WHY ARE RAPID METHODS AN ALTERNATIVE TO STANDARD METHODS?

When microorganisms contaminate pharmaceutical products, standard methods are performed to quantify, detect, and identify the numbers and types of microorganisms present in a given pharmaceutical batch [6,7]. Standard methods are based upon the enrichment, incubation, and isolation of microorganisms from pharmaceutical samples. Because of the long incu- bation times, continuous manipulation, and time-consuming procedures, results are normally obtained within 6–8 days. It has been recently reported that standard methods underestimate the microbial communities present in pharmaceutical environments [8–11]. This has been demonstrated in samples of water, contact plates, and air samples from different pharmaceutical manufacturing facilities and clean room environments. Adenosine triphos- phate (ATP) bioluminescence, direct viable counts, deoxyribonucleic acid (DNA), and polymerase chain reaction (PCR) technology have demonstrated that a nonculturable portion of the microbial community in pharmaceutical environments is viable and undetectable by compendial methods. Therefore, these new technologies provide a higher resolution and discrimination be- tween microbial species. Accurate information of the types and numbers

of microorganisms in pharmaceutical environments will lead to the imple- mentation of processes that minimize microbial distribution, viability, and proliferation.

Furthermore, identification of several environmental isolates from pharmaceutical environments using standard identification procedures is proven to be incorrect [8,11,12]. When identification is performed by bio- chemical, lipids, and DNA analyses, DNA analysis provides the best repro- ducibility, sensitivity, accuracy, and resolution. To develop the proper corrective action when out-of-specification (OOS) results are obtained, ac- curate microbial identification is needed if the contamination source has to be determined and tracked. A corrective action is not effective if the wrong in- formation is used.

On the basis of these studies, it is evident that in some cases standard methods are not accurate and precise to optimize process control leading to faster releasing time, sample analysis, and high-throughput screening of samples. Although standard methods are valuable and do provide informa- tion on the numbers, microbial genera, and species, they were developed for the identification of microorganisms from clinical samples [13]. Most clinical samples originate from human fluids or tissues, which are rich in nutrients and exhibit temperatures of 35–37jC. Environmental samples, e.g., raw materials, finished products, air, water, equipment swabs, and contact plates, taken from production facilities are not rich in nutrients (oligotrophic) and tem- perature fluctuates below and above ambient temperature. Low water activity and dramatic changes in pH also contribute to microbial stress. Furthermore, manufacturing of pharmaceutical products comprises physical processes such as blending, compression, filtration, heating, encapsulation, shearing, tab- leting, granulation, coating, and drying [14]. These processes expose micro- bial cells to extensive environmental stresses.

Microorganisms survive under those conditions by adapting to the lack of nutrients and other environmental fluctuations by undertaking different survival strategies [15]. Microorganisms are not always metabolically active and reproducing. For instance, gram-positive bacteria such as Bacillus spp. and Clostridium spp. develop dormant structures called spores. On the other hand, gram-negative bacteria such as Escherichia coli, Salmonella typhimu- rium, and other gram-negative rods undergo a viable, but not culturable, stage. Furthermore, bacterial cells that do not grow on plate media but retain their viability going through the viable but culturable stage are still capable of causing severe infections to humans. Several studies have shown that mi- crobial cells in pharmaceutical environments have changed the cell size as well as the enzymatic and physiological profiles as a response to environmental fluctuations [16–18]. These responses are considered stress-induced, which allow the microbes to repair the damage caused. Similar responses have been reported by bacteria exposed to drug solutions where significant morpho-

logical and size changes are observed. Bacterial cells spiked into different types of injectables products have shown different changes in their metabo- lism, enzymatic profiles, and structural changes that interfered with their identification using standard biochemical assays. Furthermore, bacteria un- dergoing starvation survival periods are capable of penetrating 0.2/0.22 Am rated filters which are supposed to retain all bacterial species.

Therefore, using enzymatic and carbon assimilation profiles, e.g., bio- chemical identification, to discriminate and identify microorganisms from environmental samples might in some cases yield unknown profiles that will not provide any significant information on the microbial genera and species. In pharmaceutical environments, information on the genera and species of a microbial contaminant will provide valuable information on the possible sources of the contamination allowing the implementation of effective cor- rective actions.

It has been also shown that the recovery of microorganisms from en- vironmental samples including clean room environments is enhanced by using low nutrient media [19]. The recovery of microorganisms from pharmaceu- tical water samples has been shown to be increased by the use of a low nutrient media, R2A [8,10]. A recent study has also shown that the majority of bacteria present in a pharmaceutical clean room environment are recovered and counted by using a low nutrient media [9]. Similar results are observed for other environmental samples when low-nutrient medium is used [20]. The need for a stress recovery phase is demonstrated by longer incubation times and low nutrient media. In the case of heat-damaged bacterial spores, re- covery and growth is based upon media composition, pH, incubation tem- perature, and incubation time.

Although the development and application of current good manufac- turing practices (cGMP) has improved process control in pharmaceutical environments, microbial contamination is still one of the major causes for product recalls worldwide. Some of the reasons for the lack of compliance with cGMP guidelines are:

_{Poor sanitization practices
Lack of personnel}

_{Lack of training
Lack of resources}

Inadequate facilities for quality control testing

Absence of process validation

Absence of process documentation

Lack of understanding of basic microbiological principles

When products are contaminated, microbial growth will have a negative impact on product integrity creating a serious health threat to consumers. Therefore, there is a need to develop and implement rapid microbiological

methods. Rapid methods have proven to be sensitive, accurate, robust, and provide faster results that might indicate problems in processes and systems used in pharmaceutical environments. Earlier detection of microbial con- tamination allows rapid implementation of corrective actions resulting in the minimization of manufacturing losses and optimization of risk assessment. Current good manufacturing practices (cGMPs) are a dynamic and ongoing process based on applying the latest technological advances to the manu- facturing of pharmaceutical products to provide effective and safe products. Quality control analysis is one of the most important aspects of pharma- ceutical process control. Therefore, reducing testing time, increasing throughput, with faster product release optimize process control.