WATER SYSTEM VALIDATION

Water System Validation

Demystifying the microbiology
By Anthony Grilli SGS U.S.Testing Co.

EARLY EVERY PHARMACEUTICAL manufacturer uses water as a raw material. Whether it is an ingredient in the final product, a component used in the manufacture of the final product, or a cleaning agent used to rinse final product from the manufacturing process, water can be a vector for microbial contamination. Bacteria will proliferate in water with very little encouragement. For this reason, microbiological analysis is a very important part of the validation of any water purification system. At the same time, specific guidance on the how, what, and when of the analysis is scattered and scarce. The compendia and the regulatory guidelines offer some direction, but they dont provide all the answers. To make matters worse, the terminology often seems too flexible for a regulatory validation; this is a world of most probable numbers, colony-forming units, and indicator organisms. This article will answer some common questions that this contract microbiologist has encountered over the years.

as well. Your contact laboratory should provide you with sample containers used by its TOC analyzers autosampler. This will minimize the potential of contamination during transfer of water from container to vial. Although TOC is actually a chemical analysis, the hold time becomes important, as any microbes in the sample can fix carbon dioxide into organic carbon, thereby artificially increasing TOC over time. Ask your laboratory for data validating TOC sample hold time and methodology.

What Method Should I Run for Bioburden?

A bioburden value is the enumeration of all viable eukaryotes in the water sample. Although water can be considered a raw material, the analysis should be conducted according to USP <61> Microbial Limits. Waters low nutrient level and unique microflora are better tested with different media, temperatures and times. The media and times used should encourage fastidious gram negatives. The typical approach is to use a high nutrient medium (Plate Count Agar) and incubate it at elevated temperatures (3035C) for 48 to 72 hours. This scenario is ideal for isolating human pathogens that are accustomed to a warm, nutrient-rich environment. A different approach utilizes a low nutrient medium (R2A) and incubates at lower temperatures (2025C) for longer periods of time (5 to 7 days). This approach is better suited for waterborne organisms. A sample analyzed with the higher nutrient medium will often result in a bioburden lower than the same sample analyzed with the low nutrient medium. So why does USP <1231> Water for Pharmaceutical Purposes recommend the high nutrient scheme as generally acceptable for monitoring pharmaceutical water systems? The risk associated with the increased time needed for incubation might outweigh any benefit derived from the higher Anthony Grilli is laboratory director for SGS Life Sciences. He can reached at anthony_grilli@sgs.com.

How Should Samples Be Sent to the Lab?

Although it seems obvious, actual experience would seem to indicate otherwise: You must obtain leak-proof, sterile sampling materials. If part of your sampling includes disinfected source water, use a sample container supplemented with sodium thiosulfate to neutralize the chlorine. You must also train your staff on how to collect samples aseptically. Your contract laboratory should be able to provide sample containers and training. Collect at least 100300 mL of sample (dependent upon the number of tests to be performed). Sample volumes <100 mL are unrepresentative and therefore unacceptable.1 Document the date and time the samples are taken. Seal the samples well, and send them to the lab in a cooled insulated shipper. Your contract laboratory should conduct bacterial testing within 24 hours of sample time2, and should therefore document the time they conduct their tests. Total Organic Carbon (TOC) sampling has its considerations

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WATER SYSTEM VALIDATION

plate count. If the data arent available for over a week, and the water has been long used by the time the data are available, how useful was the data? As stated by USP <1231>: The advantages of recovering injured microbes, slow growers, or fastidious bacteria should be balanced against the need to have a timely investigation and to take corrective action, as well as the ability of these microorganisms to detrimentally affect products or processes3. The more sensitive the product (parenteral, inhalant), the more sensitive the test should be.

Figure 1: P. Cepacia-related product recalls Date March 2000 August 2000 March 2004 Recall Class II recall of moisturizing lotion Class II recall of contaminated baby wipes Voluntary recall of contaminated 12-hour nasal spray Voluntary recall of sublingual CO2 sensors stored in contaminated buffered saline.

February 2004 Class II recall of aloe vera lotion sold to hospitals May 2004

What Pathogens Should I Assay For?

The USP indicates that pharmaceutical raw materials should be free from objectionable organisms. What makes an organism objectionable? Just about any organism can be objectionable under the right circumstances. The solution is to begin with the end in mind: What are you manufacturing? Who is the intended consumer? Is the product a solid oral dosage with low water activity? If so, this is a lower risk product that would not encourage microbial proliferation. Is the product a topical or ocular drug with a high water activity? Is it intended for immuno-compromised individuals? If so, the risks of infecting the consumer are higher. In this case, a screen for Pseudomonads, or gram negative non-fermenting bacteria, is recommended. Gram negative non-fermentors are ubiquitous in water, but can also be biocide-resistant, biofilm-forming, and pathogenic. One example is Burkholderia cepacia; formerly Pseudomonas cepacia, these bacteria are frequently isolated from water and have been implicated in several product recalls (see Figure 1). Gram positive organisms and fungi are rarely isolated from water. When recovered, it might indicate contamination in sampling or laboratory and subsequent retraining (although there have been reports of mold shed from gaskets in water purification systems). water was potable and that the purification system would only further clean it up. Neither assumption was true. If water is acquired from a public utility, one can ask for the testing data that shows the water is potable. However, its important to understand that a certain amount of coliforms are detected even in public drinking water. The EPA set a legal limit of no coliforms in more than 5% of samples taken in each month.5 One way of looking at this is that drinking water can contain coliforms one day a month and still comply with federal regulations. In addition, the water utility is only testing at the purification site. One cannot be sure of the integrity of the distribution between purification and use unless one tests for coliform at point of use.

How Long Should Sampling Continue?

A typical system validation might include daily monitoring for a month. But monitoring will continue after that at least once a week. A water purification system is dynamic and changing. As the seasons change, the microflora in the source water change. Witness the Cryptosporidium outbreak in Minnesota in 1993, which hospitalized 4,000 people and left 50 dead. This outbreak was seasonally influenced, occurring in the summer when water levels were low and turbidity hindered the disinfection process. Even within the plant, the purification system is changing all the time, as filters age, biofilms build, and materials corrode. Weekly monitoring allows for timely identification of alert levels, well before shutdown action levels occur. In the end, no compendium or regulatory guideline can detail an off-the-shelf microbiological validation scheme for water purification systems. There are too many different situations; the number of possible scenarios is an echo of the variety and adaptability of microbial life. Herein lies the solution: begin the validation process with the end product in mind. Focusing on the finished product discards unlikely microbial contaminants, and thereby focuses the methodology. References 1. FDA Guide to Inspections of High Purity Water Systems, 2 . Standard Methods for the Examination of Water and Wastewater, 18th Edition, American Public Health Association, Washington DC 2005 3. USP 27 <1231>Water for Pharmaceutical Purposes 4. US EPA 40CFR141.21 Total Coliform Rule 5. ibid.

Is a Coliform Test Necessary?

Water used in the manufacture of drug products must minimally be potable. The EPAs Total Coliform rule set a health goal for potable water at zero coliforms4. Coliforms are an artificial collection of bacteria devised by microbiologists and are used as an indicator of fecal contamination. They are categorized as gram negative lactose fermentors. Not all coliforms are pathogenic, but the presence of coliforms in water indicates the water is potentially unsafe. There are nonbacterial microbes of fecal origin that can make water unsafe: enteroviruses and protozoa, for example. It is not practical to screen for these organisms. Coliforms are used to indicate whether any other fecal pathogens might be present. Water collected from an onsite well must be tested for coliforms. The water is coming into the factorys purification system raw, and there is no evidence that it is coliform-free. A solid oral dosage manufacturer once found Klebsiella pneumonia in its final product and could not figure out where it was coming from. The company sampled the active, the excipients, the air and the personnel in search of the source. Eventually, the source was identified as the water used to clean the equipment. The manufacturer made the false assumption that the well

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