110 CONTRACT PHARMA • October 2004 www.contractpharma.com

WATER SYSTEM VALIDATION

NEARLY EVERY PHARMACEUTICAL manufacturer uses wateras a raw material. Whether it is an ingredient in thefinal product, a component used in the manufacture of

the final product, or a cleaning agent used to rinse final prod-uct from the manufacturing process, water can be a vector formicrobial contamination. Bacteria will proliferate in water withvery little encouragement. For this reason, microbiologicalanalysis is a very important part of the validation of any waterpurification system. At the same time, specific guidance on thehow, what, and when of the analysis is scattered and scarce.The compendia and the regulatory guidelines offer some direc-tion, but they don’t provide all the answers. To make mattersworse, the terminology often seems too flexible for a regula-tory validation; this is a world of “most probable numbers,”“colony-forming units,” and “indicator organisms.” This articlewill answer some common questions that this contract micro-biologist has encountered over the years.

How Should Samples Be Sent to the Lab?Although it seems obvious, actual experience would seem toindicate otherwise: You must obtain leak-proof, sterile sam-pling materials. If part of your sampling includes disinfectedsource water, use a sample container supplemented with sodi-um thiosulfate to neutralize the chlorine. You must also trainyour staff on how to collect samples aseptically. Your contractlaboratory should be able to provide sample containers andtraining. Collect at least 100–300 mL of sample (dependentupon 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 thesamples well, and send them to the lab in a cooled insulatedshipper. Your contract laboratory should conduct bacterial test-ing within 24 hours of sample time2, and should therefore doc-ument the time they conduct their tests.

Total Organic Carbon (TOC) sampling has its considerations

as well. Your contact laboratory should provide you with samplecontainers used by its TOC analyzer’s autosampler. This willminimize the potential of contamination during transfer of waterfrom container to vial. Although TOC is actually a chemicalanalysis, the hold time becomes important, as any microbes inthe sample can “fix” carbon dioxide into organic carbon, therebyartificially increasing TOC over time. Ask your laboratory fordata validating TOC sample hold time and methodology.

What Method Should I Run for Bioburden?A bioburden value is the enumeration of all viable eukaryotesin the water sample. Although water can be considered a raw material, the analysis should be conducted according toUSP <61> Microbial Limits. Water’s low nutrient level andunique microflora are better tested with different media, tem-peratures and times. The media and times used should encour-age fastidious gram negatives.

The typical approach is to use a high nutrient medium (PlateCount Agar) and incubate it at elevated temperatures (30–35°C)for 48 to 72 hours. This scenario is ideal for isolating humanpathogens that are accustomed to a warm, nutrient-rich envi-ronment. A different approach utilizes a low nutrient medium(R2A) and incubates at lower temperatures (20–25°C) forlonger periods of time (5 to 7 days). This approach is better suit-ed for waterborne organisms. A sample analyzed with thehigher nutrient medium will often result in a bioburden lowerthan the same sample analyzed with the low nutrient medium.So why does USP <1231> Water for Pharmaceutical Purposesrecommend the high nutrient scheme as generally acceptablefor monitoring pharmaceutical water systems?

The risk associated with the increased time needed for incu-bation might outweigh any benefit derived from the higher

Anthony Grilli is laboratory director for SGS Life Sciences. Hecan reached at anthony_grilli@sgs.com.

Water System Validation

Demystifying the microbiologyBy Anthony GrilliSGS U.S.Testing Co.

112 CONTRACT PHARMA • October 2004 www.contractpharma.com

WATER SYSTEM VALIDATION

plate count. If the data aren’t available for over aweek, and the water has been long used by the timethe data are available, how useful was the data? Asstated by USP <1231>: “The advantages of recover-ing injured microbes, slow growers, or fastidiousbacteria should be balanced against the need to havea timely investigation and to take corrective action,as well as the ability of these microorganisms todetrimentally affect products or processes”3. Themore sensitive the product (parenteral, inhalant), themore sensitive the test should be.

What Pathogens Should I Assay For?The USP indicates that pharmaceutical raw materials should befree from objectionable organisms. What makes an organismobjectionable? Just about any organism can be objectionableunder the right circumstances. The solution is to begin with theend in mind: What are you manufacturing? Who is the intend-ed consumer? Is the product a solid oral dosage with low wateractivity? If so, this is a lower risk product that would notencourage microbial proliferation. Is the product a topical orocular drug with a high water activity? Is it intended forimmuno-compromised individuals? If so, the risks of infectingthe 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 canalso be biocide-resistant, biofilm-forming, and pathogenic. Oneexample is Burkholderia cepacia; formerly Pseudomonas cepacia,these bacteria are frequently isolated from water and have beenimplicated in several product recalls (see Figure 1).

Gram positive organisms and fungi are rarely isolated fromwater. When recovered, it might indicate contamination insampling or laboratory and subsequent retraining (althoughthere have been reports of mold shed from gaskets in waterpurification systems).

Is a Coliform Test Necessary?Water used in the manufacture of drug products must mini-mally be potable. The EPA’s Total Coliform rule set a healthgoal for potable water at zero coliforms4. Coliforms are an arti-ficial collection of bacteria devised by microbiologists and areused as an indicator of fecal contamination. They are catego-rized as gram negative lactose fermentors. Not all coliforms arepathogenic, but the presence of coliforms in water indicates thewater is potentially unsafe. There are nonbacterial microbes offecal origin that can make water unsafe: enteroviruses and pro-tozoa, for example. It is not practical to screen for these organ-isms. Coliforms are used to indicate whether any other fecalpathogens might be present.

Water collected from an onsite well must be tested for col-iforms. The water is coming into the factory’s purification sys-tem “raw,” and there is no evidence that it is coliform-free. Asolid oral dosage manufacturer once found Klebsiella pneumoniain its final product and could not figure out where it was com-ing from. The company sampled the active, the excipients, theair and the personnel in search of the source. Eventually, thesource was identified as the water used to clean the equipment.The manufacturer made the false assumption that the well

water was potable and that the purification system would onlyfurther clean it up. Neither assumption was true.

If water is acquired from a public utility, one can ask for thetesting data that shows the water is potable. However, it’simportant to understand that a certain amount of coliforms aredetected even in public drinking water. The EPA set a legallimit of no coliforms in more than 5% of samples taken in eachmonth.5 One way of looking at this is that drinking water cancontain coliforms one day a month and still comply with fed-eral regulations. In addition, the water utility is only testing atthe purification site. One cannot be sure of the integrity of thedistribution between purification and use unless one tests forcoliform at point of use.

How Long Should Sampling Continue?A typical system validation might include daily monitoring fora month. But monitoring will continue after that at least once aweek. A water purification system is dynamic and changing.As the seasons change, the microflora in the source waterchange. Witness the Cryptosporidium outbreak in Minnesota in1993, which hospitalized 4,000 people and left 50 dead. Thisoutbreak was seasonally influenced, occurring in the summerwhen water levels were low and turbidity hindered the disin-fection process. Even within the plant, the purification systemis changing all the time, as filters age, biofilms build, and mate-rials corrode. Weekly monitoring allows for timely identifica-tion of alert levels, well before shutdown action levels occur.

In the end, no compendium or regulatory guideline candetail an off-the-shelf microbiological validation scheme forwater purification systems. There are too many different situa-tions; the number of possible scenarios is an echo of the varietyand 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 microbialcontaminants, 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 Purposes4. US EPA 40CFR141.21 Total Coliform Rule5. ibid.

Date

March 2000

August 2000

March 2004

February 2004

May 2004

Recall

Class II recall of moisturizing lotion

Class II recall of contaminated baby wipes

Voluntary recall of contaminated 12-hour nasal spray

Class II recall of aloe vera lotion sold to hospitals

Voluntary recall of sublingual CO2 sensors storedin contaminated buffered saline.

Figure 1: P. Cepacia-related product recalls