Validating and testing high purity water systems
New technologies are emerging that may one day permit the real-time detection of viable and non-viable organisms in water samples.
By Richard Prince
The validation of purified water (PW) and water for injection (WFI) systems is a basic GMP requirement for plants that manufacture sterile dosage forms, active pharmaceutical intermediates (APIs) associated with some manufacturing steps and processes, or non-sterile dosage forms that are susceptible to microbial adulteration. In the Guide to Inspections of High Purity Water Systems, the FDA has provided guidance to industry in terms of evaluating and, ultimately, validating high purity water systems. Useful information can also be found in USP 23, Fifth Supplement, <1231>, Water for Pharmaceutical Purposes, and in (draft) Guidance for Industry: Manufacture, Processing or Holding of Active Pharmaceutical Ingredients, pages 19-20, August 1996. This column will discuss some microbiological requirements and observations pursuant to the validation and testing of high purity water systems.
When designing the validation (i.e., performance qualification) of a high purity water system, it is critical that the appropriate microbiological specifications be established and written into the protocol. For WFI, the microbiological bioburden count must be <10 colony forming units per 100 mL, and, for PW, the microbiological bioburden count must be <100 colony forming units per mL. Note that the PW Action Level should be lowered if products such as antacids, which are intrinsically nutritive, are manufactured from the high purity water. WFI systems must thus harbor bioburden levels that are, minimally, 1,000 times lower than those levels found in PW systems. WFI systems must also demonstrate acceptable control of endotoxin levels, i.e., <0.25 Endotoxin Units per mL.
The exact quantitative relationship between bioburden counts and predicted endotoxin levels is not currently known. At any point in time, the levels of endotoxin are likely a direct function of the total numbers of gram negative bacteria present, relative purity of the system`s bacterial population, and rate of endotoxin liberation. Since it is unarguably true that the complete absence of microbes in a high purity water system precludes the presence of any associated endotoxin, one can hypothesize that a bioburden level/profile may be predictive in determining whether unacceptable levels of secondary endotoxin in water systems has also occurred. This “combination” microbiological test would be akin to the Total Organic Carbon test that recently became a compendial requirement, and which replaced the need to perform a series of inorganic and less sensitive analytical tests.
As an example to illustrate this point, in a PW system that this author recently validated for a client, over 2,500 water samples were tested for microbiological attributes during a 12-month validation time-frame. A total of 100 percent of the samples gave acceptable counts of <100 colony forming units per mL. A total of approximately 100 samples were tested for endotoxin levels. In all cases, the endotoxin levels were <0.25 Endotoxin Units per mL. These data clearly demonstrate that counts of >100 colony forming units per mL were needed to generate endotoxin levels in excess of 0.25 Endotoxin Units per mL in this particular high purity water system. A contrarian might argue that cumulative levels of endotoxin can build up and shed over time which could lead to discrepant results that are independent of bioburden counts at a given point in time.
The established and preferred method for determining water bioburden is to perform membrane filtration (MF) testing using 100 mL water samples, planting the filters on a low nutrient medium, and then incubating for at least 48 hours at 30 to 35 degrees Celsius. Variations of this testing approach are pervasive within the industry in terms of the type of medium and the incubation conditions used in generating the results. Some firms still perform water testing using 1 mL and 10 mL direct plate counts (PC). Both the MF and the PC techniques require the incubation of samples for several days to visually detect colony forming units. This delay in getting results frustrates many in the industry, particularly if out-of-specification (OOS) results are eventually obtained. Since water samples represent point-in-time determinations, originating from the pulling of samples in fluid or dynamic systems, it is not possible to literally pull another sample of the “same water.” This of course assumes that the facility does not pull real-time back-up samples during the sampling process. So, when the OOS results are first reported, what should be done to determine if the water system is in fact operating properly? Firms will perform a re-test using either the back-up sample or a sample from the system point pulled several days later. Should the original OOS result be “confirmed,” corrective action is supposed to be taken such as re-sanitizing the system.
In the validated PW system that was discussed previously, a total of 99.7 percent of the samples exhibited freedom of so-called objectionable organisms. Of the 0.3 percent samples that generated OOS results, only 19 percent of these OOS results were confirmed following re-testing at the same point at a later point in time. From a Barr Labs vs. USA court decision perspective, one could interpret this to mean that the presence of the objectionable organism could not be confirmed and, thus, there is no underlying problem. Of course, this is ridiculous given that organisms are not found in normal distributions in nature nor in pharmaceutical manufacturing settings. This underscores the dubious efforts of attempting to microbiologically “confirm” point-in-time water determinations. New technologies are emerging that may one day permit the real-time detection of viable and non-viable organisms in water samples. This will help industry efforts in addressing and correcting water system deficiencies within hours, as opposed to the current standard of addressing and correcting water system deficiencies within days.
If objectionable organisms are determined to be present in the water sample, it is prudent to always check the microbiological attributes of the API or product manufactured from the system water that is suspected of being of questionable quality from a microbiological perspective. The rationale for what the firm considers to be objectionable organisms should be discussed in the relevant SOP. Most groupings that this author has seen include some or all of the following organisms: i) Pseudomonas aeruginosa, ii) Escherichia coli, iii) Salmonella sp., iv) Staphylococcus aureus and v) Burkholderia cepacia. It seems logical to classify organisms as “objectionable” if their presence is known to cause morbidity or mortality in man. Since it is possible that “opportunistic” organisms may become pathogenic in immuno-compromised or diseased patients, it is not an easy matter to arbitrarily decide what is considered to be an objectionable organism in a high purity water system. The key thing here is if an objectionable organism is detected in a water sample, that the plant take immediate steps such as re-sanitization and product testing, as reflected in an SOP, to remediate the problem. CR
Richard Prince, Ph.D., is president of Richard Prince Associates, Inc. (Short Hills, NJ), a pharmaceutical-based consultancy. Prince has been providing contract testing and, now, consulting services to the pharmaceutical and allied industries for 12 years. He can be reached at (973) 564-8565 or E-mail: rpaincorp@ aol.com.