Bacterial action and alert limits

It is important to note that, with the exception of the reference to sterility for packaged waters, there are no bacteria specifications contained in the USP Official Monographs for pharmaceutical waters. Drinking Water has been defined. The EPA’s NPDWR references specific guidelines related to the absence of coliform bacteria. However, the USP Purified Water and Water for Injection Official Monographs merely state that the feed water to the water purification system producing Purified Water or Water for Injection must meet the NPDWR (or other appropriate European, Japanese, or World Health Organization standards). Subsequently, it is extremely important to point out that information associated with bacterial levels is outlined in USP General Information Section <1231> Water for Pharmaceutical Purposes. While this section provides valuable information to support the Official Monographs, material contained within this section, as implied, is for general information.

Suggested maximum total viable bacteria levels as well as the enumeration method are included directly in the EP Purified Water and Water for Injection Monographs.

In reviewing the USP General Information section with regard to alert and action levels for bacteria, the following definitions are provided:

Alert Levels are events or levels that, when they occur or are exceeded, indicate that a process may have drifted from its normal operating condition. Alert Level excursions constitute a warning and do not necessarily require a corrective action. However, alert level excursions usually lead to the alerting of personnel involved in water system operation as well as QA. Alert level excursions may also lead to additional monitoring with more intense scrutiny of resulting and neighbouring data as well as other process indicators.

Action Levels are events or higher levels that, when they occur or are exceeded, indicate that a process is probably drifting from its normal operating range. Examples of kinds of Action Level events include exceeding the Alert Levels repeatedly; or in multiple simultaneous locations, a single occurrence of exceeding a higher microbial level; or the individual or repeated recovery of specific objectionable microorganisms. Exceeding an Action Level should lead to immediate notification of both QA and personnel involved in water system operations so that corrective actions can immediately be taken to bring the process back into its normal operating range.

While it should be indicated that the definition stated above are directly from the USP General Information section, a “guideline” to support the balance of the material in USP, they provide an excellent description of Alert and Action Level definition and corrective action.

The Action and Alert Levels are further defined with regard to product purity as follows:

A specification excursion may trigger an extensive finished product impact investigation, substantial remedial actions within the water system that may include a complete shutdown, and possible product rejection.

The selection of Alert and Action Levels must consider the product being manufactured as well as the method of manufacturing the product. While the levels should be extremely conservative, ensuring the safety of the public, it should be pointed out that regulatory authorities generally consider exceeding an Action Level as an out-of-specification (OOS) condition, requiring an evaluation of the potential effects on the quality of the product (from a microbiological standpoint) and an Incident Evaluation Report.

The USP General Information section indicates that the maximum suggested action levels are as follows:

Drinking Water: 500 cfu/mL

Purified Water: 100 cfu/mL

Water for Injection: 10 cfu/100 mL

The EP suggested maximum limits for Purified Water and Water for Injection are similar but the indicated microbial enumeration methodologies are different. As discussed within this text, actual bacteria Alert and Action Limits for Purified Water and Water for Injection systems are generally lower than the indicated values.

The recommended USP enumeration methodologies in the General Information section are as follows:

Drinking Water: Method: pour plate or membrane filtration Minimum sample volume: 1 mL Growth medium: plate count agar Incubation time: 42–72 hours minimum Incubation temperature: 30–35 0 C

Purified Water: Method: pour plate or membrane filtration Minimum sample volume: 1 mL Growth medium: plate count agar Incubation time: 48–72 hours minimum Incubation temperature: 30–35 0 C

Water for Injection: Method: membrane filtration Minimum sample volume: 100 mL Growth medium: plate count agar Incubation time: 48–72 hours minimum Incubation temperature: 30–35 0 C



Some of pharmacopeias, such as EP, reference different bacteria enumeration methods than USP. The EP Monograph requirements are indicated as follows:

Purified Water: Method: membrane filtration Minimum sample volume: chosen in relation to the expected results Growth medium: R2A agar Incubation time: 5 days Incubation temperature: 30–35 0 C

Water for Injection: Method: membrane filtration Minimum sample volume: 200 mL, minimum Growth medium: R2A agar Incubation time: 5 days Incubation temperature: 30–35 0 C



While the USP General Information section and the EP Monograph contain specific methods for enumeration of bacteria, it is suggested that the selected method, including culture media, incubation time period, and incubation temperature be established for the specific system, process, product, and operating conditions. Ideally, selection should provide a result as quickly as possible. However, bacteria, particularly in a low-nutrient environment, may require a number of days to culture. Rapid microbial techniques may be coupled with conventional techniques to meet the indicated requirements.

SOPs associated with the methodology used for bacterial monitoring should clearly state that the analyst inspect plates every 24 hours (as a minimum) until the recommended incubation time period is reached. This would allow the analyst to report excursions in bacterial levels as quickly as possible, minimizing the amount of product manufactured with bacterial levels exceeding the Action Limit. If performed in an adequately air-filtered environment, the effects of atmospheric bacteria, during observation of colonies prior to the specified incubation time, should be minimal.

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