Microbiology

In determining the appropriate microbial limit, the USP Microbiology Expert Committee considers such matters as the route of administration, the form of the product, and the source material. For example, it is never appropriate to have any microorganisms in a product intended for injection intravenously. Therefore, you will never see a microbial limit listed for such products. Instead, those products must meet the test for Sterility <71>. Other products may be in a form possessing extremely low water activity, such that microbial growth could not occur. You may not see microbial limits provided for some of those products. Many other products, such as those intended for oral administration, will have limits provided. Those limits are selected such that the risk of harm to the consumer is extremely low, while being reasonable from a manufacturing and quality control perspective. Products from botanical sources may have higher limits due to the larger bioburden associated with the raw materials. Again, the limits are set such that consumer risk is minimized without creating an untenable situation for the manufacturer. Consideration is also given to the likelihood of product spoilage. Since some products are more prone to spoilage due to microbial contamination, limits may be set lower for these. The issues discussed above also influence which, if any, specific organisms must be tested.

USP Chapter <643> intentionally says nothing about how often the system suitability test (SST) should be run. The reasoning is that this frequency depends on the stability of the Total Organic Carbon (TOC) instrument response and other factors associated with the water quality and risk. If the TOC of a quality water system is very low, say <20 ppb, then many opt to reduce the frequency of testing due to less risk. The stability of different TOC measurement technologies may vary over extended periods of time. The instrument manufacturer can advise the user on this matter and user experience can also be valuable in determining a suitable frequency. Another factor is the risk of a non-conforming system suitability test result since the Rs-Rw result used in this calculation is the limit response for the instrument, the crucial pass/fail value for the TOC test. If a non-conforming system suitability test is obtained, it implicates the accuracy of all TOC test results since the previous successful system suitability test. For this reason, many users choose to perform the system suitability test more frequently than the stability of the TOC instrument response might suggest, just to minimize the impact of a possibly non-conforming result. This is why a typically low OTC water system is at less risk, even with a failed SST. If the SST fails, some remediation is necessary to re-adjust the instrument, replace a lamp, or some other means of instrument improvement. But even a 50% error will have little impact on the past TOC readings (since the readings, even with this error, are so low relative to the Limit). On a high TOC water system, the failure of the SST is possibly more critical. This is up to the risk the user is willing to assume, knowing the historic stability of their instrument and other factors. Therefore, the Total Organic <643> is silent on the frequency of performing the system suitability test because it is up to the user to decide what is appropriate.

There is a "target limit response" of 500 ppb carbon. The true limit is the response of the TOC measurement system to a 500 ppb sucrose solution, Rs, corrected for the response to reagent water, Rw. This is equal to Rs–Rw. The actual number will vary based upon your reference standard solution, your equipment, background carbon, etc. You will determine compliance using the USP Reference Standards.

Where USP is silent on storage conditions and the stability of prepared reference standard solutions, the implication is that the solutions should be prepared fresh with every use, starting with the solid material provided by USP. Only with suitable stability studies performed over various periods using controlled storage conditions can a user’s storage conditions be justified as suitable. It should be noted that a number of factors can influence the stability of the reference standard solutions used for the system suitability test in the <643> Total Organic Carbon (TOC) test. These include concentration, microbial decomposition, adsorption to the container surface, and the degradative effects facilitated by oxygen, light, and temperature. The development of turbidity, additional color, or performance variability relative to freshly prepared solutions are indicators of instability. If the user wishes to store and re-use these prepared reference standard solutions or solution concentrates, then the user should have stability data for these solutions under their own storage conditions. If working standard solutions are purchased from a third party supplier, then that supplier should certify the suitability of their materials based on stability studies they have performed. Otherwise, the user should prepare fresh solutions each time the system suitability test is performed. Most of the suppliers of solutions specify expiry dates. But as a practical matter, concentrated reference standard solutions of Sucrose last 3-6 months, and analogous solutions of 1,4 Benzoquinone (pBQ) last about 2 months, assuming they are stored at appropriate temperatures in appropriate containers and protected from light (for pBQ). It is recommended to use refrigeration since this slows down all degradation.

In Stage 3, a neutral electrolyte (KCl) is added to accurately measure the pH of the solution. If the ionic strength of the solution is not increased, the pH measurement will be highly unstable and inaccurate. So KCl is added so that there is minimal concentration gradient across the pH electrode’s membrane between the water and sensor’s internal electrolyte. As a neutral salt, KCl does not impact the pH. It impacts the conductivity of the water, but you do not measure the conductivity again at this stage of the test, only a pH measurement. The method says to use this pH result and the conductivity result from Step 4 (Stage 2), and go to the Stage 3 table. If the measured conductivity from Stage 2 is less than the value in the Stage 3 table (at the measured pH), then you pass.

The <645> Stage 1-3 must be done sequentially, otherwise the user will reach wrong conclusions.

There is nothing in <643> that says how often to run the system suitability test. You need to run the system suitability periodically. The frequency would depend on the quality of water and typical results of system suitability testing. Once the limit has been established (Rs–Rw) from your last system suitability test, you would then determine the frequency for system suitability testing.

Because of the various uses of these waters, microbial requirements are not included in these monographs since this would require some uses to adhere to meaningless and/or inappropriate requirements. Microbial guidelines are provided under the non-mandatory informational chapter Water for Pharmaceutical Purposes <1231>. We recommend that you implement Alert and Action Levels no higher than, and preferably lower than, those listed in Water for Pharmaceutical Purposes <1231> based on the normal microbial performance trends in your water system. The purpose of Alert and Action Levels is to trigger additional, rather than routine microbial measures. These additional control measures should avoid objectionable levels and types of microorganisms for the water’s use.

Yes, additional FAQs on these chapters are available in the Harmonization section of the web site.