DUALITY CONTROL
`
`ANALYTICAL METHODS
`
`The Essentials of United States
`Pharm,acopeia Chapter <51>
`Antim,icrobial Effectiveness Testing
`
`AND ITS APPLICATION IN PHARMACEUTICAL COMPOUNDING
`
`Nicole Vu and
`Thomas C. Kupiec
`are affiliated with
`Analytical Research
`Labarataries, Inc.,
`Oklahoma City, Okla(cid:173)
`homa. Kevin Nguyen
`is aPharmD Candi(cid:173)
`date and is affiliated
`with the Oklahoma
`University Health
`Science Center, Okla(cid:173)
`homa City, Oklahoma.
`
`PHOTO SOURCE:
`ANALYTICAL
`RESEARCH
`LABORATORIES
`& PICKENS
`PHOTOGRAPHY
`
`Nicole Vu, PhD
`Kevin Nguyen
`Thomas C. Kupiec, PhD
`
`INTRODUCTION
`Antimicrobial preservatives are excipi(cid:173)
`ents added to multi -dose formulas ofboth
`sterile and nonsterile drug products for
`inhibition of microbial growth. Microbial
`contamination may occur during nonsterile
`processing or during the period of use due to
`the repeated withdrawal of individual doses
`from multi-dose containers.1 Multi-dose
`pharmaceutical products containing preser(cid:173)
`vatives offer several advantages over single(cid:173)
`dose packages. Multi-dose drugs minimize
`product wastage and allow flexibility for
`dosage adjustments; repeated doses may be
`obtained from the same container without
`concerns for microbial growth during use;
`and their packaging is reduced because mul(cid:173)
`tiple doses are supplied in a single con(cid:173)
`tainer.2 It is general knowledge that
`unit-dose packaging is the most optimal
`with respect to the maintenance of sterility,
`but it is not efficient and cost effective as
`preserved multi -dose preparations.
`Antimicrobial preservatives can be
`microcidal, microstatic, and sporicidal.
`
`ABSTRACT Antimicrobial preservatives are excipients
`added to multi-dose containers of both sterile and non(cid:173)
`sterile drug products. Antimicrobial preservatives are
`used primarily to inhibit growth of microbial contamina(cid:173)
`tion occurring during the period of use. Demonstration of
`antimicrobial preservative effectiveness is required for
`
`these functional excipients. This article reviews key factors
`for consideration in the selection of preservatives, princi(cid:173)
`ples of the preservative-effectiveness test, and the signifi(cid:173)
`cance of requirements for preservative-effectiveness
`testing in the compounding practice.
`
`www.IJPC.com
`
`International Journal of Pharmaceutical Compounding
`Vol.lB No.2 I March 1 April l2014 123
`
`Page 1 of 8
`
`SENJU EXHIBIT 2124
`INNOPHARMA v. SENJU
`IPR2015-00903
`
`

`
`.----~ Oualitv Control
`
`They interfere with various mechaJlisms in microbial cells causing
`cellular damage or cell lysis. The mechanisms for antimicrobial
`effects are not always specific and can be difficult to elucidate.
`Some preservatives may act at the cell wall, others may target the
`cytoplasmic membrane or cytosolic components. Their activities
`may lead to irreversible cell membrane damage, precipitation of cel(cid:173)
`lular proteins, or inhibition of critical pathweys for signal induction
`and cellular transport. Preservatives may also act synergistically
`with other preservatives or with other components of the formula
`to enhance the total effects for microbial control. Due to the cyto(cid:173)
`toxic effects they exert against microbial cells, these preservatives
`can not be regarded simply as inactive ingredients. Their inclusion
`in pharmaceutical preparations should be at a concentration that is
`effective but nontoxic to humans.3 An ideal preservative should be
`active against a broad spectrum of microorganisms but nontoxic to
`human cells and should be tolerable by the intended patient groups;
`it must also be stable and compatible with the other components of
`the drug product to be effective. Activities of common]y used anti(cid:173)
`microbial agents, which are relatively safe for use in pharmaceuti(cid:173)
`cal compounding, will be discussed in the following sections. The
`principle of antimicrobial effectiveness testing and its require(cid:173)
`ments in the compounding practice will also be discussed.
`
`GENERAL CONSIDERATIONS IN THE
`SELECTION OF ANTIMICROBIAL
`PRESERVATIVES
`Most viable cells function optimally within a narrow pH range
`around neutrality, and growth is slow at pH beyond 6 or S:' This pH
`range may not always be optimal due to solubility and stability of for(cid:173)
`mulation ingredients. Hence, the pH of a formula is often adjusted to
`enhance product quality. In terms of solubility, the optimum pH for
`formulation ingredients can be deduced from their dissociation con(cid:173)
`stants (piQ and their oil-water partition coefticients (LogP 0,ro.).
`Both parameters are related to their aqueous solubllity, where the
`antimicrobial effect is required, and their concentration in different
`phases of a multiphasic system. However, the relationship between
`pH and antimicrobial activities is more complex. For example, the
`antifungal activity ofbenzoic acid is less susceptible to pH than its
`antibacterial activity. Similarly, sorbic acid has significant antifun(cid:173)
`gal but little antibacterial activity at pH 6.0.4
`Antacid formulations and multi phase systems are more difficult
`to preserve than simple aqueous formulas. Such products require
`additional ingredients that have a high potential for interactions.
`Interactions of preservatives with formulation ingredients and con(cid:173)
`tainers may compromise product stability and preservative effi(cid:173)
`cacy. Interactions do not always lead to structural modification of
`the preservatives but may occur in the form of complex formation,
`precipitation, or adsorption to surfaces. Incompatibility among
`components occurs in the presence of strong oxidizing agents, or
`between a strongb&Be and acidic preservatives. Cationic preserva-
`
`tives are incompatible with anionic surfactants, and non-ionic sur(cid:173)
`factants (e.g., polysorbate 80) are incompatible with some alcohol
`phenolic preservatives. The parabens, benzoic acid, chlorobutanol,
`m-cresol, etc. are relatively volatile and can be lost during process(cid:173)
`ing and storage. Preservative precipitation in the presence of poly(cid:173)
`valent cations was observed with sorbic acid, butylated
`hydroxyanisole, chlorhexidine, etc. Additionally, reconstitution of
`Activase, Proleukin, and Leukin.e with diluents containing preser(cid:173)
`vatives may denature protein and peptide molecules.4.S Lab tech(cid:173)
`niques such as size exclusion chromatography (SEC), dynamic light
`scattering (DLS), fourier transform infrared {FTm), electron
`microscopy, histologic BllBlysis, and immunological assay have been
`used to characterize interactions in small-molecule drug products
`and in biopharmaceuticals.2
`In addition to in vitro formulation issues, in vivo adverse effects
`mi!,Yfurther limit the availability of suitable agents for preserved
`products. As previously discussed, most preservatives are cytoxic
`to microbial cells, and their use mi!,Y impart unintended side effects
`in patients. Notably, benzyl alcohol is not recommended in neona(cid:173)
`talparenteral products, as it has been linked to fatal toxic syn(cid:173)
`drome in premature neonates. Irritants, such as parabens, were
`determined unsuitable for ophthalmic preparations, and benzalko(cid:173)
`nium chloride may not be appropriate for soft contact lenses solu(cid:173)
`tions. Concerns over neurotoxicity haw lead to the declined usage
`of organomecuric compounds in parenteral products, and hexa(cid:173)
`chlorophene in topical products.M
`The above discussion highlights formulation and external factors
`that must be considered in the preparation of preserved products.
`Optimization of the preservative system is often conducted during
`pre-formulation studies, which are not usually performed for com(cid:173)
`pounded preparations, thW! emphasizing the requirements for the
`demonstration of antimicrobial efficacy in dispensing and beyond(cid:173)
`use dating (BUD).
`
`COMMONLY UTILIZED ANTIMICROBIAL
`PRESERVATIVES
`In a review conducted by Meyer, et al, 2 the authors observed that
`macromolecular biotech products such as peptides and proteins
`usually contain phenol and benzyl alcohol as preservatives.
`Whereas a combination ofparabens are found in small molecule
`parenterals, and phenozyethanol is often found in vaccines. M-ere(cid:173)
`sol and chlorobutanol are present in fewer products, and older prod(cid:173)
`ucts may also contain thimerosal or phenylmercurric salts although
`they are no longer preferred agents in new formulas. Most intraoc(cid:173)
`cular and intrathecal products are preservative-free because of
`safety considerations. 7
`AliBt of common antimicrobial preservatives with proven perfor(cid:173)
`mance characteristics in various dosage forms is provided in Table
`1. Although limited in content, the table contains historical data
`that may be useful as a quick reference in a busy pharmacy environ-
`
`Ildeimatl.ozaal.JC1111'DA1 ofPharmaoeutloalCompcnuullzlg
`124 Vol IS No.2 I Maroh I ~il l 2014
`
`Page 2 of 8
`
`

`
`Quality Control ~
`
`mem.l118~1.1f~Bm~!.racompzehell­
`elveaoliiC&of clr;tadeec:rlb!n,rphllllcochemlcal properlleall!ld
`llafe1Jprolllecotaftllobleelld,p!e~~lll,illcludlllgAiltlml.ci'Ob!alpre­
`~r-ead-.da:rea:redlreGtl!d tolhllraflnrl.oetor
`addl11oml.inlbrmosioll.
`
`ANTIMICROBIAL EFFECTIVENESS TESTING
`(UNITED STA'TES PIIARifACOPEIA
`CHAPTER <51>)
`SUMMARY OF TEST
`The USPChllptar <51> .Antlmlorob!ai.Blfemi:vea.a. Teltlllooll(cid:173)
`d1ic18db7'~IIP'I<'Ifled~la.dlridullytothelelt
`produortatrelativelybighoo-atioJIJitoaimulaleooi!:IAmiDJ.(cid:173)
`tlon. Th&produortlthllldtor 28clqa, clurliii'91!Uab.Umethe8dded
`mlerooi'IIIGIImotare«<l'UUIIllnadatd6Adint>el:val.etodetlil:rmllu>
`aa;,oh.u&em~-J!:I.Il1~mioxoorpaiml.t
`IDala.d&~~~ll{lw,~cflli,~­
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`<tritz:ria ~Ue gpeni6ed lor each dn!J p:mdu!lt aat.eJOriec. In lll'll.l:nll. •
`
`1 to3lej~zedu.c1:1onlnbacteriailrom.lbe lmt:!.al.lmll.llhouldOCC'IIfln
`cm.etotwoweolat, with no ft.lrtlulr ;n......., in ba<:to:ri&thereaftac at
`28 dqs. P'or)'OIMI;&Ildmold. no ll!..-trom1bell!.lllallllO<l'll!wn
`!8ni.U. J)GIUll1tedalaD.14111lpliDIJ~
`
`PRODUCT CATEGORIES AND SPECIFICATIONS
`Pharmllce\llieoal ~- cl!.uledllllo&w~ buod
`o:n product rilk.' .Mihown m Tallie :~.t wrtet aamplillg illtetvaia
`dluiqa28.ciqperlod,.admme.lltrlqllmtarll:atiaareUICH!Ial8d
`wilh~lproduclll,wblchincludea~~in
`8q\leo\l.l bue orem:ulaio:oJI (~ DUeotio:n.r, ol:iil ptOduj)br, oplllhll.(cid:173)
`mlo~naaal.~).~~-·ati.Diliaindioa•ecl
`'bJ110tt-Win11111d3lej~redlaodolllll be.atarlaloownilrom.1be
`lnltlal-nlue r.tci11y1&1ldda'v14.~.Sitb~ • ...,...
`tialooutaat~28allouldDOtino:teMeftom-at~:W.. LeG
`~ Gl'!lm'laue-wllacl. to topSoil and oral. pzodaolllllloatetto(cid:173)
`rlee 2 ULd a For oral &lld toplc&lp:rocluc:lll,atlel&t llol {o,..J prod(cid:173)
`..W) ud 2lol (topioalproduota) ~u..m.iaitl81 baetotial
`00UIItatda'vl41hould b&oblernd,.aDC!DOillateue~to
`dQ--14 COIUillll al.dl0'·28teatlnl. Anladd prodtu:taarequalliied bF
`
`Ml
`
`Do you need guidance on
`complying with USP <797>?
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`• Mt!dia Fill
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`lltat_,.....IMfngOanclmlll,...._..ra or --845-'S478
`
`Page 3 of 8
`
`

`
`.----~ Oualitv Control
`
`TABLE 1. Common Pharmaceutical Preservad.ves.
`
`PRBSBRVATIVB
`4-Chlorocresol
`
`FORMULATION
`OraL Topical
`
`CON'CENTRATION
`(PERCENTAGE)
`Up to 0,2
`
`OPTIMAL
`pB
`
`<9.0
`
`4-ChloroxyJenol
`
`Topical
`
`0.1 to 0.8
`
`Benzalkonium
`
`Benzethonium
`chloride
`
`Benzoic add
`
`OraL Ophthalmic,
`Topical
`
`0,01 to0,02
`
`4to10
`
`Topical, Ophltlalmic
`
`Up to 0.5
`
`4to10
`
`OraL Parentera~
`Topical
`
`0.1 to 0.2
`
`2.5 to 4.5
`
`Benzyl alcohol
`
`Oral Parenteral
`
`Up to2.0
`
`Boric acid
`
`Ophthalmic, Topical
`
`<5.0
`
`3.5 to 4.1
`
`Cetrimide
`
`Ophthalmic. Topical
`
`• Ophthalmic: 0.005
`• Topical: 0.1 toto
`
`Neutral or
`slightly alkalne
`
`Chlorhexidine
`
`Ophthalmic
`
`0,01
`
`St:o7
`
`Chlorobutanol
`lmidUI'INI
`
`Parenteral
`Topical, Ophltlalmic
`
`Up to 0.5
`0,03 to 0,5
`
`m-Cresol
`
`Parenteral
`
`0.15t:o0.3
`
`Methylparaben
`
`Oral Parenteral
`
`0.0018
`
`Phenols 0.5%
`
`Parenteral
`
`0.01
`
`Phenoxyethanol
`
`Parenteral Topical
`
`O.Sto 2.2
`
`Potassium sorbate
`
`Oral Topical
`
`0.1 to 0.2
`
`Propionic acid
`Propylparaben
`
`OraL Topical
`Oral Parenteral
`
`0.0002
`
`Sodium benzoate
`
`OraL Parenteral
`
`Sorbic acid
`
`OraL Topical
`
`• Oral: 0.02 to 0.5
`• Panenteral: 0.5
`0.05 to 0.2
`
`<5.5
`3 to9
`
`<9.0
`
`4to8
`
`<9
`
`<7
`
`<6
`
`3.9
`4to8
`
`2 to 5
`
`4.5
`
`Thimerosal
`
`Ophthalmic,
`Parenteral
`
`0.001 to 0.01
`
`7to8
`
`Ildeimatl.ozaal.JC1111'DA1 ofPharmaoeutloalCompcnuullzlg
`126 Vol IS No.2 I Maroh I ~il l 2014
`
`SPEC'l'IWM
`• Bacteria, spores, molds, and yeasts
`• Active in adcfiC media
`• Gram(+) bacteria
`• Less active vs Gram (-) bacteria
`• Synergistic with EDTA
`• Gram(+)> Gram(-) bacteria
`• Ineffective vs resistant P. aeruginosa strains
`• Minimal activity vs bacterial endospores, acid-fast bacteria
`• Bacteria, fungi, and molds
`• Synergistic with ethanol
`• Reduced efficacy by soaps and other anionic surfactants
`• Moderate activityvs Gram(+)< Gram(·)
`• Moderate activity vs fungal
`• Moderate activity vs mold
`• Moderate activityvs Gram(+)< Gram(·)
`• Effective vs molds and yeasts
`• Weak bacteriostatic
`• Weak fulgistatic
`• Gram(+)> Gram(·) bacteria
`• Synergistic with alcohols
`• Variable activity vs fungi
`• Synergistic with EDTA vs resistant strains of P. aeruginosa,
`A. niger, C. albicans
`• Gram(+)> Gram(-)
`• Weak activity vs Proteus and Pseudomonas
`• Inactive vs acid-fast bacili
`• Weak activity vs molds, yeasts
`• Activity Gram(+), Gram(·). and some fungi
`• Broad-spectrum antibacteria
`• Some antifungal properties
`• Synergistic with parabens vs fungi
`• Moderately Gram(+)> Gram(-)
`• Weak activity vs yeasts and molds
`• Broad spectrum antimicrobial activity
`• Most effective vs yeasts and molds
`• Moderate activityvs Gram(+)< Gram(-)
`• Weak activity vs yeasts and molds
`• Antibacterial vs P. aeruginosa < Proteus vulgaris
`• Weak activity vsGram (-)
`• F111quently uslld in combination with other pres1rvativ1s
`• Predominantly antifungal
`• Moderate a ntlbacterlal
`• Bacteria, fungi, and molds
`• Activity vs yeasts and molds > bacteria
`• Gram(+)> Gram(-) bacteria
`• Bacteriostatic
`• Antifungal
`• Primarily antifungal
`• Weak antimicrobial
`• Synergy with glycol
`• Bactericidal at acidic pH
`• Bacteriostatic and fungistatic at alkaline or neutral pH
`• Ineffective vs spore-forming organisms
`
`Page 4 of 8
`
`

`
`Oualitv Control
`
`separate criteria in Category 4 due to the inherent issues with this
`product (e.g., high pH, interactions of preservatives with formula(cid:173)
`tion ingredients). Effective antimicrobial activities in antacid prod(cid:173)
`ucts are indicated by no increase in bacterial, yeast, and mold from
`initial counts when tested at day 14 and day 28. No increase or no
`change is equivalent to not more than a 0.5log change from the
`initial inoculum level to account for variability of the test.
`
`TEST ORGANISMS AND PREPARATION OF
`STANDARDIZED CELL SUSPENSIONS
`A panel offive challenge organisms are used in USP <51>, includ(cid:173)
`ing Candida albicans (yeast), Aspergillus niger (mold), Escherichia
`coli (Gram-negative enterobacillus), Pseudomonas aeruginosa
`(Gram-negative bacillus), and Staphylococcus aureus (Gram-posi(cid:173)
`tive coccus). Fresh cultures of each organism are harvested in
`sterile saline and standardized to about 108 colony forming units
`per mL (cfu/mL). Extensive propagating of microbial cells is dis(cid:173)
`couraged because it could lead to changes in phenotypic expression
`and antimicrobial susceptibility. Therefore, seed-stock techniques
`are recommended for long-term storage, and stock cultures of each
`
`organism are limited to no more than five passages removed from
`the original seed stock.1•9
`The microbial enumeration test is performed to determine the
`number of viable cells in each cell suspension. Bacteria are grown at
`30°C to 35°C on Soybean-Casein Digest Agar, while yeast and mold
`are grown at 20°C to 25°C on Sabouraud Dextrose Agar. Table 3
`describes the culture conditions for the preparation of standardized
`cell suspensions and microbial recovery study.
`
`CHALLENGE TEST
`The standardized cell suspensions are added to the test product
`in five separate containers, one container for each challenge
`organism. The concentration of challenge organisms in product
`Categories 1 through 3 is between 105 and 106 cfu/mL. The prod(cid:173)
`ucts in Category 4 (antacids) contain between 103 to 104 cfu/mL of
`each challenge organism. The inoculum volume should not exceed
`1% of the total volume of the product to be tested. Inoculated
`samples are incubated at 20°C to 25°C for 28 days. The microbial
`enumeration test is performed at days 7, 14, and 28 by the vali(cid:173)
`dated method.
`
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`
`www.IJPC.com
`
`International Journal of Pharmaceutical Compounding
`Vol.18 No.2 I March 1 April l2014 127
`
`Page 5 of 8
`
`

`
`.----~ Oualitv Control
`
`METHOD SUITABILITY TEST
`The antimicrobial preservatives in the
`drug product must be neutralized to recover
`viable cells in the microbial enumeration
`test. This neutralization can be accom(cid:173)
`plished by neutralizing agents, membrane
`filtration, dilution, or any combination of
`these methods. Neutralization conditions
`must be validated for efftciency and suit(cid:173)
`ability by the counting method. All organ(cid:173)
`isms used in the challenge test must be
`included in the validation of methods. The
`
`validation protocol should follow guidelines
`elaborated in USPGeneral Chapters <61>
`and <1227>. Briefly, the validation study
`mwrt show that recovery of an inoculum
`containing goo cfu of the challenge organ(cid:173)
`ism is not inhiliited by the test sample and
`by the neutralization method. This is
`accomplished by comparing recovery
`results for three treatment groups:
`
`1. Thetestgroup: Neutralized product
`inoculated with 100 cfu of the challenge
`organism
`
`2. The peptone control group: The same
`treatment as in the test group but peptone
`is used instead of the test product
`3. Inoculum control containing 100 cfu of
`the challenge organism, but no neutral(cid:173)
`ization and no product present
`
`The validation study is conducted in
`three independent experiments. In each
`experiment, average recovery of viable cells
`in the test group should be at least 70% rela(cid:173)
`tive to the inoculum control.
`
`TABLE 2. Four Categories ofDnJg Products aDd SpeoiftcatlODS for
`Antimicrobial Eftica.ey.1
`
`PRODUCT
`CA.TBGORY DBSCRIPTION
`
`CBITBBIA FOR
`BA.CTBRIA
`
`CRITBRIA FOB YEAST
`AJIIDMOLD
`
`1
`
`2
`
`3
`
`4
`
`• Parenterals (Injections,
`emulsions)
`• Otic, ophthalmic. and
`sterile nasal products
`in aqueous base
`
`• Topical products in
`aqueous base
`• Nonsterlle nasal
`products
`• Nonsterile emulsions
`• Products for mucosal
`application
`
`Or11 products in 1queous
`base (excluding antacids)
`
`No Increase at days 7, 14, and 28
`relative to Initial count
`
`• ~1.0 log reduction at day
`7 relative to Initial count
`• :1:3.0 log reduction at day
`14 relative to initial count
`• No increase at day 28
`relative to day·14 count
`
`• :22.0 log reduction at day No increase at days 7, 14, and 28
`14 relative to Initial count
`relative to Initial count
`• No Increase at day 28
`relative to day-14 count
`
`• ~1.0 log reduction at day
`14 relative to initial count
`• No incr1ase at day 28
`relative to day·14 count
`
`No increue at days 7, 14, 1nd 28
`relative to initial count
`
`Antacids in aqueous base No increase at days 14 and 28 relative to initial count
`
`TABLE 3.Incubation Temperature and. Incubation Time for Preparation
`of Standardized Cell SuspensioDs aDd Microbial Recovery Study.1
`
`ORGANISM
`E. coli
`
`P. aeruginosa
`
`s. cwreus
`
`C:albicans
`
`A. niger
`
`ctJLTVBB
`MEDIUM
`
`Soybean-Ciseln
`Digest (broth, agar)
`
`Soybean-<:asein
`Digest (broth, agar)
`
`Soybean-<:aseln
`Digest (broth, agar)
`
`Sabou raud Dextrose
`(broth, agar)
`
`Sabou raud Dextrose
`(broth, agar)
`
`TEMPBRATURE
`("C)
`30 to35
`
`TIMB
`(CELL
`SUSPENSION)
`
`18 to 24 llours
`
`TIMB
`(RECOVERY)
`3to 5 days
`
`JO to35
`
`30 to35
`
`20 to25
`
`20 to25
`
`18 to 24 hours
`
`3to 5 days
`
`18 to 24 nours
`
`3to s days
`
`44 to 52 hours
`
`3to 5 days
`
`6 to 10 days
`
`3to 7 days
`
`Ildeimatl.ozaal.JC1111'DA1 ofPharmaoeutloalCompcnuullzlg
`128 VollS No.2 I Maroh I ~il l 2014
`
`COMPARISON AMONG
`COMPENDIA MICROBIAL(cid:173)
`EFFICACY TESTS
`Procedures for antimicrobial efficacy
`determination are described in three
`major compendia: the USP (Chapter <51>
`Antimicrobial Effectiveness Testing), the
`European Pharmacopoeia (EP) (Chapter
`<5.1.3> Efficacy of Antimicrobial Preser(cid:173)
`vation), and the Japanese Pharmacopoeia
`(JP) (Chapter <19> Preservative Effective(cid:173)
`ness Test). These chapters are essentially
`harmonized in principles, but minor dif(cid:173)
`ferences exist with respect to the challenge
`organisms, test intervals, and acceptance
`criteria. 9 In situations where compliance
`to three compendia. are required, these dif(cid:173)
`ferences should be incorporated into the
`test protocol.
`
`PREPARATION OF CHALLENGE
`MICROORGANISMS
`TheEPdoes not include E. coli in the
`panel of challenge microorganisms, but
`does allow supplementing the panel with
`additional species "that may represent
`likely contaminants," and recommends the
`addition of E. coli for all oral prepara.tions.10
`USP <51> listed only the strains of challenge
`organisms sourced from American Type
`Culture Collection (ATCC), while both the
`EP and JP recognize additional. source
`strains besides those listed in USP <51>.
`The incubation temperatures of subcul(cid:173)
`tures are harmonized, but the incubation
`durations are slightly varied for yeast and
`mold. To comp]y with three compendia, C.
`
`Page 6 of 8
`
`

`
`Oualirv Control
`
`TABLE 4. Comparison of Acceptance Criteria for Parenteral Products
`by JP, USP, and EP.
`
`JP (%REDUCTION)
`
`USP (LOG REDUCTION)
`
`EP (LOG REDUCTION)
`
`TIME
`
`BACTERIA
`
`FUNGAL
`FUNGAL
`AND MOLD BACTERIA AND MOLD
`
`FUNGAL
`BACTERIA AND MOLD
`B
`
`A
`
`B
`
`A
`
`albicans should be harvested at about 48
`hours, and A. niger should be harvested
`after 6 to 7 days "when good sporulation is
`obtained." Standardized cell suspensions
`should be used within 8 hours, and stored at
`2°C to 8°C when not in use.9
`
`TEST INTERVALS AND
`ACCEPTANCE CRITERIA
`
`6 Hours
`
`24 Hours
`
`NT
`
`NT
`
`7 Days
`
`NT
`
`14 Days
`
`0.1% or less
`
`28 Days
`
`reduction
`~ at day 14
`
`The acceptance criteria expressed in the
`EP are the most stringent compared to the
`USP and JP. The EP has two criteria (A
`and B) for products in Categories 1 (paren(cid:173)
`teral intrauterine, intramammary prepara(cid:173)
`tions) and 2 (ear, nasal, inhalation,
`cutaneous preparations). The A criteria
`are "the recommended efficacy to be achieved," and "In justified
`cases where the A criteria cannot be attained ... , the B criteria must
`be satisfied." The EP Category 1-A has approval criteria at 6 hours
`and 24 hours in addition to days 7, 14, and 28. The JPhas accep(cid:173)
`tance criteria expressed as a percentage recovery for days 14 and
`28.9 To comply with three compendia requirements, sampling
`intervals should start at 6 hours for products in Category 1, and
`day 2 for products in Category 2. Table 4 shows sampling frequen(cid:173)
`cies and acceptance criteria expressed by the EP, JP, and USP for
`sterile parenteral products.9·10
`
`NT
`
`NT
`
`NT
`
`~a t day 14
`
`~ at day 14
`
`NT
`
`NT
`
`1
`
`3
`
`Nl
`
`NT
`
`NT
`
`Nl
`
`Nl
`
`Nl
`
`2
`
`3
`
`NT
`
`NT
`
`NR
`
`NT
`
`1
`
`3
`
`NT
`
`Nl
`
`NT
`
`NT
`
`2
`
`NT
`
`Nl
`
`NT
`
`NT
`
`NT
`
`1
`
`Nl
`
`Nl =no increase; NR = no recovery; NT= not tested
`EP = European Pharmacopoeia; JP = Japanese Pharmacopoeia; USP = United States Pharmacopeia
`
`Antimicrobial preservative effectiveness should be demonstrated
`during development, during scale-up, and throughout the shelf(cid:173)
`life ... , although chemical testing for preservative content is the
`attribute normally included in the specification.
`
`SIGNIFICANCE OF ANTIMICROBIAL(cid:173)
`EFFECTIVENESS TEST
`The purpose of USP <51> is to provide a guide to antimicrobial(cid:173)
`effective testing. Preservatives are not meant to replace but to com(cid:173)
`pliment current good manufacturing processes. USP <51> testing
`ensures the efficacy of pharmaceutical products containing preser(cid:173)
`vatives in original, unopened containers made and distributed by
`the manufacturer. Measurement of preservation during in-use is
`outside the scope of the current protocol and requires different
`experimental designs (e.g., broaching study designs). In addition,
`the panel of five organisms employed in the challenge study does
`not represent resistant phenotypes that have acquired the ability to
`withstand the activity of the preservative. The standard preserva(cid:173)
`tive test may then be insufficient to demonstrate the survival capac(cid:173)
`ity in pharmaceuticals of strains adapted to low-nutrient
`environment and low storage temperatures.U
`
`WHEN ANTIMICROBIAL-EFFECTIVENESS
`TEST IS PERFORMED
`The antimicrobial-effectiveness test is often performed during
`drug development for optimization of formulation ingredients. The
`International Conference on Harmonization (ICH) requires12:
`
`Validate the proficiency
`of your technicians and
`pharmacists per USP <797>
`
`I"'TC?.liiHiirif.l¥1 fmr ~ o ~~o~O
`HardyVal@ HardyDiagnostics.com I Phone: 800-266-2222, ext. 5609 I Fax: 805-361-2735
`
`www.IJPC.com
`
`International Journal of Pharmaceutical Compounding
`Vol. 18 No. 2 I M arch 1 April ! 2014 129
`
`Page 7 of 8
`
`

`
`Quality Control
`
`The ICH QaA further specifies12:
`
`The testing for antimicrobial preserva(cid:173)
`tive content should normally be per(cid:173)
`formed at release. Acceptance criteria for
`preservative content should be based
`upon the levels of antimicrobial preser(cid:173)
`vative necessary to maintain microbio(cid:173)
`logical quality of the product at all stages
`throughout its proposed usage and shelf
`life. The lowest specified concentration
`of antimicrobial preservative should be
`demonstrated to be effective in control(cid:173)
`ling microorganismJI by using a pharma(cid:173)
`copeia! antimicrobial preservative
`effectiveness test.
`
`The ICH further states13;
`
`A single primary stability batch of drug
`product should be tested for antimicrobial
`preservative e1fectiveness (in addition to
`preservative content) at the proposed
`shelfllfe for verification purpose.
`
`In pharmaceutical compounding, USP
`Chapter <51> forms apart of the product
`quality test for preserved preparations due
`to limited p re-formulation data. Preserva(cid:173)
`tive content and effectiveness testing should
`be apart of a stability program for BUD of all
`preserved preparations, "when such a test is
`performed, the results sball support the
`BUD assigned to the oompoundedprepara(cid:173)
`tions."10ne strategy is to prepare formulas
`with 100% and 70% of the label concentra(cid:173)
`tion for the preservative (limit for BBBay ±
`209f, of preservative label content). Preser(cid:173)
`vative effectiveness and content are estab(cid:173)
`lished for these samples at the initial time
`point, and then content testing will be con(cid:173)
`ducted for the remainder of the stability
`time intervals. It is also prudent to confirm
`the preservative effectiveness at the BUD
`according to USPChapter <51>. Based on
`the stability results, only the content test
`will need to be conducted for future batches
`unl.esa fundamental changes occur in the
`formulation or compounding procedure.
`The above discussion pertains to antimicro(cid:173)
`bial-preservative testi.ng only and does not
`address other testing requirements for com(cid:173)
`pounded preparations.
`
`CONCLUSION
`The USPCha.pter <51> Antimicrobial
`Effectiveness Testing is a culture-based
`method and accuracy of results is depen(cid:173)
`dent upon adequate neutralization of anti(cid:173)
`microbial activities in test samples for
`enwneration testing. The efficiency of the
`neutralization method employed must be
`validated for all ftve challenge orgaD:i.&I!lll.
`Alternative methods can be substituted if
`proven to be equivalent to compendia testiJlg.
`USP <51> does not address contamination
`by end UBers. Evaluating the efficacy of the
`preservative system for these in-use condi(cid:173)
`tions requires different experimental
`designs that simulate in-use. The three
`major compendia (,EP, JP, USP) are harmo(cid:173)
`nized in principles but different in some
`aspects, which must be incorporated into
`the test protocol for compliance.
`
`REFERENCES
`1. United States PhllnDllt:Opeial Convention,
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`2. Meyer BK, Ni A, HuB et aL Antimicrobial
`preeervative use in parenteral products:
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`3. Rowe RC, Sheskey PJ, Quinn ME, eds.
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`4. Elder DP, Crowley PJ. Antimicrobial Pre(cid:173)
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`comfFeatured-Articles/38885-Antimicro(cid:173)
`bial-Preservativea-Part-Two-Choosing-a(cid:173)
`Preservalive. Accessed January16, 2014.
`5. Akers, MJ. Excipient-drug interactions in
`parenteral formulations. J Pham&Sci
`2002; 91(11): 2288.
`6. ElderDP, CrowleyPJ.AntimicrobfaZPre-
`8ei'V<Uives.Part TMee: C1uJ1lenges Facing
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`ceutical Review Website.] Januaryl, 2012.
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`calreview.corn/Featured-Articles/38874-
`Antimicrobial-Preservatives-Part-Three-
`
`lllhlnlatioaal.JOIU'II&l ofl'harmaoellt:loCompcnuullzle
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`
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`Accessed Ja.nuary 16, 2014.
`7. Elder DP, CrowleyPJ.AnnmicrobialPre(cid:173)
`Ber'I/Oiives Part OM: Choosing a.Preseroa(cid:173)
`nve System [AmeriaanPhannaceutica.l.
`Review Website.] Januaryl, 2012.Avail(cid:173)
`able at: www.americanpharmaceuticalre(cid:173)
`view.com/Featured-Articles/38886-
`Antimicrobial-Preservatives--Part-One(cid:173)
`Choosing-a-Preservative-System.
`AcceiiiM!dJanuary16,2014.
`B. SuttonS. GMP and Compounding Phar(cid:173)
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`at: www.americanphannaceuticalreview.
`comjFeatured-Articles/135985-GMP(cid:173)
`and-Compou.nding-PharmaciM.Accessed.
`January 16,2014.
`9. Moser CL, Meyer BK. Comparison of com(cid:173)
`pendia! antimicrobial effectiveness tests:
`Arevisw.AAPSPhllrmSciTech 2011; 12(1):
`222-226.
`10. EuropeanPhannacopoeUI. EP 7.0-
`0]/2011:50103 <5.1.3> Efficacy of antimi(cid:173)
`crobial preservatives.
`11. Charnock C, Otterholt E. Evaluation ofpre(cid:173)
`servativeefficacyinphannaceuticalprod(cid:173)
`ucts: The use ofpsychrotolerant,
`low-nutrient preferrinimicrobea in chal(cid:173)
`lenge tests. JClinPham& Ther2012; 37(5):
`558-564.
`12. International Conference on Harmonisa(cid:173)
`tionofTechnicalRequirementsforRegis(cid:173)
`tration ofPha.rma.ceuticalB for Human
`Use. ICH Harmorrised Trll)(.l11ite ~­
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`acceptcmce criteria/orne~~~ dnlg sub(cid:173)
`Bfmlees and fi6W drug produces: chemical
`substances QGA (4). October 1999.
`13. International Conference on Harmonisa(cid:173)
`tion of Technical Requirements for Rsgis(cid:173)
`tration ofPb.arrnaceuticals for Human Use.
`ICHHcmnomaed Tripartite Guideltne. Sta(cid:173)
`bility tetrting ofniNJ drug substancBs and
`productB Q1A (R2). February2003.
`
`Address correspondence to Nicole Vu, Ph.D,
`AnalyticalllesearchLaboratoriss, lfiC., 840
`ResearchPtlrkway, Suite 546, Oklahoma
`City, OK73l04. E-mail: nw@arlok.com
`
`Page 8 of 8