Review Article ISSN: 0974-6943

Poonam Kushwaha et al. / Journal of Pharmacy Research 2010, 3(1),124-131

Available online through www.jpronline.info

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 ABSTRACT 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. INTRODUCTION Pharmaceutically active products (drug products) are expected to be efficacious, however; the presence of microorganisms in these products may have adverse effects on their efficacy. The severity of the effects that microorganisms may have on any particular drug product is a function of the nature of the product, its intended use, and the nature of the microorganism concerned. At one end of the spectrum, microbial contamination of a sterile parenteral product may, on injection into a debilitated patient, result in fatality; at the other, patients may refuse to begin or continue a course of medication because of aromas, off-flavors, or discolorations of microbial origin. In either situation, or in any related situation, the presence of microorganisms ought to be avoided in drug products [1]. Manufacturers of human and veterinary products, medical devices, processed food and cosmetics are required to establish quality systems to help ensure that their products consistently meet applicable requirements and specifications. In the United States, current Good Manufacturing Practice (cGMP) regulations are issued by the U.S. Food and Drug Administration (FDA) as the minimum requirements for quality systems for FDA-regulated products such as food, drugs, biologics and devices. One requirement of cGMP regulations is the monitoring of microbiological contamination. Microorganisms can be transferred into an aseptically-filled product directly by contaminated raw ingredients or indirectly through condensation, aerosols, lubricants, packaging materials or workers [2]. *Corresponding author.
Poonam Kushwaha Faculty of Pharmacy, Integral University;Lucknow-226026 (India) Tel.: + 91-9451144299 E-mail: poonm1@yahoo.co.in

Incoming raw materials from pharmaceutical ingredients, chemical compounds, vitamins, minerals, herbs and even food ingredients are tested for the presence of undesirable microbes according to the current USP (United States Pharmacopeias) methods. All finished products have to be tested for the presence of undesirable microbes as well. This ensures that all processes are clean and micro free [3]. The pharmacopoeial requirement of sterility test for all products intended for parental administration and ophthalmic preparation, irrespective of whether they are prepare by end -sterilization process or produced under conditions, provides an adequate level of control for such preparation. Many other products, especially liquid preparations and creams for topical application to severely injured skin, large open wounds or mucus membrane which are liable to bacterial, mould and fungal contamination from the atmosphere ( or less frequently from the contaminated equipments during manufacture) should be controlled for microbial contamination. Few materials are self sterilizing but many products capable of supporting microbial growth requires the addition of suitable antibacterial or antifungal agents, if microbiological spoilage of the product is to be completely avoided. Certain materials, which are particularly prone to microbial contamination, may constitute a health hazard unless they are carefully controlled. These are mainly substances of natural origin, which are known to be liable to contamination usually with specific organisms. The most satisfactory control therefore is one which set a requirement for freedom from specified microbial contamination. This is the preferred method of control in the United Kingdom. In some countries reliance is place on a limit of total viable count of microorganism present. This method of control, is however, is less satisfactory as growth of contaminating micro-organisms may occur on storage of the product [4].

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and the ambient temperature.i. The goal of the investigation could be to determine the nature of primary contamination and the causes. in each manufacturing environment a unique microbial community “natural-micro flora” will be present since the local conditions tend to select for a particular assemblage of microorganisms.Raw materials/ excipients a) Components Natural vs. water used in the processing.. Contamination originating from raw materials can contaminate hands of workers and then be transferred to the product and equipment. soil q Fungi yeast personnel q Mold (filamentous Environment. the product and environmental factors.3. of raw materials or Extrinsic. 3(1). senna. When investigating microbial contamination in an industrial setting. aerosolized product).e. etc.i. it is important to have an idea of the possible contaminants (i.Equipment and process Design Material/Surface Unprotected storage tanks Back flow Perforated heat exchangers Unsanitary pumps Carbon filters Membrane filters Valves Seals/gaskets 3-Environment/Facility Undesirable facility design Inadequate air handling systems Inadequate construction materials Inadequate sanitization procedures Inadequate maintenance (standing water. Once the contaminating organism has been identified. samples of the contaminated material should be sent to a reputable laboratory for analyses. The significance of contamination in a processing industry is determined by a number of factors as well as properties of the microorganism(s) concerned. For non-sterile products it is a balance between the cost of reducing contamination against possible risk to the consumer and product [5]. dust. These factors determine what microorganism(s) would be the dominant contaminant(s) and the level of spoilage of the product or raw material. . The first thing is to have a walkthrough of the plant and analyze in detail the stages of processing or production and using microbiological knowledge identify the stage(s) in the entire production process where possible contamination is likely to occur. rust.Issue 1. the “natural-flora”).Poonam Kushwaha et al. Various sources of microbial contamination include [6. / Journal of Pharmacy Research 2010. Generally. soil Sources of Microbial Contamination: Micro-organisms will contaminate formulations from a wide variety of sources. microorganisms of concern are often present in small numbers as part of the natural micro flora of raw materials and could not be totally eliminated. synthetic products Water activity level Containers (cardboard boxes) b) Water Systems (Purified Water.Freedom from pseudomonas: dried AlOH3. microbiological analysis of the product/raw material and the air within the processing area is recommended.e. 7]: 1. All aspects of formulation and manufacture should try to minimize contamination. soil Gram (+) cocci –personnel Gram (-) rods -water. The primary contamination may be: Intrinsic.) Ineffective EM Program 4-Personnel (Major potential source of microbial contamination) Journal of Pharmacy Research Vol.. available nutrients (e.Freedom from salmonellae: acacia. Factors contributing to multiplication of microorganisms to unacceptable levels may include improper storage conditions. WFI) Biofilm 2. likely sources of contamination and the stages in the processing where contamination of the product or raw material is most likely to occur. tregacanth .g.. This is critical for sterile products. q Bacteria fungi) Gram (+) rods -environment.January 2010 124-131 . Similarly.124-131 Contamination from microorganisms is a big problem for all formulations containing moisture but it can be a bother in solid dosage forms also if some natural polymers are used because many natural polymers are fertile sources of microorganisms [5]. it is then easy to design suitable sampling strategies to trace the source of contamination [6]. during or after processing Since contamination of raw materials or the product may originate from contaminated air. improper handling by the workers.Freedom from escheria coli: sterculia .. equipment or even the workers handling the product/raw materials. The source or the cause of primary contamination requires to be identified for appropriate action to be taken.e. Recommended control of microbial contamination in natural pharmaceutical substances: [4] Types of microbial contamination [7] If contamination involves the product or raw material. Environmental factors may include pH and water activity of the product or raw material. dried aluminum phosphate.

0 IU/mg Microbiological testing of nonsterile pharmaceuticals has thus far been performed only in special cases. Differential media uses specific media for the detection of microbial contaminants. oils) Exposed Hair Exposed Clothing Dirt Cosmetics Tobacco smoke Behavior Types of Microbial Contamination in Biological / Sterile Products: There are three possible origins of microbial contaminants: cross contaminants.01 IU/mg NMT 10. This is because the amount of microbial contamination in the biological product can be very low. Environmental contaminants are micro-organisms that are present in the production facility.Poonam Kushwaha et al. This method is especially useful. / Journal of Pharmacy Research 2010. microbial contaminants are detected by the formation of colonies. specific amino acids. Companies should put plans in place immediately for this work and show consistent progress toward this goal.” It must be stressed that FDA’s concern about objectionable organisms” is not the same as the compendial tests for “specified” organisms. For rich media. The introduction of these three harmonized chapters is likely to require some revalidation of existing methodologies. and their growth in the presence of a large quantity of biological product can be limited or inhibited. The manufacturer is responsible for the quality and safety of the product marketed. the “absence of objectionable microorganisms”) from the product.January 2010 . Commonly-known microbial contaminants are well-known to frequently cause contamination in biological products [8]. The National Formulary monograph requirement for the absence of specific organisms is a minimal requirement and should not be taken as proof that the product is suitable for sale from a microbiological perspective. environmental contaminants.00 IU/mg NMT 55. Some of these tests are described in subsequent paragraphs [7]. 124-131 Journal of Pharmacy Research Vol.” which is in agreement with the FDA expectation for absence of “objectionable” organisms [11. 12]. and can detect relatively low levels of microbial contaminants. List of some Drugs containing Endotoxin and their Limit [16] Drug Amoxicillin sodium buserelin Carbenicillin sodium desmopressin fosfomycin Heparin sodium insulin oxytocin somatropin Tetracyclin hydrochloride Vanomycin xylitol Limit NMT 0.5 IU/mg NMT 500. or other specific chemicals or nutrients [9]. species. microbial contaminants are detected by the presence of colonies that have different morphologies from those of the biological products. Cross contaminants are biological products that are manufactured at the same manufacturing facility.124-131 Microbial Contamination in Nonsterile Products: Exposed skin (flakes. 15].50 IU/mg NMT 0.0 IU/mg NMT 0. and it is FDA’s clear expectation (as described in CFR) that this will include a determination of the microbial safety (i. For selective media. An immunofluorescence assay can be used to determine the presence of microbial contamination. such as by Polymerase Chain Reaction (PCR).0 IU/mg NMT 05. The suspected colonies can be further evaluated.25 IU/mg NMT 1. Some of the specific media support the growth of essentially all micro-organisms (referred to as rich media).0 IU/mg NMT 0. and commonlyknown microbial contaminants. resulting in false negative results. Though these controls are not as stringent as those for aseptic manufacture.083IU/mg NMT 0.0IU/mg NMT 300. the US Code of Federal Regulations (21 CFR) clearly requires the manufacturer to produce products free of” objectionable organisms. The US Pharmacopoeia and the US Food and Drug Administration are in agreement about the question of the microbial quality of nonsterile pharmaceuticals: the product must be safe for use.25 IU/mg NMT 4. when the type. Detection of Microbial Contamination in Biological/ Sterile Products: Many tests can be used to detect microbial contamination of biological products. The composition of these media is sometimes modified to limit or inhibit the growth of biological products. consideration must be given to the promotion of hygiene and safety as well as to the feasibility of application in GMP [17]. The internationally harmonized chapters provide a strong framework for this assurance [10]. which the sponsor is aware of in these microorganisms prior to production [8]. and strains of the microbial contaminants are known. 3(1). ) [14. The harmonized microbial limits tests only address the “absence of specified microorganisms” and leave the determination of the “absence of objectionable microorganisms” in the capable hands of each company’s appropriately educated and well-trained microbiology group [13]. Harmonized Chapter (1 11 1) recommends the determination of the risk associated with “other organisms. In formulating such regulations. Accordingly. and so the finished product should also be evaluated for the presence of “objectionable” while in quarantine (costing money for warehousing and not being sold.3. Nonsterile products are subject to process controls from a microbiological perspective. Other media support the growth of specific micro-organisms (referred to as selective media). in order to improve the sensitivity of detecting microbial contamination.Issue 1. regulations concerning the permissible level of nonpathogenic microorganism in non sterile drugs exist in only a few countries..e. Microbiological control of such products is necessary until now. this assay should be validated to detect cross contaminants and commonly-known contaminants. Components that are sometimes used to limit or inhibit the growth of the biological products can be specific antibiotics.

19].3. designed to prevent objectionable microorganisms in drug products not required to be sterile. Finished product testing is at best confirmatory and in some cases may be dispensed with when manufacturing controls ensure that products are highly unlikely to become microbiologically contaminated (parametric release in its broadest sense) [1]. This may involve obtaining samples at each stage of production for the presence of micro-organisms unrelated to the biological products. Regular hygiene monitoring of a plant and equipment. product-contact packaging components. it is unusual to find valid statistical sampling and testing being done at batch release. these assurances are obtained by applying controls that protect materials. It is secondarily concerned with minimizing the potential for any microorganisms that may have contaminated drug products to increase to levels that may risk the efficacy of the product. The sampling procedure in particular should be designed with special care. that contamination of protected areas and materials (production) is excluded. designed to prevent microbio124-131 Journal of Pharmacy Research Vol. Laboratory waste must be disposed of such. manufacturing processes. or destruction [1]. by a bioburden test. glassware. washing. 11 citations in FDA Warning Letters were caused by a lack of control of microbiological contamination (21 CFR 211.124-131 Steps for avoiding Microbial Contamination: There are some steps that should be taken to avoid microbial contamination. All downstream production processes (eg. filling) must be performed aseptically inside a Class 100 laminar flow hood. as well as personnel monitoring. In most cases the implementation of appropriate hygiene programmes and measures have been implemented as an essential part for the manufacturing of pharmaceutical products. personal hygiene and beauty products. equipment.Issue 1. performance in a Laminar-Air-Flow Cabinet to reduce the contamination risk [13]. The facility should include features for control and containment of environmental and cross contamination of microorganisms. Such conditions include examination to take place in a dedicated room i. Control of Microbial Contamination: Microbial control of pharmaceuticals is primarily concerned with minimizing the opportunities for drug products to be contaminated by microorganisms. (b) Appropriate written procedures. secure containment of biological products. Contamination especially in food industry can result to not only human morbidity and mortality but also to business losses [6].113). The testing of product samples for compliance with microbiological standards is only one small part of this. growth medium. strict separation of production workflow from laboratory workflow and of flow of personnel and materials etc. inactivation. In 2005. these findings have been in the Top Ten List of FDA Warning Letters. do require stringent measures i. e.113 Control of microbiological contamination states that: (a) Appropriate written procedures. precludes product testing in this particular room. manufacturing facilities. during. and equipment. All microbial examinations must be performed under conditions designed to avoid accidental contamination of the product and of the product sample to be examined. Further measures include dedicated gowns and other protective clothing. with rinse and swab samples fully analyzed for confirmation according to validated cleaning procedures.e. as a consequence.e. for the detection of micro-organisms unrelated to the biological products. physically and logistically separated from production areas by various means: by location and by restriction of access. and processes from sources of microbiological contamination. The control of microbiological contamination is an outstanding integral part of inspection findings. 211. At the end of each production. The integrity of the filters used for media sterilization should be tested before and after production. or detection of microorganisms after extensive incubation) to ensure a lack of contaminating microorganisms. The overall goal of such a system is to prevent microbiological contamination of the pharmaceutical product [18. microbial monitoring of samples of purified water.January 2010 . a separate heating/ ventilating/air-conditioning system for each suite. and so forth. Processing industries such as pharmaceutical.g. Environmental monitoring should be performed before. and packing materials should be sterilized prior to use. an air pressure cascade system. etc. food (including dairy) have microbial contamination control procedures in place. microbiological contaminants are also routinely controlled by removal. The laboratory has to be monitored on viable count at appropriate and constant intervals. A series of regulations address the subject of microbiological facility control. The execution of testing such as on growth promotion or on the efficacy of antimicrobial preservation in a given room. by individual air supply and exclusion of air flow from laboratory to production. 3(1). and microbiological sampling of products and raw materials are invaluable in detecting potential sources and routes of contamination. formulation. The growth medium and other components related to fermentation and production should be tested (eg. By and large. The production areas should undergo environmental monitoring. All equipment (fermentor. By and large. sterility test. In recognition of the frailty of protective measures in all but the most extreme circumstances. In-process controls should be performed to prevent and identify contaminants. and equipment thoroughly cleaned and sterilized. microbiological test methods are product-destructive and.).Poonam Kushwaha et al. 21CFR Sec. the production suite should be thoroughly cleaned . A variety of tests. laboratory. Examples are multiple suites or operations for different products. shall be established and followed. unless such analyses were carried out in a LaminarAir-Flow Cabinet. and after each production [8]. centrifuge bottles. Over the last years. Minimizing the risk of microbiological contamination of drug products is assured by the application of microbiological and physical standards and controls to starting materials. / Journal of Pharmacy Research 2010.

23]. serious health hazard..113 under the section tory determination of satisfactory conformance to final specifications “Control of microbiological contamination (a)” states “Appropriate for the drug product.” There is a test in the Microbial Limits chapter to demonstrate the absence of Pseudomonas aeruginosa. for Aureus [sic]. there shall be appropriate labora. prior to release. Prior to acceptance and use.. the Agency has been absolutely clear on the concern over objectionable microoracceptance levels and/or appropriate rejection levels. If there is a monograph that requires a test ucts. repro. or vaginal adBacterial Endotoxin. shall be established and followed. specificity.Microbiological Attributes Chapter <1111> provides little specific cessed material must meet appropriate standards.Issue 1. ganisms in the product. and guidance other than “The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of any other relevant criteria [20]. For USP and FDA frequently are interested in the same thing.on the health hazard. oral liquids for E. 211. 211. such batches may be released prior to comple.” So. However. and that fact that testing to the USP chapter (e) The accuracy. It is governed by the USP monographs found in the acceptable levels and types of microbiological contamination in prodNational Formulary (NF). and articles intended for rectal. The USP monograph for a product (as provided in the (b) There shall be appropriate laboratory testing.124-131 logical contamination of drug products purporting to be sterile. / Journal of Pharmacy Research 2010. for Antibiotic/ Vitamin Potency. designed to prevent objectionable microorganactive ingredient.194 (a) (2). then chapter <51> Antimicrobial Effective. However. and the potential hazard to the user. the referee chapters in USP (those numbered under <1000>) are enforced.3.e.laboratory testing. of each batch of drug product required to be free of objectionable microorganisms. provided such testing is com. The FDA Concern (d) Acceptance criteria for the sampling and testing conducted by the FDA will enforce the GMP requirement that if your product quality control unit shall be adequate to assure that batches of drug products meet each appropriate specification and appropriate statis. Where sterility and/or pyrogen isms on drug products not required to be sterile. are not “objectionable”) [11. but it is not sufficient to demonstrate microbial methods employed by the firm shall be established and documented. this test may be required to demonstrate compliance with the mono(c) Any sampling and testing plans shall be described in written pro. and reproducibility of test might be necessary. Coli [sic]. drug products. for antimicrobial efficacy. the product.objectionable microorganisms.aeruginosa. aeruginosa and S. Through the literature and through our invesJournal of Pharmacy Research Vol. In 1970. although organisms.” The USP recommends that certain categories be routinely tested Regulatory Aspects: for total counts and specified indicator microbial contaminants.also include definitive microbial limits [12]. Dr.165 Testing and release for distribution states that: FDA’s concerns are not covered by a USP referee test method. “For a variety of reasons. A number of specific monographs tests is not driven by any concern over “Good Manufacturing Pro. (b) There shall be appropriate tion of sterility and/or pyrogen testing. for Microbial Limits etc [21]. as necessary.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. we have seen a number of prob(f) Drug products failing to meet established standards or specifica. sterility.” This is reinforced by 21 CFR 211. The statistical quality control criteria shall include appropriate you must do that.approval to market submission contained a statement that you would tical quality control criteria as a condition for their approval and re. of current National Formulary) may require “Absence of Pseudomonas each batch of drug product required to be free of objectionable micro. urethral.” 21CFR 211. specifications. In fact.the target population (i. The need for these ministration for yeasts and molds. shall be established testing are conducted on specific batches of short-lived and followed. ments are identical.165 which states radiopharmaceuticals. such written procedure shall be fol. quality [23].QC Microbiology Labs the FDA states: dance with Sec. in the 1993 instructional guide for inspections of Such validation and documentation may be accomplished in accor. The USP Reprocessing may be performed. as necessary. there is a need to have a test for nella. example natural plant. One such situation is with the CFR requirement that medicines be “free of (a) For each batch of drug product. including the identity and strength of each written procedures. Such procedures shall include validaFDA has similar.Poonam Kushwaha et al. topicals for P.January 2010 124-131 . Dunnigan of the Bureau of Medicine of the FDA commented for antimicrobial efficacy. sensitivity.“Testing and release for distribution. there are situations where the 21CFR Sec. Where the requiretion of any sterilization process. animal and some mineral products for SalmoFrom the vantage point of USP. a problem [21]. here we have pleted as soon as possible. This is purely a GMP concern. nasal solutions and inhalation products [24]. However. the nature of the product. but separate concerns. As a general guide for cess” (GMP). lowed.test the finished product by the Microbial Limits Tests that in fact lease. he said that topical preparations conness Test” is the referee test used to demonstrate that characteristic taminated with gram negative organisms are a probable moderate to [22].lems associated with the microbiological contamination of topical tions and any other relevant quality control criteria shall be rejected. 3(1).

it is widely recognized that Pseudomonas cepacia is objectionable if found in a topical product or nasal solution in high numbers. Later in the 1980’s there was a series of articles appearing in the literature describing contamination by P. The procedures in USP were designed to detect the presence of specific “index” or “indicator” organisms. as the current tests will be disappearing.6. 23] USP <61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms <1111> Microbiological Quality of Nonsterile Pharmaceutical Products EP 2.3.compromised patients. The agency classified this as a Class I recall because the product was contaminated with Pseudomonas gladioli/cepacia. 3(1). these organisms would not have been identified by testing procedures delineated in the general Microbial Limits section of the Compendia [25]. and immuno. To accomplish this. the signed-off versions have yet to be published. The health hazard evaluation commented that the risk of pulmonary infection is especially serious and potentially life-threatening to patients with chronic obstructive airway disease. Nevertheless. The care must be taken in performing these tests. Obviously.e. isolates from plate counts.. but not all objectionable organisms. The chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs. and this prompted increased attention to microbial content of non-sterile drugs. yet. 27]. an extensive text on laboratory detection of microorganisms would be required. 2005 meeting of the Pharmacopeial Discussion Group (PDG) held in Chicago. Microbial testing may include an identification of colonies found during the Total Aerobic Plate Count test. we do know that the harmonized tests do not differ greatly from the drafts published in 2003 (USP 2003a.. However. cepacia (currently Burkholderia cepacia) and its survival in disinfectants. Pharm Eur) Microbial Limits Tests are in the final stages of harmonization.6. and 25]. i. as well as enrichment testing. They were signed off to Stage 6A at the November. USP 2003b. [26. so that microbial contamination from the outside can be avoided [28]. In the late 1960’s several outbreaks of disease were traced back to pathogen contaminated medications.Issue 1. Why is this concern? To understand this we have to go back to the 1970’s.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests 2. inhalants or nasal solutions where there is a major concern for microbiological contamination.. In a one page Stimuli to the Revision Process the microbiology committee of the time states [25]: “The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive. nor does it provide procedures for detecting thousands of other potentially pathogenic organisms. Microbial Limit Tests: The microbial limit Tests are designed to perform the qualitative and quantitative estimations of specific viable microorganisms present in samples. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. there are no test methods provided in the USP that will enable the identification of the presence of this microorganism [2]. 25]. Additionally. The USP Concern The USP is on record as early as 1982 verifying that the demonstration of “absence of objectionable microorganisms” is not the intent of the chapter.Poonam Kushwaha et al. for other products such as topicals. IL USA (USP 2006a). USP 2003c). However. This lead to the addition of requirements in the 21 CFR to ensure that there are not objectionable organisms in product released to market [11. It includes tests for total viable count (bacteria and fungi) and coliform test to determine Escherichia coli. The chapter does not provide specific methods for this. For example. Cepacia – the organism requires subsequent differentiation. The Microbial Limits Tests are actually two chapters in the Journal of Pharmacy Research Vol. the USP Microbial Limits Chapter <61> provides methodology for selected indicator organisms. The importance of identifying all isolates from either or both Total Plate Count testing and enrichment testing will depend upon the product and its intended use. should be identified” [26. 27]. and so we will use those drafts as the description of the finalized test [15. The USP XXII monograph requires no microbial testing for this product. / Journal of Pharmacy Research 2010.January 2010 124-131 . the identification should not merely be limited to the USP indicator organisms.4 Microbiological Quality of Nonsterile Pharmaceutical Products public knowledge. Likewise. USP had a test for the “Bacteriological Examination of Gelatin” as early as 1942. if an oral solid dosage form such as a tablet is tested. harmonized test is not yet Table 1: harmonized chapter numbering scheme: [11. However. The classical example being the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital in Massachusetts several years ago [24. 21. Therefore. to detect all potential pathogens. cystic fibrosis. A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation Solution. Again. However.124-131 tigations.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms 5. most non-sterile medications in the US were not required to assay for microbiological quality attributes until the introduction of the Microbial Limits Tests in 1970. and the final.1. The USP and the European Pharmacopoeia (EP. This makes the description of the test a bit difficult. the present chapter does not preclude the detection of Ps. it may be acceptable to identify isolates when testing shows high levels. each company is expected to develop microbial specifications for their nonsterile products [24]. 29]. it has been shown that a variety of infections have been traced to the gram negative contamination of topical products.

3. it can only count as many as 100 CFUs/membrane.Issue 1.. 27. aeruginosa. “Appropriate written procedures. that the product should be protected (usually by its packaging) from additional contamination after release to market. of each batch of drug product required to be free of objectionable microorganisms. USP was only interested in specified organisms. USP <62> “Absence of Specified Microorganisms USP <1111> “Microbial Quality”: a new compendial consideration of “other organisms” current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c).. The method of plating can be pour plate. 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. This presents two areas of concern relevant to microbial control: first. 23. (b) There shall be appropriate laboratory testing. If the product cannot be put into a solution.January 2010 .124-131 USP harmonized chapters: [11. But. shall be established and followed. or material filtration and then placing the membrane filter on an agar plate surface. In addition to standards applying to the products themselves. Although this test may be needed to demonstrate compliance with the monograph requirements laid out in the National Formulary. these standards are concerned with the protection of the public from infection by limiting the numbers and types of microorganisms to levels that are unlikely to be harmful. this is not the major change. simple design to count the number of colony-forming units (CFUs) in a nonsterile product or raw material.. 26.113 and 21 CFR 211. 21. This will be modified in the harmonized version to mirror the European format: [23] Microbial standards: Microbial standards for drug products are published in the pharmacopoeias and/or are required by regulatory agencies for their registration. These manufacturing standards (Good Manufacturing Practices or GMPs) are intended to ensure that finished product standards are being attained consistently. 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. with (at least in the author’s opinion) significant changes from the current USP. Though membrane filtration is a good method to test a large volume of liquid. the allowance for twice the specification in observed results is significant.” Tables IIIa–c presents the existing “Microbial Limits—Absence of Specified Microorganisms” tests from the current USP and Pharm Eur. They need not be sterile but it has to 124-131 Journal of Pharmacy Research Vol. but interested readers can refer to the discussion in the US Food and Drug Administration’s Bacterial Analytical Manual. 28 and 29] USP<61>”Microbial Enumeration” The microbial enumeration test is a basic. 3(1). The membrane filtration method should only be used when few CFUs are expected to be found in the material to be tested. that the product should be formulated to prevent proliferation of any microorganisms that may have been present at tolerable levels at the time of release [1]. are not “objectionable”). It is presented as an aid to evaluation. FDA has been concerned about objectionable organisms. as necessary. The USP monograph for a product (as provided in the current National Formulary [NF]) may require the “Absence of Pseudomonas aeruginosa. industry has had a problem. 25.165) relating to the importance of “objectionable microorganisms.165 which states in the section “Testing and release for distribution. The “Control of Microbiological Contamination (a)” section of 21 CFR 211. A full description of the MPN method is outside the scope of this article. The microbial standards applying to drug products are expected to be maintained until time of use by the patient (or healthcare professional) and throughout their shelf-lives. spread plate. designed to prevent objectionable microorganisms on drug products not required to be sterile.113 states. It should be noted that this harmonized chapter represents a true compromise by all parties.” Thus. Generally. and second. FDA is bound by the concern expressed in the Code of Federal Regulations (21 CFR 211. 12.Poonam Kushwaha et al. There is a significant controversy in the United States over the intent of this evaluation. as well as the harmonized document. Before the introduction of the harmonized Chapter ‹1111›. the most probable number (MPN) method has several provisions to use.e.” This is reinforced by 21 CFR 211. Chapter ‹1111› “Microbial Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use” is a relatively short section that has a significant impact. Pharm Eur. Non-sterile preparations have less stringent requirements regarding exclusion of microbes. These organisms are specified in monographs. But. For the US reader.” This issue is addressed in the final section of this review because the harmonized Chapter ‹1111› deals with “other organisms. / Journal of Pharmacy Research 2010. and may assist in determining whether revalidation of method suitability studies is needed.” A test in the “Microbial Limits” chapter demonstrates the absence of P. there are also microbial standards applying to the conditions under which drug products are allowed to be manufactured. and JP chapters.

“Microbiological Quality of Nonsterile PharmaCONCLUSION: Unwanted and potentially dangerous microbiological contaminants in pharmaceutical manufacturing have long been an area of concern. 28. MD.technol.microbiol. 28-71.4. FDA. 2006 .org/docs/ sutton.Look mart find articles. Conflict of interest: None Declared Journal of Pharmacy Research Vol. Rockville. http://www.pdf) 16. 2002. USP. vol. Goingtomeet_com Microbial Contamination . no. page no. Applied microbiology.” Pharm Forum. requires manufacturers to employ a number of technical and scientific techniques in the manufacturing cycle.M impurities and pharmaceutical substances and their limit test. Sutton.Corrective Actions.microbial%20%imp/ controlled%20envirnment%20%20understanding%20contamination. pharmaceutical technology.H. Troubleshooting microbial contamination in an industrial environment. 2004). (http://www. Pharma Times. FDA.com/ Stability_Of_Drugs:Microbiological_Stability 6. page no. 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