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Poonam Kushwaha et al.

/ Journal of Pharmacy Research 2010, 3(1),124-131

Review Article
ISSN: 0974-6943 Available online through
Microbial Contamination: A Regulatory Perspective
Poonam Kushwaha
Faculty of Pharmacy, Integral University;Lucknow-226026 (India)

Received on: 10-09-2009; Revised on: 17-10-2009; Accepted on:05-12-2009

The microbiological attributes of pharmaceutical ingredients are often critical to final product quality. FDA expects from the manufacturers to
measure and characterize the bioburden of their products. The concern of FDA and compendia towods microbial contamination described in
this article. This article also describes the source, methods of detection, and methods of elimination of microbial contamination.

Keywords: Microbial contamination; source of contamination; control of microbial contamination; FDA concern; USP concern; microbial
limit test.


Pharmaceutically active products (drug products) are ex- Incoming raw materials from pharmaceutical ingredients, chemical
pected to be efficacious, however; the presence of microorganisms in compounds, vitamins, minerals, herbs and even food ingredients are
these products may have adverse effects on their efficacy. The sever- tested for the presence of undesirable microbes according to the cur-
ity of the effects that microorganisms may have on any particular rent USP (United States Pharmacopeias) methods. All finished prod-
drug product is a function of the nature of the product, its intended ucts have to be tested for the presence of undesirable microbes as
use, and the nature of the microorganism concerned. At one end of well. This ensures that all processes are clean and micro free [3].
the spectrum, microbial contamination of a sterile parenteral product
may, on injection into a debilitated patient, result in fatality; at the The pharmacopoeial requirement of sterility test for all prod-
other, patients may refuse to begin or continue a course of medica- ucts intended for parental administration and ophthalmic prepara-
tion because of aromas, off-flavors, or discolorations of microbial tion, irrespective of whether they are prepare by end -sterilization
origin. In either situation, or in any related situation, the presence of process or produced under conditions, provides an adequate level of
microorganisms ought to be avoided in drug products [1]. control for such preparation. Many other products, especially liquid
preparations and creams for topical application to severely injured
Manufacturers of human and veterinary products, medical skin, large open wounds or mucus membrane which are liable to
devices, processed food and cosmetics are required to establish qual- bacterial, mould and fungal contamination from the atmosphere ( or
ity systems to help ensure that their products consistently meet ap- less frequently from the contaminated equipments during manufac-
plicable requirements and specifications. In the United States, cur- ture) should be controlled for microbial contamination.
rent Good Manufacturing Practice (cGMP) regulations are issued by
the U.S. Food and Drug Administration (FDA) as the minimum re- Few materials are self sterilizing but many products capable
quirements for quality systems for FDA-regulated products such as of supporting microbial growth requires the addition of suitable anti-
food, drugs, biologics and devices. One requirement of cGMP regula- bacterial or antifungal agents, if microbiological spoilage of the prod-
tions is the monitoring of microbiological contamination. Microor- uct is to be completely avoided. Certain materials, which are particu-
ganisms can be transferred into an aseptically-filled product directly larly prone to microbial contamination, may constitute a health hazard
by contaminated raw ingredients or indirectly through condensation, unless they are carefully controlled. These are mainly substances of
aerosols, lubricants, packaging materials or workers [2]. natural origin, which are known to be liable to contamination usually
with specific organisms. The most satisfactory control therefore is
*Corresponding author. one which set a requirement for freedom from specified microbial con-
Poonam Kushwaha tamination. This is the preferred method of control in the United King-
Faculty of Pharmacy, Integral University;Lucknow-226026 (India) dom. In some countries reliance is place on a limit of total viable count
Tel.: + 91-9451144299 of microorganism present. This method of control, is however, is less
E-mail: satisfactory as growth of contaminating micro-organisms may occur
on storage of the product [4].

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Poonam Kushwaha et al. / Journal of Pharmacy Research 2010, 3(1),124-131
Contamination from microorganisms is a big problem for all formula- If contamination involves the product or raw material, samples of the
tions containing moisture but it can be a bother in solid dosage forms contaminated material should be sent to a reputable laboratory for
also if some natural polymers are used because many natural poly- analyses. Once the contaminating organism has been identified, it is
mers are fertile sources of microorganisms [5]. then easy to design suitable sampling strategies to trace the source
of contamination [6].
Recommended control of microbial contamination in natural phar-
maceutical substances: [4]
Types of microbial contamination [7]
- Freedom from salmonellae: acacia; senna; tregacanth
- Freedom from escheria coli: sterculia
q q q
- Freedom from pseudomonas: dried AlOH3, dried aluminum phos- Bacteria Fungi Mold (filamentous
phate. fungi)
Gram (+) rods -environment, soil yeast Environment, soil
Gram (+) cocci –personnel personnel
The significance of contamination in a processing industry Gram (-) rods -water, soil
is determined by a number of factors as well as properties of the
microorganism(s) concerned, the product and environmental factors. Sources of Microbial Contamination:
Environmental factors may include pH and water activity of the prod-
uct or raw material, available nutrients (e.g., aerosolized product), and Micro-organisms will contaminate formulations from a wide
the ambient temperature. These factors determine what variety of sources. All aspects of formulation and manufacture should
microorganism(s) would be the dominant contaminant(s) and the level try to minimize contamination. This is critical for sterile products. For
of spoilage of the product or raw material. Generally, in each manufac- non-sterile products it is a balance between the cost of reducing
turing environment a unique microbial community “natural-micro flora” contamination against possible risk to the consumer and product [5].
will be present since the local conditions tend to select for a particular
assemblage of microorganisms. Similarly, microorganisms of concern Various sources of microbial contamination include [6, 7]:
are often present in small numbers as part of the natural micro flora of 1- Raw materials/ excipients
raw materials and could not be totally eliminated. Factors contribut- a) Components
ing to multiplication of microorganisms to unacceptable levels may Natural vs. synthetic products
include improper storage conditions, improper handling by the work- Water activity level
ers. Contamination originating from raw materials can contaminate Containers (cardboard boxes)
hands of workers and then be transferred to the product and equip- b) Water Systems (Purified Water, WFI)
ment. Biofilm

When investigating microbial contamination in an indus- 2- Equipment and process

trial setting, it is important to have an idea of the possible contami- Design
nants (i.e., the “natural-flora”), likely sources of contamination and Material/Surface
the stages in the processing where contamination of the product or Unprotected storage tanks
raw material is most likely to occur. Back flow
Perforated heat exchangers
The goal of the investigation could be to determine the na- Unsanitary pumps
ture of primary contamination and the causes. The first thing is to Carbon filters
have a walkthrough of the plant and analyze in detail the stages of Membrane filters
processing or production and using microbiological knowledge iden- Valves
tify the stage(s) in the entire production process where possible con- Seals/gaskets
tamination is likely to occur. The source or the cause of primary con-
tamination requires to be identified for appropriate action to be taken. 3-Environment/Facility
The primary contamination may be: Undesirable facility design
Inadequate air handling systems
Intrinsic- i.e., of raw materials or Inadequate construction materials
Extrinsic- i.e., during or after processing Inadequate sanitization procedures
Inadequate maintenance (standing water, dust, rust, etc.)
Since contamination of raw materials or the product may Ineffective EM Program
originate from contaminated air, water used in the processing, equip-
ment or even the workers handling the product/raw materials, micro- 4-Personnel
biological analysis of the product/raw material and the air within the (Major potential source of microbial contamination)
processing area is recommended.

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Poonam Kushwaha et al. / Journal of Pharmacy Research 2010, 3(1),124-131
Exposed skin (flakes, oils) Microbial Contamination in Nonsterile Products:
Exposed Hair
Exposed Clothing The manufacturer is responsible for the quality and safety
Dirt of the product marketed, and it is FDA’s clear expectation (as de-
Cosmetics scribed in CFR) that this will include a determination of the microbial
Tobacco smoke safety (i.e., the “absence of objectionable microorganisms”) from the
Behavior product. The US Pharmacopoeia and the US Food and Drug Admin-
istration are in agreement about the question of the microbial quality
Types of Microbial Contamination in Biological / Sterile Products: of nonsterile pharmaceuticals: the product must be safe for use. The
internationally harmonized chapters provide a strong framework for
There are three possible origins of microbial contaminants: this assurance [10]. The introduction of these three harmonized chap-
cross contaminants, environmental contaminants, and commonly- ters is likely to require some revalidation of existing methodologies.
known microbial contaminants. Cross contaminants are biological Companies should put plans in place immediately for this work and
products that are manufactured at the same manufacturing facility. show consistent progress toward this goal. The National Formulary
Environmental contaminants are micro-organisms that are present in monograph requirement for the absence of specific organisms is a
the production facility. Commonly-known microbial contaminants are minimal requirement and should not be taken as proof that the prod-
well-known to frequently cause contamination in biological products uct is suitable for sale from a microbiological perspective. Harmo-
[8]. nized Chapter (1 11 1) recommends the determination of the risk asso-
ciated with “other organisms,” which is in agreement with the FDA
Detection of Microbial Contamination in Biological/ Sterile Prod- expectation for absence of “objectionable” organisms [11, 12]. The har-
ucts: monized microbial limits tests only address the “absence of specified
microorganisms” and leave the determination of the “absence of ob-
Many tests can be used to detect microbial contamination jectionable microorganisms” in the capable hands of each company’s
of biological products. Some of these tests are described in subse- appropriately educated and well-trained microbiology group [13].
quent paragraphs [7].
Nonsterile products are subject to process controls from a
Differential media uses specific media for the detection of microbiological perspective. Though these controls are not as strin-
microbial contaminants. Some of the specific media support the growth gent as those for aseptic manufacture, the US Code of Federal Regu-
of essentially all micro-organisms (referred to as rich media). For rich lations (21 CFR) clearly requires the manufacturer to produce prod-
media, microbial contaminants are detected by the presence of colo- ucts free of” objectionable organisms.” It must be stressed that FDA’s
nies that have different morphologies from those of the biological concern about objectionable organisms” is not the same as the
products. The suspected colonies can be further evaluated, such as compendial tests for “specified” organisms, and so the finished prod-
by Polymerase Chain Reaction (PCR). Other media support the growth uct should also be evaluated for the presence of “objectionable”
of specific micro-organisms (referred to as selective media). For se- while in quarantine (costing money for warehousing and not being
lective media, microbial contaminants are detected by the formation sold, ) [14, 15].
of colonies.
List of some Drugs containing Endotoxin and their Limit [16]
The composition of these media is sometimes modified to
Drug Limit
limit or inhibit the growth of biological products, in order to improve Amoxicillin sodium NMT 0.25 IU/mg
the sensitivity of detecting microbial contamination. This is because buserelin NMT 1.00 IU/mg
Carbenicillin sodium NMT 55.5 IU/mg
the amount of microbial contamination in the biological product can desmopressin NMT 500.0 IU/mg
be very low, and their growth in the presence of a large quantity of fosfomycin NMT 0.083IU/mg
biological product can be limited or inhibited, resulting in false nega- Heparin sodium NMT 0.01 IU/mg
insulin NMT 10.0IU/mg
tive results. Components that are sometimes used to limit or inhibit oxytocin NMT 300.0 IU/mg
the growth of the biological products can be specific antibiotics, somatropin NMT 05.0 IU/mg
Tetracyclin hydrochloride NMT 0.50 IU/mg
specific amino acids, or other specific chemicals or nutrients [9]. Vanomycin NMT 0.25 IU/mg
xylitol NMT 4.0 IU/mg
An immunofluorescence assay can be used to determine
the presence of microbial contamination. This method is especially Microbiological testing of nonsterile pharmaceuticals has
useful, and can detect relatively low levels of microbial contaminants, thus far been performed only in special cases. Microbiological con-
when the type, species, and strains of the microbial contaminants are trol of such products is necessary until now; regulations concerning
known. Accordingly, this assay should be validated to detect cross the permissible level of nonpathogenic microorganism in non sterile
contaminants and commonly-known contaminants, which the spon- drugs exist in only a few countries. In formulating such regulations,
sor is aware of in these microorganisms prior to production [8]. consideration must be given to the promotion of hygiene and safety
as well as to the feasibility of application in GMP [17].

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Steps for avoiding Microbial Contamination: testing such as on growth promotion or on the efficacy of antimicro-
bial preservation in a given room, precludes product testing in this
There are some steps that should be taken to avoid micro- particular room, unless such analyses were carried out in a Laminar-
bial contamination. The facility should include features for control Air-Flow Cabinet. A variety of tests, e.g. microbial monitoring of
and containment of environmental and cross contamination of micro- samples of purified water, do require stringent measures i.e. perfor-
organisms. Examples are multiple suites or operations for different mance in a Laminar-Air-Flow Cabinet to reduce the contamination
products, a separate heating/ ventilating/air-conditioning system for risk [13].
each suite, an air pressure cascade system, secure containment of
biological products, and so forth. All downstream production pro- Control of Microbial Contamination:
cesses (eg, washing, formulation, filling) must be performed asepti-
cally inside a Class 100 laminar flow hood. All equipment (fermentor, Microbial control of pharmaceuticals is primarily concerned
centrifuge bottles, glassware, etc.), growth medium, and packing ma- with minimizing the opportunities for drug products to be contami-
terials should be sterilized prior to use. The growth medium and other nated by microorganisms. It is secondarily concerned with minimiz-
components related to fermentation and production should be tested ing the potential for any microorganisms that may have contaminated
(eg, by a bioburden test, sterility test, or detection of microorganisms drug products to increase to levels that may risk the efficacy of the
after extensive incubation) to ensure a lack of contaminating micro- product. The testing of product samples for compliance with micro-
organisms. The integrity of the filters used for media sterilization biological standards is only one small part of this. By and large,
should be tested before and after production. In-process controls microbiological test methods are product-destructive and, as a con-
should be performed to prevent and identify contaminants. This may sequence, it is unusual to find valid statistical sampling and testing
involve obtaining samples at each stage of production for the pres- being done at batch release. Finished product testing is at best con-
ence of micro-organisms unrelated to the biological products. At the firmatory and in some cases may be dispensed with when manufac-
end of each production, the production suite should be thoroughly turing controls ensure that products are highly unlikely to become
cleaned , and equipment thoroughly cleaned and sterilized, with rinse microbiologically contaminated (parametric release in its broadest
and swab samples fully analyzed for confirmation according to vali- sense) [1].
dated cleaning procedures. The production areas should undergo
environmental monitoring, as well as personnel monitoring, for the
Processing industries such as pharmaceutical, personal hy-
detection of micro-organisms unrelated to the biological products.
giene and beauty products, food (including dairy) have microbial
Environmental monitoring should be performed before, during, and
contamination control procedures in place. Regular hygiene monitor-
after each production [8].
ing of a plant and equipment, and microbiological sampling of prod-
ucts and raw materials are invaluable in detecting potential sources
Minimizing the risk of microbiological contamination of drug
and routes of contamination. Contamination especially in food in-
products is assured by the application of microbiological and physi-
dustry can result to not only human morbidity and mortality but also
cal standards and controls to starting materials, product-contact pack-
to business losses [6].
aging components, manufacturing facilities, manufacturing processes,
and equipment. By and large, these assurances are obtained by ap-
The control of microbiological contamination is an outstand-
plying controls that protect materials, equipment, and processes from
ing integral part of inspection findings. In 2005, 11 citations in FDA
sources of microbiological contamination. In recognition of the frailty
Warning Letters were caused by a lack of control of microbiological
of protective measures in all but the most extreme circumstances,
contamination (21 CFR 211.113). Over the last years, these findings
microbiological contaminants are also routinely controlled by removal,
have been in the Top Ten List of FDA Warning Letters. In most cases
inactivation, or destruction [1].
the implementation of appropriate hygiene programmes and measures
have been implemented as an essential part for the manufacturing of
All microbial examinations must be performed under condi-
pharmaceutical products. A series of regulations address the subject
tions designed to avoid accidental contamination of the product and
of microbiological facility control. The overall goal of such a system
of the product sample to be examined. Such conditions include exami-
is to prevent microbiological contamination of the pharmaceutical
nation to take place in a dedicated room i.e. laboratory, physically and
product [18, 19].
logistically separated from production areas by various means: by
location and by restriction of access, by individual air supply and
exclusion of air flow from laboratory to production. Further measures
21CFR Sec. 211.113 Control of microbiological contamination states
include dedicated gowns and other protective clothing, strict separa-
tion of production workflow from laboratory workflow and of flow of
personnel and materials etc. Laboratory waste must be disposed of
(a) Appropriate written procedures, designed to prevent objection-
such, that contamination of protected areas and materials (produc-
able microorganisms in drug products not required to be sterile, shall
tion) is excluded. The sampling procedure in particular should be
be established and followed.
designed with special care. The laboratory has to be monitored on
viable count at appropriate and constant intervals. The execution of
(b) Appropriate written procedures, designed to prevent microbio-
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logical contamination of drug products purporting to be sterile, shall
be established and followed. Such procedures shall include valida-
tion of any sterilization process. FDA has similar, but separate concerns. Where the require-
ments are identical, the referee chapters in USP (those numbered
under <1000>) are enforced. However, there are situations where the
21CFR Sec. 211.165 Testing and release for distribution states that: FDA’s concerns are not covered by a USP referee test method. One
such situation is with the CFR requirement that medicines be “free of
(a) For each batch of drug product, there shall be appropriate labora- objectionable microorganisms.” 21CFR 211.113 under the section
tory determination of satisfactory conformance to final specifications “Control of microbiological contamination (a)” states “Appropriate
for the drug product, including the identity and strength of each written procedures, designed to prevent objectionable microorgan-
active ingredient, prior to release. Where sterility and/or pyrogen isms on drug products not required to be sterile, shall be established
testing are conducted on specific batches of short-lived and followed.” This is reinforced by 21 CFR 211.165 which states
radiopharmaceuticals, such batches may be released prior to comple- “Testing and release for distribution... (b) There shall be appropriate
tion of sterility and/or pyrogen testing, provided such testing is com- laboratory testing, as necessary, of each batch of drug product re-
pleted as soon as possible. quired to be free of objectionable microorganisms.” So, here we have
a problem [21]. The USP monograph for a product (as provided in the
(b) There shall be appropriate laboratory testing, as necessary, of current National Formulary) may require “Absence of Pseudomonas
each batch of drug product required to be free of objectionable micro- aeruginosa.” There is a test in the Microbial Limits chapter to demon-
organisms. strate the absence of Pseudomonas aeruginosa. However, although
this test may be required to demonstrate compliance with the mono-
(c) Any sampling and testing plans shall be described in written pro- graph requires as laid out in NF it does not meet the FDA concern that
cedures that shall include the method of sampling and the number of any organism in the final product be acceptable to the product and
units per batch to be tested; such written procedure shall be fol- the target population (i.e. are not “objectionable”) [11, 23].
The FDA Concern
(d) Acceptance criteria for the sampling and testing conducted by the
quality control unit shall be adequate to assure that batches of drug FDA will enforce the GMP requirement that if your product
products meet each appropriate specification and appropriate statis- approval to market submission contained a statement that you would
tical quality control criteria as a condition for their approval and re- test the finished product by the Microbial Limits Tests that in fact
lease. The statistical quality control criteria shall include appropriate you must do that. This is purely a GMP concern. However, the Agency
acceptance levels and/or appropriate rejection levels. has been absolutely clear on the concern over objectionable microor-
ganisms in the product, and that fact that testing to the USP chapter
(e) The accuracy, sensitivity, specificity, and reproducibility of test might be necessary, but it is not sufficient to demonstrate microbial
methods employed by the firm shall be established and documented. quality [23]. In fact, in the 1993 instructional guide for inspections of
Such validation and documentation may be accomplished in accor- QC Microbiology Labs the FDA states:
dance with Sec. 211.194 (a) (2).
“For a variety of reasons, we have seen a number of prob-
(f) Drug products failing to meet established standards or specifica- lems associated with the microbiological contamination of topical
tions and any other relevant quality control criteria shall be rejected. drug products, nasal solutions and inhalation products [24]. The USP
Reprocessing may be performed. Prior to acceptance and use, repro- Microbiological Attributes Chapter <1111> provides little specific
cessed material must meet appropriate standards, specifications, and guidance other than “The significance of microorganisms in nonsterile
any other relevant criteria [20]. pharmaceutical products should be evaluated in terms of the use of
the product, the nature of the product, and the potential hazard to the
Regulatory Aspects: user.” The USP recommends that certain categories be routinely tested
for total counts and specified indicator microbial contaminants. For
USP and FDA frequently are interested in the same thing. example natural plant, animal and some mineral products for Salmo-
From the vantage point of USP, there is a need to have a test for nella, oral liquids for E. Coli [sic], topicals for P. aeruginosa and S.
sterility, for antimicrobial efficacy, for Antibiotic/ Vitamin Potency, for Aureus [sic], and articles intended for rectal, urethral, or vaginal ad-
Bacterial Endotoxin, for Microbial Limits etc [21]. The need for these ministration for yeasts and molds. A number of specific monographs
tests is not driven by any concern over “Good Manufacturing Pro- also include definitive microbial limits [12]. As a general guide for
cess” (GMP). It is governed by the USP monographs found in the acceptable levels and types of microbiological contamination in prod-
National Formulary (NF). If there is a monograph that requires a test ucts, Dr. Dunnigan of the Bureau of Medicine of the FDA commented
for antimicrobial efficacy, then chapter <51> Antimicrobial Effective- on the health hazard. In 1970, he said that topical preparations con-
ness Test” is the referee test used to demonstrate that characteristic taminated with gram negative organisms are a probable moderate to
[22]. serious health hazard. Through the literature and through our inves-

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tigations, it has been shown that a variety of infections have been The USP Concern
traced to the gram negative contamination of topical products. The
classical example being the Pseudomonas cepacia contamination of The USP is on record as early as 1982 verifying that the
Povidone Iodine products reported by a hospital in Massachusetts demonstration of “absence of objectionable microorganisms” is not
several years ago [24, 25]. the intent of the chapter. In a one page Stimuli to the Revision Pro-
cess the microbiology committee of the time states [25]:
Therefore, each company is expected to develop microbial
specifications for their nonsterile products [24]. Likewise, the USP “The tests described in the Microbial Limits Tests <61> were
Microbial Limits Chapter <61> provides methodology for selected not designed to be all-inclusive, i.e., to detect all potential pathogens.
indicator organisms, but not all objectionable organisms. For example, To accomplish this, an extensive text on laboratory detection of mi-
it is widely recognized that Pseudomonas cepacia is objectionable if croorganisms would be required. The procedures in USP were de-
found in a topical product or nasal solution in high numbers; yet, signed to detect the presence of specific “index” or “indicator” or-
there are no test methods provided in the USP that will enable the ganisms. Nevertheless, the present chapter does not preclude the
identification of the presence of this microorganism [2]. detection of Ps. Cepacia – the organism requires subsequent differ-
entiation. The chapter does not provide specific methods for this, nor
A relevant example of this problem is the recall of does it provide procedures for detecting thousands of other poten-
Metaproterenol Sulfate Inhalation Solution. The USP XXII mono- tially pathogenic organisms. Individual monographs include require-
graph requires no microbial testing for this product. The agency clas- ments for limits on total aerobic counts and/or absence of one or
sified this as a Class I recall because the product was contaminated more of the four selected “indicator” organisms. The chapter on Mi-
with Pseudomonas gladioli/cepacia. The health hazard evaluation crobial Limits Tests provides methods to assure that one may test for
commented that the risk of pulmonary infection is especially serious those microbial requirements in the individual monographs... [26, 27].
and potentially life-threatening to patients with chronic obstructive
airway disease, cystic fibrosis, and immuno- compromised patients. Microbial Limit Tests:
Additionally, these organisms would not have been identified by
testing procedures delineated in the general Microbial Limits section The microbial limit Tests are designed to perform the quali-
of the Compendia [25]. tative and quantitative estimations of specific viable microorganisms
present in samples. It includes tests for total viable count (bacteria
Microbial testing may include an identification of colonies and fungi) and coliform test to determine Escherichia coli. The care
found during the Total Aerobic Plate Count test. Again, the identifi- must be taken in performing these tests, so that microbial contamina-
cation should not merely be limited to the USP indicator organisms. tion from the outside can be avoided [28].
The importance of identifying all isolates from either or both Total
Plate Count testing and enrichment testing will depend upon the The USP and the European Pharmacopoeia (EP, Pharm Eur)
product and its intended use. Obviously, if an oral solid dosage form Microbial Limits Tests are in the final stages of harmonization. They
such as a tablet is tested, it may be acceptable to identify isolates were signed off to Stage 6A at the November, 2005 meeting of the
when testing shows high levels. However, for other products such as Pharmacopeial Discussion Group (PDG) held in Chicago, IL USA (USP
topicals, inhalants or nasal solutions where there is a major concern 2006a). However, the signed-off versions have yet to be published.
for microbiological contamination, isolates from plate counts, as well This makes the description of the test a bit difficult, as the current
as enrichment testing, should be identified” [26, 27]. tests will be disappearing, and the final, harmonized test is not yet
Table 1: harmonized chapter numbering scheme: [11, 23]
Why is this concern? To understand this we have to go
back to the 1970’s. USP had a test for the “Bacteriological Examina- USP EP
tion of Gelatin” as early as 1942. However, most non-sterile medica- <61> Microbiological Examination 2.6.12 Microbiological Examination
Of Nonsterile Products: Microbial Of Nonsterile Products: Microbial
tions in the US were not required to assay for microbiological quality Enumeration Tests Enumeration Tests
attributes until the introduction of the Microbial Limits Tests in 1970. <62> Microbiological Examination of 2.6.13 Microbiological Examination of
In the late 1960’s several outbreaks of disease were traced back to Nonsterile Products: Tests for Nonsterile Products: Tests for
pathogen contaminated medications, and this prompted increased Specified Microorganisms Specified Microorganisms
<1111> Microbiological 5.1.4 Microbiological Quality
attention to microbial content of non-sterile drugs. Later in the 1980’s Quality of Nonsterile of Nonsterile Pharmaceutical
there was a series of articles appearing in the literature describing Pharmaceutical Products Products
contamination by P. cepacia (currently Burkholderia cepacia) and its
survival in disinfectants. This lead to the addition of requirements in public knowledge. However, we do know that the harmonized tests
the 21 CFR to ensure that there are not objectionable organisms in do not differ greatly from the drafts published in 2003 (USP 2003a,
product released to market [11, 21, and 25]. USP 2003b, USP 2003c), and so we will use those drafts as the de-
scription of the finalized test [15, 29].

The Microbial Limits Tests are actually two chapters in the

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USP harmonized chapters: [11, 12, 21, 23, 25, 26, 27, 28 and 29]
The microbial enumeration test is a basic, simple design to count the number of
USP<61>”Microbial Enumeration” colony-forming units (CFUs) in a nonsterile product or raw material. The
preferred method is to put the material into a solution and then plate the
aliquots to determine the CFUs/g (or mL) of initial material. If the product
cannot be put into a solution, the most probable number (MPN) method has
several provisions to use. A full description of the MPN method is outside the
scope of this article, but interested readers can refer to the discussion in the US
Food and Drug Administration’s Bacterial Analytical Manual. The method of
plating can be pour plate, spread plate, or material filtration and then placing
the membrane filter on an agar plate surface. The membrane filtration method
should only be used when few CFUs are expected to be found in the material to
be tested. Though membrane filtration is a good method to test a large volume
of liquid, it can only count as many as 100 CFUs/membrane.

USP <62> “Absence of Specified Microorganisms There is a significant controversy in the United States over the intent of this
evaluation. FDA is bound by the concern expressed in the Code of Federal
Regulations (21 CFR 211.113 and 21 CFR 211.165) relating to the importance
of “objectionable microorganisms.” This issue is addressed in the final section of
this review because the harmonized Chapter ‹1111› deals with “other organ-
isms.” Tables IIIa–c presents the existing “Microbial Limits—Absence of Speci-
fied Microorganisms” tests from the current USP and Pharm Eur, as well as the
harmonized document. It is presented as an aid to evaluation, and may assist in
determining whether revalidation of method suitability studies is needed. It
should be noted that this harmonized chapter represents a true compromise by
all parties, with (at least in the author’s opinion) significant changes from the
current USP, Pharm Eur, and JP chapters.

Chapter ‹1111› “Microbial Examination of Nonsterile Products: Acceptance

USP <1111> “Microbial Quality”: a new compendial consideration of Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical
“other organisms” Use” is a relatively short section that has a significant impact. For the US
reader, the allowance for twice the specification in observed results is signifi-
cant. But, this is not the major change.
Before the introduction of the harmonized Chapter ‹1111›, USP was only
interested in specified organisms. These organisms are specified in monographs.
But, FDA has been concerned about objectionable organisms. The “Control of
Microbiological Contamination (a)” section of 21 CFR 211.113 states, “Ap-
propriate written procedures, designed to prevent objectionable microorgan-
isms on drug products not required to be sterile, shall be established and fol-
lowed.” This is reinforced by 21 CFR 211.165 which states in the section
“Testing and release for distribution... (b) There shall be appropriate laboratory
testing, as necessary, of each batch of drug product required to be free of objec-
tionable microorganisms.” Thus, industry has had a problem. The USP mono-
graph for a product (as provided in the current National Formulary [NF]) may
require the “Absence of Pseudomonas aeruginosa.” A test in the “Microbial
Limits” chapter demonstrates the absence of P. aeruginosa. Although this test
may be needed to demonstrate compliance with the monograph requirements
laid out in the National Formulary, it does not meet FDA’s concern that all
microorganisms in a nonsterile product should be acceptable to the product and
the target population (i.e., are not “objectionable”).

current USP: Current USP <61> Microbial Limits Tests (USP 2006b) products are allowed to be manufactured. These manufacturing stan-
and <1111> Microbiological Attributes of Nonsterile Pharmaceutical dards (Good Manufacturing Practices or GMPs) are intended to en-
Products (USP 2006c). This will be modified in the harmonized ver- sure that finished product standards are being attained consistently.
sion to mirror the European format: [23] The microbial standards applying to drug products are expected to
be maintained until time of use by the patient (or healthcare profes-
Microbial standards: sional) and throughout their shelf-lives. This presents two areas of
concern relevant to microbial control: first, that the product should
Microbial standards for drug products are published in the be protected (usually by its packaging) from additional contamina-
pharmacopoeias and/or are required by regulatory agencies for their tion after release to market, and second, that the product should be
registration. Generally, these standards are concerned with the pro- formulated to prevent proliferation of any microorganisms that may
tection of the public from infection by limiting the numbers and types have been present at tolerable levels at the time of release [1].
of microorganisms to levels that are unlikely to be harmful. In addi-
tion to standards applying to the products themselves, there are also Non-sterile preparations have less stringent requirements
microbial standards applying to the conditions under which drug regarding exclusion of microbes. They need not be sterile but it has to
Journal of Pharmacy Research Vol.3.Issue 1.January 2010 124-131
Poonam Kushwaha et al. / Journal of Pharmacy Research 2010, 3(1),124-131
be shown that some specifically named organisms are not present in no. 1714–1722.
them. 12. USP, Chapter ‹1111›, “Microbiological Quality of Nonsterile Pharma-
ceutical Products,” Pharm Forum. 29 (5), 2003, page no. 1733–1735.
13. Envirnmental conditions for the microbiological examination of non-
CONCLUSION: sterile products, technical interpretation. (
Unwanted and potentially dangerous microbiological con- 14. Sutton, Scott. Microbiological attributes of nonsterile pharmaceutical
products, pharmaceutical technology, page no. 97-98. (http://
taminants in pharmaceutical manufacturing have long been an area of
concern. These contaminants can lead to unpredictable pharmaco- 15. Sutton, Scott. & Knapp, Joseph. Activities of the USP microbiology
logical effects or super- or sub-potency of the active drug substance. subcommitte of revision during the 1995-2000 rivision cycle. (http://
Worse, they may be toxic. Because of the potentially dangerous im-
16. Vishweshwar. S & Gupta.R.M. FAQ on impurity profile for the bulk drugs,
plications of these contaminants on patient safety, the FDA, through Pharma Times, vol. 34, 2002.
its Current Good Manufacturing Practices (cGMP) regulations, re- 17. Buhlmann, Xaver. Method for microbiological testing of nonsterile phar-
quires manufacturers to employ a number of technical and scientific maceuticals, Applied microbiology, vol. 16, no.2, 1968, page no. 1919-
techniques in the manufacturing cycle. These techniques share a 1923.
18. Gronostajski, D.E. Understanding control of contamination for process
common goal: to provide a high level of assurance that finished drug validation, march 2003. (http://www.microbial%20%imp/
products meet their intended and defined specifications. Compendia controlled%20envirnment%20%20understanding%20contamination.)
also provide harmonized microbial limit tests which helps in their con- 19. Microbial contamination- Corrective actions. Preventive actions. Nov
trol. 23, 2006 - Nov 24,2006. (http://www. Goingtomeet_com Microbial Con-
tamination - Corrective Actions, Preventive Actions.htm)
20. Parenteral Drug Association (PDA) Technical Report #13, “Revised
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 1.January 2010 124-131