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

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.

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.

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

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

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

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

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 [BACARA^TM] or Brilliance^TM B cereus agar [BBCA^TM]).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

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

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

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.