
`9518
`8626
`8545
`–
`–
`
`4.83
`82.118
`53.126
`–
`–
`
`10788
`–
`
`–
`–
`
`–
`–
`
`3179
`–
`
`–
`–
`
`–
`–
`
`48.72
`1431.83
`
`–
`149007
`
`13276
`13275
`3972
`1594
`9455
`
`ATCC American Type Culture Collection (USA)
`NCIMB National Collection of Industrial, Marine and Food Bacteria (Scotland)
`CIP Collection de l'Institut Pasteur (France)
`NCTC National Collection of Type Cultures (UK)
`NCPF National Collection of Pathogenic Fungi
`IP Institute Pasteur (France)
`IMI CABI Genetic Resource Collection (UK)
`
`Once a preservative has been chosen and the final
`formulation of the drug product has been established, the
`preservative levels in the drug product are chemically assayed
`at stability time intervals to assure that the preservative
`remains at effective concentrations in the drug product over
`the shelf life. It is also a regulatory requirement to measure
`the efficacy of
`the preservatives using the preservative
`effectiveness tests on the drug product in its final container
`through expiry. To establish the lower effective shelf life
`specifications, the product is formulated at 100%, 75%, and
`50% of
`the labeled preservative concentration and its
`effectiveness at these concentrations confirmed using the
`AET (4–6). Based on these findings, future marketed product
`stability testing may be conducted using the chemical assay
`and not the microbiological challenge test.
`The compendial chapters divide the types of products to
`be tested into categories such as sterile multi-dose prepara-
`tions, topical products, non-sterile oral products, etc. This
`review article focuses on the sterile multi-dose parenteral
`drug products that are category 1 products in USP <51>,
`5.1.3.1 products in Ph. Eur., and 1 in JP (4–6).
`
`Summary of Test
`
`The AET is performed by spiking a panel of challenge
`microorganisms (representing Gram-positive cocci, Gram-
`
`negative bacilli, yeast, and mold) individually into the product
`and determining the log reduction of organisms at prescribed
`time intervals to quantitatively evaluate the effectiveness of
`the antimicrobial preservative to prevent microbial prolifer-
`ation and/or kill the organisms (4–6).
`
`Preparation of Challenge Microorganisms
`
`The inocula of challenge microorganisms are typically
`prepared from fresh, recently grown stock cultures. Each
`compendia recommends the use of fresh cultures as this assures
`that log phase cells are used to challenge the product. There are
`subtle differences between the compendia with respect to how
`these fresh cultures are to be prepared, but none of the
`differences are scientifically significant with respect to growing
`healthy challenge organisms.
`The USP and JP list the use of five challenge organisms
`for the AET of sterile multi-dose parenterals (4,6). These
`organisms are Staphylococcus aureus (Gram-positive coccus),
`Pseudomonas aeruginosa (Gram-negative bacillus), Escher-
`ichia coli (Gram-negative bacillus), Candida albicans (yeast),
`and Aspergillus brasiliensis (mold). The Ph. Eur. specifies the
`same organisms, but does not make E. coli mandatory for
`sterile multi-dose parenterals and does recommend it as
`appropriate for oral liquids (5). Each compendia also lists
`the source strains for each organism from major strain culture
`
`Cultures
`
`Bacteria
`S. aureus
`P. aeruginosa
`E. coli
`Yeast
`C. albicans
`
`Mold
`
`A. brasiliensis
`
`Table II. Recommended Challenge Organism Inocula Preparation (4–6)
`
`Growth Media
`
`Incubation temperature
`
`Duration until harvest
`
`Harvesting fluid
`
`Soybean-Casein Digest
`
`Sabouraud Dextrose (JP: also states
`glucose-peptone agar and potato
`dextrose agar can be used)
`Sabouraud Dextrose (JP: also states
`glucose-peptone agar and potato
`dextrose agar can be used)
`
`30–35 C
`
`20–25 C
`
`20–25 C
`
`USP: 18–24 h
`EP: 18–24 h
`JP: 18–24 h
`
`USP: 44–52 h
`EP: 48 h
`JP: 40–48 h
`USP: 6–10 days or until
`good sporulation
`EP: 1 week or until
`good sporulation
`JP: 1 week or until
`good sporulation
`
`0.9% Saline
`
`0.9% Saline
`
`0.9% Saline with 0.05%
`polysorbate 80 to
`disperse spores
`
`Page 2 of 5
`
`

`
`224
`
`Cultures
`
`Bacteria
`S. aureus
`P. aeruginosa
`E. coli
`Yeast
`C. albicans
`
`Mold
`A. brasiliensis
`
`A. brasiliensis
`
`Moser and Meyer
`
`Table III. Recommended Inoculum Preparation (4–6)
`
`Harvest fluid
`
`Standardization level
`
`Duration of use
`
`Storage
`
`USP: Saline
`EP: Saline
`JP: Saline or 0.1% Peptone water
`
`USP: Saline
`EP: Saline
`JP: Saline or 0.1%
`Peptone water
`USP: Saline with 0.05% PS80
`EP: Saline with 0.05% PS80
`JP: Saline with 0.05% PS80 or
`0.1% Peptone water
`
`USP: 1×108 CFU/ml
`EP: 108 CFU/ml
`JP: 108 CFU/ml
`
`USP: 1×108 CFU/ml
`EP: 108 CFU/ml
`JP: 108 CFU/ml
`
`USP: 1×108 CFU/ml
`EP: 108 CFU/ml
`JP: 108 CFU/ml
`
`USP: within 2 h or
`refrigerate
`
`EP: use immediately
`
`JP: within 2 h or
`refrigerate
`
`USP: 24 h
`EP: no mention
`JP: 24 h
`
`USP: 24 h
`EP: no mention
`JP: 24 h
`
`USP: 7 days
`EP: no mention
`JP: no mention
`
`collections (Table I; 4–6). These strains are considered (8).
`However, if the American Type Culture Collection (ATCC)
`source is used, compliance with all
`three compendia is
`assured.
`Multiple subculturing and the number of passages of the
`stock cultures become an important parameter to control for
`this test, as continuously propagating cells could lead to
`changes in phenotypic expression, especially antimicrobial
`susceptibility. For that reason, both the USP and JP
`recommend using recently grown cells that are no more than
`five passages from the stock cells (4,6). The Ph. Eur. does not
`specifically state the number of passages, but does state to
`keep the cell passages to a minimum (5). A passage is
`understood as the transfer of organisms from an established
`culture to fresh medium. The USP is the only compendia to
`frozen and stored “stock” cultures
`mention the use of
`prepared from source strains such as ATCC (4). The use of
`stock frozen cultures or purchased standardized inocula is
`often the practice for laboratories especially during formula-
`tion development. Both the USP and JP also mention using
`both broth cultures and solid media cultures to prepare cells
`while the EP does not specifically mention broth cultures (4–6).
`Solid media-derived cells are easier to harvest and standardize
`as one has to harvest by centrifugation and wash the broth-
`derived cells to remove the growth media.
`Parameters for microbial growth conditions, times, and
`temperatures, as well as the recommended media are defined
`in each compendium. The purpose of these parameters is to
`
`Table IV. USP Criteria for Tested Microorganisms (4)
`
`For category 1 (sterile parenteral) products
`
`Bacteria
`
`Yeast and molds
`
`Not less than 1.0 log reduction
`from the initial calculated count at
`7 days, not less than 3.0 log reduction
`from the initial count at 14 days,
`and no increase from the 14 days'
`count at 28 days.
`No increase from the initial
`calculated count at 7, 14, and 28 days.
`
`grow healthy viable cells for challenging the preservative in
`the product. There are minor differences between compendia,
`none of which are significant. A single preparation strategy
`can be employed that satisfies all requirements (see Table II).
`For all compendia, bacteria are grown at 30–35°C for 18–24 h
`on Soybean-Casein Digest medium. This period of time
`assures the cells to be viable and growing in the log phase,
`minimizing the amount of dead cells harvested and standard-
`izing the response to antimicrobial agents. Yeast is grown at
`20–25°C which is a suboptimal temperature and requires a
`longer period of time. The USP states 44–52 h, the Ph. Eur.
`states 48 h, and the JP states 40–48 h (4–6). Anytime around
`48 h is appropriate but should not exceed 52 h (4–6). Molds
`are grown “until good sporulation is obtained” (4–6). It takes
`approximately 6–7 days to observe a copious lawn of black A.
`brasiliensis spores on solid media. Since mold spores are used
`for the microbial challenge, attempts are made to harvest as
`many spores as possible. The spore cells can be harvested
`anytime between 6 and 10 days depending upon visual
`observation of the culture (4–6). The culture medium for
`growing yeast and mold is usually Sabouraud Dextrose Agar
`although other media can be used as specified by the JP
`(4–6).
`
`HARVESTING THE CELLS
`
`Each culture is “harvested” after it has been grown for
`the appropriate amount of time. After harvesting, the cell
`suspensions are then “standardized” to provide an inoculum
`of approximately 108 colony forming units (CFU)/ml (4–6).
`The USP states to standardize to approximately 1×108 CFU/
`ml, while the EP and JP are less specific and state the
`standardization to be to approximately 108 CFU/ml (see
`Table III) (4–6). For this test,
`there is no significant
`difference. All
`three compendia state that
`the cultures
`should be grown on solid media and, except
`for A.
`brasiliensis, harvested using sterile saline (0.9% saline)
`(4–6). Harvesting cells from solid media is performed by
`adding some diluent (saline) to the media and scraping the
`cells from the surface with a sterile tool. The cells are then
`diluted to the approximate 108 CFU/ml level using the same
`diluent (i.e., sterile saline) (4–6). Usually spectrophotometric
`measurements are used to standardize to the use level with
`
`Page 3 of 5
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`

`
`Comparison of Compendial Antimicrobial Effectiveness Tests
`
`225
`
`Cultures
`
`Temperature Duration USP
`
`Duration EP
`
`Table V. Incubation Conditions (4–6)
`
`30–35°C
`
`3–5 days
`
`3–5 days (refers to the microbial limits test)
`
`Duration JP
`≤3 days (refers to the microbial limits test)
`
`Bacteria
`S. aureus
`P. aeruginosa
`E. coli
`Yeast
`C. albicans
`Mold
`
`20–25°
`
`20–25°
`
`3–5 days
`
`5–7 days (refers to the microbial limits test)
`
`≤5 days (refers to the microbial limits test)
`
`3–7 days
`
`5–7 days (refers to the microbial limits test)
`
`≤5 days (refers to the microbial limits test)
`
`transmittance or
`turbidity readings (percentage of
`absorbance). When harvesting A. brasiliensis mold spores,
`the diluent contains 0.05% polysorbate 80 that is added to
`help to disperse the spore cells and prevent clumping (4–6).
`The JP also mentions that 0.1% peptone water can be used in
`place of sterile saline (6). The use of sterile saline vs. the
`0.1% peptone water has no impact on the preservative
`effectiveness test.
`Once the cells have been harvested and standardized, the
`cells should be used within the specified amount of time to
`assure a healthy viable cell challenge to the product. Two
`objectives are to be met during the test. The first objective is
`to enumerate the cells for a baseline CFU number against
`which the product preservative effectiveness is measured. The
`other objective is to spike the healthy cells into the product
`for the actual AET. The USP and JP state to use the cells
`within 2 h or refrigerate for up to 24 h (4,6). The EP states to
`use the cells immediately and mentions no storage conditions
`(5). The USP states that the harvested A. brasiliensis spores
`can be stored at 2–8°C for up to 7 days (4). The EP and JP
`are silent with respect to A. brasiliensis spore storage (5,6).
`Once the cells are harvested and standardized, all enumera-
`tion testing and product spiking should occur within an 8 h
`period of time and the cells should be stored at 2–8°C when
`not in use. A. brasiliensis spores have been successfully stored
`at refrigerated temperatures for 7 days without
`losing
`viability.
`
`ENUMERATING THE CELLS
`
`A zero time baseline enumeration result for the entire
`challenge cell cultures used for spiking product must be
`established. The organisms are diluted to the level where they
`are theoretically within the countable ranges. This is
`performed by making dilutions (usually tenfold) of each
`organism in diluting fluid (i.e., sterile saline) and using the
`pour plate technique to quantitatively establish the enumer-
`
`ation of the working stock culture in terms of colony forming
`units per milliliter. The organisms are diluted from the
`108 CFU/ml stock to a countable number (25–250 CFU for
`bacteria and yeast, 8–80 CFU for mold; 4–6). A 1-ml aliquot
`of the diluted organism is added to a standard 100×15-mm
`Petri plate. Media tempered to approximately 45°C is then
`added to the plate (see Table IV). Laboratories may perform
`variations of this enumeration method, for example, spread
`plates or membrane filtration methods, but the result would
`be the same. The temperature of incubation and duration of
`microbial growth on the Petri plates for each organism is
`defined in each compendia and differs only slightly in wording
`(see Table V; 4–6). Organisms must be grown sufficiently to
`be counted visually as colonies. ATP bioluminescence, flow
`cytometry, or other rapid methods have been used that will
`detect colonies before they can be counted by the naked eye.
`In general, growth for longer periods of time is of no
`detriment as colonies will not “disappear.” However, mold
`may need to be “precounted” 1–2 days early as these colonies
`tend to sporulate and spread thus making colony
`differentiation difficult
`if
`the plate is overgrown. Each
`colony forming unit is theoretically derived from one cell.
`Even though small differences exist between the differ-
`ent compendia in the wording for the duration of plate
`incubation, good scientific practice is to incubate the plates
`long enough for all cells to have propagated into colonies that
`can be counted by the naked eye. As mentioned before, rapid
`microbiological methods have been developed and may be
`used to detect cells before they can be seen with the naked
`eye.
`
`PERFORMING THE ANTIMICROBIAL
`EFFECTIVENESS TEST
`
`All compendia prefer that the test be executed on the
`product in its marketed container. The rationale is that the
`final container test best represents “real world” contamina-
`
`Table VI. EP Criteria for Tested Microorganisms (5)
`
`6 h
`
`2
`–
`–
`–
`
`Log reduction
`
`24 h
`
`3
`1
`–
`–
`
`7 day
`
`14 day
`
`28 day
`
`–
`3
`2
`–
`
`–
`–
`–
`1
`
`NR
`NI
`NI
`NI
`
`Bacteria
`
`Fungi
`
`A
`B
`A
`B
`
`NR no recovery, NI no increase, A recommended, B mandatory
`
`Page 4 of 5
`
`

`
`226
`
`Moser and Meyer
`
`Table VII. JP Criteria for Tested Microorganisms (6)
`
`For category 1A (sterile parenteral) products
`
`Bacteria
`
`Yeast and Molds
`
`14 days: reduction of 0.1% of
`inoculum count or less
`28 days: same or less than
`level after 14 days
`14 days: same or less than
`level after 14 days
`28 days: same or less than
`level after 14 days
`
`tion. Often this is neither possible nor practical. Enough
`material must be present in the final container to allow one to
`remove aliquots at the time intervals for enumeration. For
`this reason, all of the compendia do allow for product to be
`removed from its final container, pooled, and placed into a
`suitable vessel for testing (4–6). The amount of inoculum or
`standardized challenge organism added to challenge the
`preserved product should not be a volume that dilutes out
`the product and its preservatives changing their concentra-
`tions. The volume of
`inoculum should be negligible in
`comparison to the total volume of the test product. All
`compendia state that this volume should not exceed 1% of
`the total volume of the product to be tested and should result
`in a 105 to 106 CFU/ml product challenge (4–6). All
`compendia require that
`the challenged contaminated
`product be stored at 20–25°C (ambient room temperature)
`for the duration of the test period irrespective of the storage
`conditions of the product. Aliquots of contaminated product
`are to be removed at specified time intervals for enumeration
`(4–6). The Ph. Eur. and JP also state that the challenged
`product containers be protected from light during this time,
`but the USP is silent on this parameter (4–6). The USP and
`JP also mention recording obvious signs of microbial
`contamination and proliferation such as changes in color,
`odor, and appearance while the EP is silent on this parameter
`(4–6). In practice, most microbiologists store the test material
`in a 20–25°C incubator for the duration of the test.
`
`The Ph. Eur. (see Table VI) has the most strict accept-
`ance criteria in that it requires a log reduction at 6 and 24 h
`for “Criteria A” preservative effectiveness (5). These criteria
`are difficult
`to achieve with many preservative systems.
`Often, the level of preservative added to achieve these results
`has detrimental effects on the product and/or is at toxic levels.
`The Ph. Eur. also has “Criteria B” that
`is considered
`mandatory by EU regulatory agencies and is more achievable
`(5). By 24 h, the preservative is expected to achieve at least a
`1 log reduction and prevent proliferation throughout the
`28 day period (5). The USP (see Table IV) does not have
`criteria for acceptance until day 7, and the JP (see Table VII)
`begins criteria at day 014 (4,6). The USP also defines “no
`increase” in proliferation as not more than 0.5 log higher than
`the previous measured level that corresponds to the expected
`plate count variability (4). Quality sterile multi-dose prepa-
`rations should have a preservative that can rapidly prevent
`cell proliferation and destroy any microorganisms inadver-
`tently introduced by multiple withdrawals of product through-
`out the product's use period. The USP chapter is an official
`test method and although AET is not a product release
`specification, the FDA requires products to meet the log
`reduction requirements in the chapter.
`
`CONCLUSION
`
`The antimicrobial effectiveness or preservative effective-
`ness test is described in USP <51>, EP 5.1.3 and JP 19 for
`sterile parenteral multi-dose formulated products (4–6). The
`execution of the test is essentially harmonized with respect to
`inoculum preparation and test execution. Harmonization has
`not been achieved with respect to acceptance criteria. One
`test can be performed that satisfies all of the compendia and
`their acceptance criteria.
`
`ACKNOWLEDGMENTS
`
`We thank Anthony Cundell for a critically reviewing this
`manuscript.
`
`TEST INTERVALS AND ACCEPTANCE CRITERIA
`
`REFERENCES
`
`The acceptance criteria for the product type determine
`the time intervals at which the samples are enumerated for
`log reduction over the initial time zero inoculum levels. The
`three compendia are not harmonized with respect to accept-
`ance criteria in terms of challenge reduction and the
`significance level that is expressed (4–6). However, one test
`can be executed that satisfies all time intervals and compen-
`dial acceptance criteria. The major difference for sterile
`multi-dose formulations is that the Ph. Eur. has 6- and 24-h
`time interval criteria while the USP and JP do not have
`criteria until days 7, 14, and 28 (4–6). Given these different
`criteria, enumerations performed at 6 h, 24 h, 7 days, 14 days,
`and 28 days after the initial microbial challenge satisfy all
`compendial requirements (4–6). These plate counts are
`converted to log10 and compared to the time zero enumera-
`tion performed on the saline control inoculum levels (4–6).
`
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