Preservation of Pharmaceutical Dosage Forms



Preservation of Pharmaceutical Dosage Forms


Patrick J. Crowley

David P. Elder



Shelf lives of medications are invariably measured in years, and product usage can be intermittent in some cases. Removing/replacing a product closure during use, a common practice with multidose aqueous liquid products, can inadvertently introduce microbial contaminants. Proliferation of microbes that are present in input materials or introduced during product manufacture or use can also lead to contaminated product, such risks being inflated in products containing ingredients that support microbial growth. The inclusion of a suitable microbicide in products that are aqueous in nature mitigates such risks. However, choosing a preservative is not a simple task. The diversity of the organisms mandated for preservative efficacy testing, the differing regulatory requirements, and seemingly progressive narrowing of choice of suitable preservatives, along with the lack of new preservatives present formidable challenges in dosage form design and preservation. Such challenges and possibilities for their resolution are discussed in this chapter.


BACKGROUND AND CURRENT STATUS

Historically, the risks of microbial contamination in products were manifold. Many active components as well as excipients such as suspending agents were derived from plant or other vegetable sources: They were animal extracts in some cases. Methods of purification were often rudimentary or nonexistent. Preparations of agents such as extracts and tinctures for medicinal use were usually alcohol based, which probably helped limit microbial contamination. The advent of therapeutic agents prepared by chemical synthesis provided opportunities for purification by crystallization and contributed to purer drug substances and excipients. Good Manufacturing Practice (GMP) requirements and associated standards and testing that are now common have also greatly reduced possibilities for microbial contamination. These, along with tests and controls for microbial contamination in source drugs and excipients, provide reasonable assurance of the microbial cleanliness of the final manufactured product. Focus has therefore shifted to protecting against microbial contamination in pharmaceutical products that are aqueous in composition and not manufactured as single-dose sterile preparations. Nevertheless, product recalls due to microbial contamination are becoming increasingly prevalent and concern both sterile (parenteral) and nonsterile dosage forms.1 Regulatory agencies are accordingly devoting increased resource and attention to the microbial quality of excipients, drug substances, and drug products. Lack of sterility in purportedly sterile products is a prime concern. Fungal and bacterial contamination of ostensibly sterile products prepared in a compounding pharmacy has caused deaths and illness on administration to patients, being attributable to using nonsterile ingredients and noncompliance with GMP standards.2

The excipients and active ingredients used in pharmaceutical products must meet pharmacopoeial limits for microbial quality. Most are not microbe free and, although products may be manufactured and packaged in appropriately clean facilities using controlled operating procedures, microbial growth is possible during subsequent product storage. Inadvertent contamination of multidose liquid products during patient use can also lead to microbial growth and risk to the patient. Hence, it is necessary to include a preservative in products that are not manufactured as sterile unless another product component or mixture of components provide an antimicrobial effect that meets pharmacopoeial standards.

Products manufactured as sterile and contained in single-dose presentations do not require a preservative. Examples are not only single-dose parenteral products but may also include single-dose products for ophthalmic instillation. Sterile products in multidose containers must
contain a preservative or components that provide the requisite antimicrobial effects.

Risk assessment tools are described in various compendia and industry documents to help identify potential sources of microbial contamination in nonsterile drug products. These guidelines include



  • International Council for Harmonisation Quality Risk Management Q9 (ICH Q9)3


  • Fault Tree Analysis (FTA)4


  • Failure Mode and Effect Analysis (FMEA)5


  • Hazard Analysis Critical Control Point (HACCP)6


REGULATORY AND PHARMACOPOEIAL REQUIREMENTS

The US Food and Drug Administration (FDA) requires that all ingredients used in a licensed product must meet pharmacopoeial standards for purity and quality. Any preservative used shall be sufficiently safe so that the amount present in the recommended dose of the product will not be harmful to the recipient. It is also essential that the impact of preservatives on other product quality attributes is considered in dosage form design. The US Code of Federal Regulations (CFR §610.15[a]) directs that “preservatives be examined in the context of the overall product, including the recommended dose, and the requirement that patient safety is not compromised at the level included.” The European Medicines Agency (EMA) has comparable guidelines for product components, preservatives, and antioxidants and adopts a similar approach when reviewing, assessing, and approving their inclusion in medicinal products.7 The EMA also states that the choice of a preservative is supported by the following justifications:



  • why the material (preservative) is included


  • proof of its effectiveness


  • the method and standards for its control in the product


  • labeling details on the product


  • relevant safety information

If the product is contained in a multidose container and does not contain a preservative because it is intended for single use only, is self-preserving, or is an oil-based formulation, the applicant may justify the absence of preservative(s). The EMA indicates that this requirement emphasizes the risk associated with using nonpreserved materials and highlights the absence of a preservative. The original intent was to stress the need for risk/balance in decision making, bearing in mind that preservatives (being biocidal) can be considered as “toxic” and pose a safety risk in some situations or in vulnerable populations, such as pediatrics or the elderly. It is also feasible that nonpreserved formulations, intended for single use but which might be used in a multidose fashion in certain situations, can pose risks of microbial contamination and affect patient safety. Subsequently, EMA broadened the guidance to include all excipients in a product.8

Products available in multidose presentations, being vulnerable to contamination during use, may benefit from the presence of a preservative. Examples include



  • Orally dosed aqueous multidose products. These may be formulated as solutions or suspensions at the outset or are constituted as aqueous liquids at time of dispensing with limited in-use periods in the liquid state.


  • Multidose parenteral products such as vaccines for mass immunization programs. Some may be constituted at time of use. Multidose parenterals may also be used to dose patients in health care settings.


  • Multidose ophthalmic medications


  • Topical products that are aqueous based (creams, lotions etc) for application to the skin and mucosal tissues


  • Other aqueous products such as aqueous based liquid metered dose inhalers or nebulized medications that are delivered to regions such as nasal passages, the oro-laryngeal space, the pleural region by inhalation, or to wounds/skin abrasions

The demographics of some users of liquid and semisolid medications (elderly and pediatrics), the cumbersome operations that are sometimes associated with administration or application, and the diversity of microbes in the dosing or application environment can lead to microbes being transferred to the medication during use. Growth during subsequent product storage, particularly when use is only occasional, can compromise quality. Preservation is necessary to prevent such microbial growth and risk to the patient.

Single-dose parenteral products have become increasingly prominent, aided by the advent of technologies, materials, and therapeutic agents that are dosed less frequently, possibly even just once (eg, novel immunotherapeutic agents or vaccines for infectious conditions). Engineered systems are also being developed that make it possible to use multidose presentations that can prevent product contamination during use. Such systems usually incorporate sophisticated technologies, manufacturing operations, and complex packaging. These can inflate costs and be expensive or impractical for operations such as mass vaccination programs. Products in single-dose forms such as sachets for oral dosage can be cumbersome when administering or constituting as liquids; spillage can compromise dose accuracy. Consequently, multidose products are unlikely to be replaced, wholesale in the foreseeable future, and their preservation will remain a prominent requirement in dosage form design.

A preservative may not be required in a product that possesses intrinsic antimicrobial activity. Examples include products containing high levels of sucrose or of polyols such as glycerol or propylene glycol that provide high osmotic pressure or reduced water activity, or products
that contain an active ingredient with anti-infective properties, present in solution at levels conferring the requisite preservative activity. In some cases, such an ingredient might not provide the full antimicrobial spectrum for preservation. For example, a preservative with antifungal activity might be required in a medication containing a broad-spectrum antibacterial that has no or limited antifungal activity.

Transdermal patch systems delivering a fixed dose of drug to or through the skin can be considered single dose and, as such, might not seem to warrant a preservative if manufactured such that the system is microbe free. However, if the delivery system is aqueous, microbial growth could occur throughout the application period, where delivery/application is prolonged and the skin is rich in microorganisms. The limited number of transdermal medications including transmucosal patches that are available in the United States are, with three exceptions, preservative-free. However, the preservative-free products have nonaqueous vehicles, whereas the systems that are aqueous contain preservatives. In essence, a single-dose product of this nature does not necessarily obviate the need for a preservative where inclusion may be warranted. There has been much interest in using low-level electric current (iontophoresis) to provide prolonged transdermal delivery of medications, the technique being facilitated by the availability of microsized power (battery) units. Such presentations are single dose but may warrant the inclusion of a preservative. Drug delivery incorporating an ionizable preservative could pose complications such that the presence of preservative may affect drug delivery, or the ionizable preservative might be transported to or beyond dermal barriers causing inflammation, irritancy, or other unwanted effects. Such possibilities warrant consideration during dosage form design.


PERFORMANCE STANDARDS FOR ANTIMICROBIAL QUALITY


Preservative Performance Standards

The diversity of microbes that could be present in a nonsterile product or in the environment during dosing or application requires a preservative system that possesses broad-spectrum antimicrobial activity. Compendial test methods and requirements reflect this, requiring that activity be demonstrated against defined strains of gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacteria as well as Candida and Aspergillus, a yeast and mold respectively. The British Pharmacopoeia (BP) and European Pharmacopoeia (Ph. Eur.) recommend that these test organisms be supplemented where appropriate by additional strains/species. Examples are defined strains of Escherichia coli in liquid oral preparations and Zygosaccharomyces rouxii in liquid oral preparations containing high concentrations of sugar. The United States Pharmacopeia (USP) also requires that efficacy be shown against E coli. Antiviral activity is not a requirement in any pharmacopoeia.

The pharmacopoeial requirements can be broadly summarized as requiring that the preservative provides a biocidal effect (following deliberate inoculation of the product) during the early incubation period (reduced microorganism counts) and a biostatic effect at later time intervals. Standards reflect the product type, the most stringent concerning parenteral and ophthalmic products. Specific requirements are also provided in the USP for oral antacid products. Liquid antacid products such as those containing aluminum hydroxide and magnesium and calcium hydroxides are difficult to preserve due to their capability to adsorb preservatives such as sodium benzoate and aminobenzoate esters. The relatively high pH of some such products can also cause hydrolysis of aminobenzoates.9,10,11 The less stringent requirements in the USP for preservation of antacids reflect such challenges. Other pharmacopoeias do not make allowances for antacid-containing products.

Test methods and performance standards for determining the antimicrobial capabilities of preservatives in pharmaceutical products are detailed in current USP, Ph. Eur., BP, and Japanese Pharmacopoieas (JP). At the time of writing, the International Pharmacopoeia does not contain testing methods nor performance requirements but states that these are defined by the relevant national authority. The requirements of the BP are harmonized with and identical to those in the Ph. Eur. All pharmacopoeial monographs caution that a preservative must not be used as a substitute for GMP.

Protocols to determine antimicrobial effectiveness involve inoculation of defined concentrations of a range of cultured microorganisms to samples of the test product which is then stored at 20°C to 25°C (representing ambient conditions). Microbial counts are performed at times that are product specific over a maximum of 28 days to provide a measure of microbial kill or growth inhibition over time. The performance standards for different products in the various pharmacopoeias are summarized in Table 39.1.

Ph. Eur. stipulates that bactericidal activity be demonstrated over the first 14 days of the incubation period: Fungicidal requirements are slightly less stringent. The USP standards are less demanding, requiring a fungistatic rather than fungicidal effect, while the bactericidal requirements are also less stringent. Such differences can complicate dosage form design programs, particularly with novel therapeutic agents. Developing a single formulation for all regions where the product is to be made available reduces the complexity of dosage form design and clinical evaluation programs. If the dosage form used in pivotal clinical evaluation programs becomes the commercial product, the risks of biological or clinical nonequivalence
are reduced. This is an important consideration where a conventional bioequivalence study is not possible or is complex, such as with topical or ophthalmic presentations. Regulatory agencies invariably defer to local/national compendial standards when reviewing information in an application for a new product approval. Products developed to meet USP preservative standards might not meet the more stringent Ph. Eur. performance requirements. A higher preservative inclusion level might be required for a product to be used in Europe than for a product formulated to USP standards. Conversely, if a product were to be developed to meet Ph. Eur. standards, it could be considered to be at variance with the generally accepted requirement that preservative levels be minimal so as to reduce possibilities for adverse reactions. Such differing compendial standards could require that two formulations with differing preservative levels be developed, adding complexity to the development program and postapproval supply chain management. There is no evidence that the less stringent USP requirements leads to poorly preserved products. A useful summary of other subtle differences in pharmacopoeial requirements is provided elsewhere.12








TABLE 39.1 Summary of pharmacopoeial preservative efficacy test requirements












































































































































































Pharmacopoeia


Product Type (Aqueous)


Organisms


Criteriaa


Log10 Reduction (Minimum)


6 h


24 h


48 h


7 d


14 d


28 d


Ph. Eur./BP


Parenteral and ophthalmic


Bacteria


A


2


3





No recovery


Fungi


B



1



3



No increase from previous reading


A





2



B






1


Oral, oromucosal, and rectal


Bacteria







3


No increase from previous reading


Fungi







1


Ear, nasal, cutaneous (topical), and inhalation products


Bacteria


A




2


3



No increase from previous reading


B






3


Fungi


A






2


B






1


USP/JPb


Parenteral and ophthalmic


Bacteria






1


3


No increase


Fungi






No increase from initial


Oral, other than antacids


Bacteria







1


No increase


Fungi







No increase from initial


Antacids


Bacteria







No increase from initial


Fungi







Topical, nonsterile nasal


Bacteria







2


No increase


Fungi







No increase from initial


Abbreviations: BP, British Pharmacopoeia; JP, Japanese Pharmacopeia; Ph. Eur., European Pharmacopoeia; USP, United States Pharmacopeia.


a A criteria are the recommended performance standards. B criteria may be acceptable, where higher preservative levels might risk adverse reactions. Use of B criteria must be justified.

b The JP does not contain requirements for antacid products.


An additional inconsistency in test and performance protocols can concern the formulation of antibacterial products, for instance β-lactam antibiotics. Most of these have limited stability in aqueous solution or suspension. Oral formulations for pediatric dosage are accordingly constituted as multidose liquids prior to use. Such liquid products have limited use periods, being constrained by the time that the product in the liquid state retains acceptable drug content, usually 7 to 14 days. Such limitations are usually acceptable because most pediatric infections are cleared within this time frame. But pharmacopoeial standards require that preservative efficacy be sustained over 28 days, a longer period than the dosing interval. Degradation products generated by β-lactam antibiotics can cause product pH to change over the 28-day test period, possibly affecting preservative efficacy. Buffering agents to stabilize pH cannot always be incorporated in such products because of catalytic effects on drug
degradation or adverse effects on taste.13 pH drift over the 14- to 28-day period could reduce the efficacy of the preservative. A requirement that preservative efficacy be sustained for 28 days when product usage time is shorter is difficult to defend. Some oral antibacterial preparations may also require refrigerated storage following constitution as liquids. Microbial growth or inhibition under such conditions may differ from that under pharmacopoeial test conditions (20°C-25°C) and not reflect in-use performance or labeling requirements.

In essence, there are inconsistencies in pharmacopoeial requirements for preservative performance. In practice, it may be impossible to define testing protocols and requirements applicable to every product form but some of the aforementioned anomalies could merit case-by-case decision making by regulatory assessors when reviewing product approval applications.


Preservative Screening

The compendial tests for preservative efficacy are laborintensive, complex, and time-consuming. Duration of incubation is 28 days, and culture preparation and microbial counts during and postincubation along with data analysis all contribute to significant resource requirements and prolonged testing cycle times. It may also be necessary, based on findings during dosage form design, to change the preservative or adjust its content to meet requirements. This can necessitate sequential tests that can delay a product development program. It may be prudent therefore to employ faster screening techniques at the outset such as determination of minimum preservative concentrations that inhibit or kill the test microorganisms in relevant media and testing conditions.14,15 Such tests are used in antimicrobial research and in preservative evaluation programs in other disciplines to determine biocidal and biostatic effects. They can usually be completed in a matter of days and, provided that methodology such as culture media and test conditions are appropriate for the organism and formulation, the findings could increase the probability of subsequent success using compendial tests.






FIGURE 39.1 Fishbone diagram for assuring microbial quality of a preserved product. Abbreviations: API, active pharmaceutical ingredient; HVAC, heating, ventilation, and air conditioning; NCE, novel chemical entity.


Other Considerations

Microbial contamination in a pharmaceutical product can emanate from materials, processes, and other operations listed in Figure 39.1. Such diversity and complexity can mean that a product that otherwise meets compendial preservative efficacy requirements may not be fully protected from contamination and microbial proliferation. To avoid such possibilities, potential contamination from sources such as materials, processes, containers, etc should be proactively considered during dosage form and process design so that particularly troublesome microbes can be identified and controlled appropriately. Such microorganisms may differ from those employed in compendial tests
and are termed objectionable in that their presence in a product can cause illness or product degradation. Parenteral products that were contaminated in this way caused fatalities attributable to contamination with microorganisms such as Aspergillus fumigatus and Exserohilum rostratum in the United States,16 whereas Bacillus cereus-contaminated total nutrient product caused three fatalities in the United Kingdom.17

An objectionable organism has been identified as18



  • A microorganism that, due to its numbers and pathogenicity, can cause infection, allergic response, or toxemia in patients receiving the product


  • A microorganism that can adversely affect the appearance, physicochemical attributes, or therapeutic effect of a nonsterile product

Possibilities for contamination by microorganisms such as Burkholderia cepacia and B cereus elicit close attention from regulatory agencies, particularly the FDA in the light of their prominence in recalled and other contaminated products. The FDA and other regulatory agencies submit that it is the responsibility of the pharmaceutical manufacturer to show that any microorganisms that may be present in nonsterile medicinal products do not pose a safety risk.18 The FDA has a well-established record of enforcing product recalls because of objectionable organisms. During the 8-year period, 1998-2006, nearly 90% of microbial-related recalls for nonsterile dosage forms were attributable to such contamination.18,19,20 Numbers approaching 75% were recorded over the period 2004-2011.1

Figure 39.2 outlines an approach to managing issues related to objectionable microorganisms. A more comprehensive decision tree is provided in a report by the Parenteral Drug Association (PDA).20 The issue facing manufacturers is complex. Some level of microbial presence in materials used in nonsterile drug products is inevitable. Microbial limits for such input materials are accordingly included in pharmacopoeial monographs because input materials are not required to be sterile. A control strategy can accordingly be considered as being within the remit of GMP procedures for controlling less desirable organisms, that is, opportunistic pathogens that do not typically cause infections in the normal, healthy population.1 However, exclusion of objectionable organisms from nonsterile products can be complex, being viewed as an undefined, critical quality attribute. There are no mandated tests nor limits at this time, and control strategies can be difficult to define. The PDA conducted a benchmarking survey to establish the scope of the issue. Respondents indicated that industry has no consistent practices to determine whether a nonspecified microorganism, isolated from a nonsterile product formulation is truly objectionable.21 The PDA has also published guidance seeking to define risk management associated with manufacture and storage of nonsterile oral products.
Suggestions are made on how to identify microbes that engender significant concern on the part of regulatory agencies.21 The PDA report does not list specific objectionable organisms because this could cause undue focus on specific microbes at the expense of an overarching, comprehensive review by the manufacturer. Instead, microbes commonly involved in product contamination/recalls are highlighted along with common opportunistic pathogens. Waterborne opportunistic pathogens, particularly Pseudomonas species and related organisms such as Burkholderia are most frequently cited in product recalls.1 Many do not grow well under defined compendial test conditions and, are not readily identifiable and can complicate test findings. Alternative detection techniques can be used if similar or superior methodologies are shown to be appropriate for any suspect microorganisms.22,23 Compliance with GMP processing requirements is also likely to constrain the presence of problem organisms. Examples of potentially objectionable organisms are provided in Table 39.2.18,24 Microorganisms associated with the greatest number of major health care-associated outbreaks of infection (linked with nonsterile dosage forms) in rank order were B cepacia > P aeruginosa > Serratia marcescens > Ralstonia mannitolilytica > B cereus > Klebsiella pneumoniae > Enterobacter cloacae > Serratia liquefaciens > Paecilomyces lilacinus > Enterobacter species.
In contrast, the objectionable organisms that were involved in the greatest number of product recalls were B cepacia > unspecified fungal organisms > B cereus > P aeruginosa > Elizabethkingia meningoseptica > Enterobacter gergoviae > Pseudomonas putida > Pseudomonas species > Salmonella species. There was particular focus on B cepacia and B cereus in such reports. B cepacia is an opportunistic waterborne pathogen typically infecting immunocompromised people such as cystic fibrosis sufferers. There is accumulating evidence that it is a common contaminant in cosmetics, disinfectants, and in preserved multiuse pharmaceuticals.26 A review of 16 representative product recalls identified B cepacia as the causative organism.25,26 The primary causes of contamination were cited as






FIGURE 39.2 Decision chart for managing objectionable organisms.








TABLE 39.2 Examples of objectionable organismsa




























































































Bacteria


Reported Sources


Aerobacter species


Counterfeit herbal and medicinal products


Bacillus species


Counterfeit herbal and medicinal products


Brucella species


Hospitals


Burkholderia cepacia


Hospitals, recalled products


Campylobacter jejuni


Hospitals, recalled products


Clostridium botulism


Hospitals, recalled products


Corynebacterium diphtheriae


Hospitals


Enterobacter species


Counterfeit herbal and medicinal products


Escherichia coli


Hospitals


Klebsiella species


Contaminated nonsterile products


Legionella species


Emerging pathogen in hospital setting


Micrococcus species


Contaminated nonsterile products


Mycoplasma species


Emerging pathogen in hospital setting


Proteus species


Contaminated nonsterile products


Pseudomonas species


Hospitals, recalled products


Salmonella species


Hospitals, recalled products


Staphylococcus species


Hospitals, recalled products


Streptococcus species


Hospitals, recalled products


Yeasts/Molds



Absidia species


Counterfeit herbal/medicinal products


Aspergillus


Recalled products and counterfeit herbal/medicinal products


Candida albicans


Hospitals and counterfeit medicinal/herbal products


Colletotrichum dermatium


Counterfeit herbal/medicinal products


Mucor


Counterfeit herbal/medicinal products


Penicillium species


Product recalls and counterfeit herbal/medicinal products


Pneumocystis jirovoci


Emerging pathogen in hospital setting


Rhizopus


Counterfeit herbal/medicinal products


Verticillium


Counterfeit herbal/medicinal products


aAdditional examples can be found in Sutton18 and US Food and Drug Administration.25




  • Poor design of water systems (eg, the system did not prevent stagnant water formation) that can rapidly lead to biofilm formation


  • Poor control of water systems (eg, failure to validate operations and processes, lack of scheduled repair and maintenance programs, and improper sanitization procedures)


  • Inappropriate quality of water (eg, use of potable water to clean and/or rinse equipment)


  • Inadequate cleaning and cleaning procedures (eg, inadequate equipment drying times, inadequate sterilization of finished product, inappropriate storage of intermediates, and inappropriate time/temperature/relative humidity controls)


  • Inadequate testing and inappropriate specifications (eg, incomplete or incorrect antimicrobial effectiveness testing, use of contaminated input materials, and inadequate microbiological analysis)


  • Inadequate environmental validation of equipment handling processes or product contact surfaces

The prominence of B cepacia may be attributable to its prevalence in water and other environments, resistance to many common preservatives, adept biofilm formation, and possession of several efflux pump and other mechanisms. Some strains can even grow in distilled water in the temperature range 12°C to 48°C, highlighting the resilience and adaptability of this organism.26,27,28,29,30

The FDA has long had concerns regarding B cepacia contamination in pharmaceutical products. The agency recognizes that there are currently no reliable methodologies for its identification in multiuse products but states that “pharmaceutical companies bear the responsibility to monitor their components, processes, and products to prevent contamination of objectionable organisms.” The FDA cautions that manufacturers should not be overly reliant on preservatives for such control but use validated in-process controls such as effective cleaning, disinfection, and drying of pharmaceutical manufacturing equipment as part of GMP-related practices. This is aligned with FDA policy that preservatives should not be surrogates for poor GMPs. Overall, the hazards presented by organisms such as B cepacia can be summarized as



  • Potential to cause infections in some patient populations25,26


  • Resistance to chemical preservatives and capability to readily share this resistance factor29


  • Detection difficulties when using conventional microbiological techniques30


  • Ability to grow in hostile low-nutrient conditions28,29

The many possibilities for contamination, the challenges associated with detection, and the resilience of B cepacia suggest that a risk-based assessment is preferable for determining the probability of this opportunistic pathogen being present in a medicinal product rather than a compendial “test and limit” approach. Factors germane to such a risk assessment could include



  • the route and method of product use


  • the patient population (eg, children, immunocompromised patients)


  • the type of product and its propensity to support microbial growth

B cereus is a gram-positive aerobic, facultative anaerobic spore-forming bacterium, claimed to be one of the more prevalent spore-forming microbes in soil, dust, sediments, food, and plants.30 Consequently, it can be consumed in diet and is normally present in human intestinal flora. Its toxicity is closely related to production of tissue-destructive toxins. The Bacillus group comprises several closely related bacterial species (eg, B cereus sensu stricto, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, and Bacillus cytotoxicus).34,35,36 Such heterogeneity may contribute to the technical difficulties associated with their identification, but these could be mitigated using specific media (eg, rapid agar-AES chemunex [BACARATM] or BrillianceTM B cereus agar [BBCATM]).22,23 In addition to association with food poisoning and eye infections, these organisms are also linked with clinical conditions such as anthrax-like progressive pneumonia, sepsis, and central nervous system (CNS) infections, predominantly in immunosuppressed patients and neonates. They are also associated with postsurgical wound infections especially when catheters are used.31,32,33,34 B cereus is the spore-forming bacterium most often associated with contaminated nonsterile materials.35 Contaminated alcohol swabs used in a surgical center in the United States lead to several fatalities.36 B cereus contamination can be caused by inadequacies associated with:37



  • inadequate cleaning and disinfection procedures


  • inadequate controls for air-handling systems


  • inadequate microbial controls for input materials


  • inadequate contamination control strategies such as deficient environmental monitoring programs


The FDA has indicated that accepting a level of sporeforming microbes as part of the standard environmental flora was a deficiency in such cases, implying that these organisms be totally absent rather than adopting a riskbased assessment process to their presence.38

Pitt et al39 stated that little if any information is available in the literature concerning B cereus infections in any body site that could be linked with use of personal care products. The authors conclude that



  • Low levels of B cereus spores may occasionally be present in near-eye cosmetics and that these products have been used by consumers for many years


  • Exposure to B cereus is more likely to occur through other routes (eg, dust-borne contamination) due to its ubiquity and the resistance of its spores


  • The organism has also been recovered from the eyes of healthy individuals


  • Although there may be a potential hazard, the risk of severe eye infections consequent to exposure through contaminated near-eye cosmetics is judged to be vanishingly small

In cases where nonsterile oral products have been microbiologically tested in a clinical setting and shown to be contaminated (eg, with Bacillus, Klebsiella, or Candida species), the cause has been attributable to poor handling of the pharmaceutical products during dispensing or repackaging, rather than GMP inadequacies.40 B cereus has also been linked with contamination of sterile-labeled products in aseptic manufactured products. It was the causative agent in 23 cases of sepsis in babies (including 3 fatalities) being administered intravenous total parenteral nutrition in the United Kingdom during 2013-2014. The source of the strain identified in 19 patients was subsequently traced to environmental samples from the aseptic area on the day of product manufacture. Investigations by the Medicines and Healthcare Products Regulatory Agency (MHRA) found no evidence to suggest that individual ingredients, product components, or materials caused the contamination. Airborne contamination was considered to be the source of the outbreak. The recalled supplies were found to be contaminated with B cereus.17

The question has been posed whether B cereus warrants categorization as an objectionable microorganism.1,18 For sterile products, the answer was an unequivocal “yes” (as with any microbe), but nonsterile oral products may require a more nuanced attitude. Regarding the related question of it being a significant concern, responses to its presence in a pharmaceutical manufacturing environment may depend on its ubiquity. Opinions have been advanced to the effect that the issue resides with the class as a whole (ie, Bacillus species rather than constraining it to B cereus).18 However, B cereus is one of the more virulent members of the class. The high resistance of spore-forming bacteria such as Bacillus species and Clostridium species to cleaning, disinfection, and drying makes it difficult to eradicate them from a manufacturing environment. Facility design, personnel practices, and compliance as well as cleaning and disinfection procedures must necessarily take account of such challenges.20 In summary, meeting compendial requirements for preservative efficacy does not necessarily guarantee that microbial contamination and growth will not occur. Product and facility-based risk assessment programs allied to GMP practices, along with validating and securing the supply chain, can all contribute to maintaining microbial cleanliness of the pharmaceutical product.

So-called hurdle technology can be a useful means of developing and implementing what might seem to be daunting risk mitigation programs.41,42 The tool was originally developed for preservation of spoilable foodstuffs, but it can be equally applicable to pharmaceutical products. Identifying the factors that optimize microbial proliferation (pH, temperature, presence of water, oxygen concentration, nutrient levels, etc) can suggest approaches for impeding microbial growth by creating nonoptimal microbial proliferation conditions (“hurdles”) to contribute to microcidal or microstatic environments in materials handling, storage, formulation, product manufacture, packaging, etc and an effective control strategy. Possibilities might include



  • Adjust product pH to more acidic or basic regions to attenuate microbial growth.


  • Raise temperature during processing or reduce during transit and/or storage.


  • Reduce water content/activity in the product by including humectants such as polyols or nonaqueous solvents such as propylene glycol.


  • Reduce oxygen concentration during manufacture or replace by nitrogen in product headspace, and otherwise minimized during transport and storage by suitable packaging.


  • Include a preservative, possibly more than one or increase its concentration.

It may not always be feasible to implement each and every one of the above possibilities. Product and process-related considerations can decree otherwise. Nevertheless, the concept of hurdle technology merits exploration as a contributor to product preservation.


FACTORS INFLUENCING PRESERVATIVE CHOICE

The prime requirement for preservative performance is to provide the requisite antimicrobial activity for product preservation. There are also usually additional, product-specific considerations that need to be incorporated in the target product profile for dosage form design. A program to design a preservation system for a specific
product should take account of the physicochemical properties of the preservative, of the drug substance, and of other product components (excipients) as well as the mode of product manufacture, dosage, and use. Preservatives permitted in pharmaceuticals and foodstuffs can be constrained by specific attribute requirements of the pharmaceutical product and its mode of use/administration (Table 39.3). Properties associated with the preservative such as irritancy (topical, oral, parenteral, ophthalmic), odor, and taste (oral liquid, inhalation, intranasal products)
can influence user acceptability, compliance, and consequently product efficacy. A thorough awareness of relevant properties of potential preservatives along with those of the active ingredient and the other product components can avoid much iterative trial and error studies that could otherwise prolong a development program. The compendial standards for antimicrobial effectiveness that were summarized earlier are demanding. For instance, few, if any, therapeutic anti-infectives have both antibacterial and antifungal activity. Yet, a preservative is required to be effective against a diversity of microbes. A target product profile to guide preservative selection might accordingly comprise the following:








TABLE 39.3 Allowable preservatives in food and pharmaceuticals in the European Communitya

























































































































































































E Number


Name


Food


Pharmaceutical


E200


Sorbic acid


Yes


Yes


E202


Potassium sorbate


Yes


Yes


E203


Calcium sorbate


Yes


Limited—less soluble than other salts


E210


Benzoic acid


Yes


Yes


E211


Sodium benzoate


Yes


Yes


E212


Potassium benzoate


Yes


Yes


E213


Calcium benzoate


Yes


Limited—less soluble than other salts


E214


Ethyl p-hydroxybenzoate


Yes


Yes


E215


Sodium ethyl p-hydroxybenzoate


Yes


Yes


E218


Methyl p-hydroxybenzoate


Yes


Yes


E219


Sodium methyl p-hydroxybenzoate


Yes


Yes


E220


Sulfur dioxide


Yes


No—toxicity considerations


E221


Sodium sulfite


Yes


No—toxicity considerations


E222


Sodium hydrogen sulfite


Yes


No—toxicity considerations


E223


Sodium metabisulfite


Yes


No—toxicity considerations


E224


Potassium metabisulfite


Yes


No—toxicity considerations


E226


Calcium sulfite


Yes


No—toxicity considerations


E227


Calcium hydrogen sulfite


Yes


No—toxicity considerations


E228


Potassium hydrogen sulfite


Yes


No—toxicity considerations


E234


Nisin


Yes


No—toxicity considerations


E235


Natamycin


Yes


No—toxicity considerations


E239


Hexamethylene tetramine


Yes


No—toxicity considerations


E242


Dimethyl dicarbonate


Yes


No—toxicity considerations


E243


Ethyl lauroyl arginate


Yes


No


E249


Potassium nitrite


Yes


No—toxicity considerations


E250


Sodium nitrite


Yes


No—toxicity considerations


E251


Sodium nitrate


Yes


No—toxicity considerations


E252


Potassium nitrate


Yes


No—toxicity considerations


E280


Propionic acid


Yes


Yes


E281


Sodium propionate


Yes


Yes


E282


Calcium propionate


Yes


Limited—less soluble than other salts


E283


Potassium propionate


Yes


Yes


E284


Boric acid


Yes


Yes


E285


Sodium tetraborate; borax


Yes


No


E1105


Lysozyme


Yes


No


aFrom EU-approved additives and E numbers: https//www.food.gov.uk/business-guidance/eu-approved-additives-and-e-numbers.43

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Preservation of Pharmaceutical Dosage Forms
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