A discussion of the pitfalls associated with microbial control in Pharmaceutical Water Applications
1. Enumeration Techniques

Bacteria enumeration techniques must be established for all systems. Enumeration method, culture media, incubation temperature, and incubation time period must all be considered. While the General information Section of USP provides suggested methodology, the European Pharmacopeia presents specific criteria for the indicated enumeration variables. It is suggested that multiple parallel testing be established to determine the culture media and incubation temperature used for a given monitoring program. This test program should be established as a validation protocol. Upon execution of the protocol, enumeration parameters shall be selected. These parameters, as indicated, should demonstrate the ability to quantitatively detect bacteria, when present, in a reasonable incubation time. This program will minimize the effect of a microbial excursion while insuring the quality of product.
2. Sample Port "Preparation"

Occasionally, sampling programs for Total Viable Bacteria (TVB) employ the use of isopropyl alcohol (IPA) preparation (spraying, rinsing, etc.). Since IPA will destroy Gram negative bacteria, its use should be limited to certain applications such as the following:

- TVB sampling from valves in a water purification system in a non environmentally controlled or humid physical location.

- TVB sampling from point-of-use valves in a non environmentally controlled production area.

- TVB sampling from point-of-use valves in an environmentally controlled area where airborne or water borne bacteria may be present during normal operation.

If IPA is employed to "prepare" sample valves, a protocol should demonstrate that IPA is not present in the TVB sample. This can be demonstrated by collecting a Total Organic Carbon sample immediately before IPA preparation and immediately prior to TVB sample collection. If the pre-TVB sample indicates a statistically higher TOC value, additional flushing prior to sampling is required. The successfully executed protocol should be used to generate a formal TVB Sampling Standard Operating Procedure.
3. Presence of Residual Disinfecting Agent in TVB Samples – Ozone or Hydroxyl Radical

The General Information Section of USP clearly states that Purified Water and Water for Injection must not contain an anti-microbial agent. The presence of anti-microbial agents can mask the presence of bacteria. Destruction of bacteria by an anti-microbial agent does not occur instantaneously. Table 1 demonstrates the "contact time" required to destroy bacteria for various disinfecting agents. Bacteria may exist in the presence of an anti-microbial agent. However, when a TVB sample is collected, the time interval between sampling and start of enumeration may be long enough to allow complete destruction of bacteria. Subsequently, samples from systems containing anti-microbial agents often indicate the complete absence of bacteria.

Ozonated USP Purified Water System points-of-use may contain trace concentration of ozone for several reasons. These include, but are not limited to, inadequate/inappropriate ozone destruct inline ultraviolet unit selection, excessively high storage tank dissolved ozone levels, and inaccurate dissolved ozone sensing/monitoring systems.

The hydroxyl radical, while a more powerful oxidant than ozone, is relatively unstable with short chemical life. However, USP Purified Water Systems employing "oversized" inline ultraviolet units with lamps operating at a wave length of 185 nanometers can produce the hydroxyl radical. If these units are positioned in the recirculating USP Purified Water Distribution System, the radical will exists in water at points-of-use. TVB results will not indicate the presence of bacteria. This is a serious situation since the water not only contains an anti-microbial agent but also contains a powerful oxidant which can negatively impact reagents and subsequently, product.
4. Presence of Residual Disinfecting Agent in TVB Samples – Chloramines

A high percentage of municipal water treatment facilities with feed from a surface water source, introduce ammonia into chlorinated water prior to distribution. This process reduces the concentration of halogenated disinfection byproducts that are known carcinogens. Over the last 5 years the number of municipalities using ammonia and chlorine has increased significantly as the regulated disinfection byproduct concentration has been reduced.

Residual chlorine reacts with ammonia producing chloramines. The chemistry of disinfecting chloramines is different than that of chlorine. Specifically, removal of residual chloramines by granular activated carbon is significantly different than that removal of residual chlorine. Activated carbon units employed for chloramines removal must be designed with both lower "face" velocity and volumetric flow rate than activated carbon units for residual chlorine removal. While specialty activated carbon media can provide longer operating time before chloramines "breakthrough" is noted, a conservative media replacement frequency is every six months.

Chloramines are not effectively removed by ion exchange resin or reverse osmosis membranes. As a result, they can be present in USP Purified Water as an anti-microbial agent. As indicated in Table 1, chloramines destroy bacteria at a slower rate than ozone or the hydroxyl radical discussed in Item 3. Unfortunately, this attribute allows bacteria to be "masked" during TVB measurement. This can result in unacceptable point-of-use actual TVB levels beyond established Alert and Action Limits.
5. Coliform Bacteria in Public Water Supplies

The United States Environmental Protection Agency's "National Primary Drinking Water Regulations" (NPDWR) include a limit for Total Coliform Bacteria. This measurement is employed as the primary indication of microbial contamination of Drinking Water although specifications for Cryptosporidium, Giardia lamblia, Viruses (enteric), and Legionella are included. The NPDWR state that Coliform is "Not a health risk in itself; it is used to indicate whether other potentially harmful bacteria may be present". Every positive sample for Total Coliform must be analyzed for either Fecal Coliform or E. coli. If the sample indicates the presence of E. coli or Fecal Coliform or if two consecutive Total Coliform results are positive, even if only one indicates the presence of E. coli or Fecal Coliform, the water does not meet Drinking Water requirements.

The USP Purified Water Official Monograph for both Purified Water and Water for Injection states that the feed water to the system must meet the "Drinking Water" Standards. Subsequently, Total Coliform monitoring of the feed water to these systems is required. While this requirement may be achieved using municipal water data for public supplies, private supplies must be monitored for Total Coliform. It is suggested that feed water periodic (monthly) Total Coliform sampling and analysis be considered for all USP Purified Water and Water for Injection Systems
6. Coliform Bacteria – Private Water Supplies

As indicated above, a Total Coliform monitoring program must be established for systems with feed water from a private source. The Total Coliform monitoring program must be consistent with criteria set forth in the NPDWR including the "Surface Water Treatment Rule" (SWTR). Specifically, the number of samples collected each month determines the actual "specification" for Total Coliform. Assuming that <40 samples are obtained and analyzed each month, no greater than a single sample may indicate the presence of Coliform in a 100 ml. sample. Further, Fecal Coliform or E. coli testing must be performed for any positive Total Coliform result. If Fecal Coliform or E. coli is detected, the water source does not meet Drinking Water requirements.
7. Activated Carbon Unit – Microbial Control

Microbial control in pretreatment equipment upstream of a reverse osmosis system, distillation unit, or other unit operation removing impurities for production of USP Purified Water and/or Water for Injection is critical. Sampling provisions should be included after each unit operation.

Activated carbon units remove residual disinfecting agent from water. As water passes through an activated unit, bacteria will proliferate. The highest water purification system microbial levels are noted in product water from an activated carbon unit (or reducing agent introduction) process. Periodic chemical sanitization with a 4% sodium hydroxide provides effective microbial control but is cumbersome to perform. Periodic steam sanitization is ineffective. Excellent microbial control can be achieved by periodic hot water sanitization and media replacement. Generally, hot water sanitization at a temperature of 90°C for a period of 2 hours and frequency of 2-3 weeks, provides acceptable product water TVB levels ( <500 cfu/ml). The suggested activated carbon media replacement frequency is 6 months for feed water from a surface source or feed water containing chloramines. Media replacement may be increased to every 12 months if the feed water is from a ground water source (not "influenced" by a surface source).
8. Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly undesirable organism. In addition to Total Coliform monitoring of feed water supplies, specific tests to verify the absence of P. aeruginosa in feed water should be considered. Further, P. aeruginosa should not be present in any samples collected throughout the water purification system or in water at points-of-use. As indicated, activated carbon unit product water will generally exhibit the highest system-wide microbial levels as well as the broadest flora of bacteria. It is suggested that a responsive microbial monitoring program should include periodic verification of the absence of P. aeruginosa in activated carbon unit product water. Further, microbial identification techniques should be established to verify the absence of P. aeruginosa in point-of-use water samples for USP Purified Water Systems.
9. "Other" Sources of Total Coliform, Pseudomonas aeruginosa, and Other Pathogens

The General Notices Section of USP defines a "Foreign Substance and Impurity". Within a water purification system producing compendial water, anything added to the system must not ultimately result in the addition of a "Foreign Substance or Impurity", not present in the feed water supply, including microbial contamination. This could include items such as water softening salt with an "iron removal agent", reducing agents employed for residual disinfecting agent removal, and replacement media (activated carbon, sand, anthracite, and ion exchange resin) containing inorganic or organic material that is eluted into the water.

A potential source for introduction of undesirable pathogens is mixed resin rechargeable ion exchange canisters. The canisters are frequently used in USP Purified Water Systems. While the potential for microbial contamination is reduced when "virgin" or "dedicated" resin is used, the potential for contamination during resin transfer, canister purity and handling is a concern. For non-dedicated "float" resin, the concern extends to the quality of resin, previous use, and regeneration procedures. It is suggested that TVB samples should be collected immediately upon placing the canisters into operation. Although bacteria results will not be available for 48-72 hours (incubation time period) the consequences of microbial introduction from the canisters (TVB and undesirable organisms) can be minimized with sampling.
10. Water Softening System Microbial Control

Water softening units contain cation exchange resin operated in the sodium form. Units are periodically regenerated with a concentrated sodium chloride solution. Unlike regeneration of a deionization unit with bacteria destroying acid or caustic, regeneration with sodium chloride does not provide bacteria destruction. In fact, bacteria may be introduced from tubing, valves, salt storage tank, etc. Selection of a "dry" salt storage system as opposed to a "wet" salt storage system can minimize bacteria proliferation in the salt storage tank. Periodic cleaning of the salt storage tank with a sodium hypochlorite solution will remove not only bacteria but particulate matter often present in regenerant salt. Finally, periodic addition of a small amount of sodium hypochlorite to the salt storage tank should be considered.
11. Microbial Control – Reducing Agents

Reducing agents, such as sodium bisulfate, can be used to remove residual disinfecting agents present in feed water. Many systems using a reducing agent exhibit high pre reverse osmosis unit TVB levels and resulting reverse osmosis membrane bacteria fouling. The selection of reducing agent tank storage volume, solution preparation/change frequency, injection pump sizing, and length of delivery tubing are all critical factors for controlling microbial growth.
12. Water Purification System Dead Legs and Operating Temperature

The presence of dead legs in USP Purified Water and Water for Injection Distribution Systems is generally considered during system design and installation. However, the presence of dead legs in the water purification portion of a system, including pretreatment, is important. Further, since chemical sanitization is, or should be employed, for microbial control in the water purification portion of the system, any crevice, threaded connection, piping tee, etc. is a dead leg. It is suggested that items such as the use of diaphragm valves, sanitary connections in plastic (PVC, polypropylene, etc.) piping, sanitary pressure gauges or use of diaphragm isolators can be employed to reduce microbial levels. Finally, recirculation of water purification components, including pretreatment components, at a temperature ? 20°C can provide an order of magnitude reduction in TVB levels. Table 2 provides TVB data (presented in the physical sequence of component arrangement) for an operating system. Pretreatment piping is CPVC, with reverse osmosis unit product tubing, and subsequent component tubing, 316L Stainless Steel. Pretreatment water temperature ranges from 5 to 20°C. Sanitization is performed every 6 months with sodium hypochlorite (pretreatment) and a 1% solution of hydrogen peroxide and peracidic acid ("RO/EDI Loop"). The Table A data where obtained about 6 years after initial system start-up.
13. Microbial Control in Ion Exchange Systems

Ion exchange may be employed in USP Purified Water Purification Systems and USP Water for Injection distillation unit feed water systems. When ion exchange is employed as the primary ion removal operation the use of separate anion and cation exchange unit should be considered. Cation, in the hydrogen form (low pH) and anion in the hydroxide form (high pH) will provide good microbial when compared with mixed ion exchange units operating at a neutral pH. Further, ion exchange vessels with top inlet and bottom outlet should be considered to minimize potential low flow or stagnant areas associated with vessels equipped with top inlet and outlet connections.
14. Reverse Osmosis System Microbial Control

There are several factors that should be considered to control product water TVB levels in reverse osmosis product water. A well designed, operated, and maintained reverse osmosis unit should be capable of providing product with a TVB level < 100 cfu/100 ml. The following factors will affect system performance:

- Feed water microbial control

- Pre-filter change frequency (time versus pressure drop)

- Constant recirculation (high and low recovery modes)

- Minimization/elimination of waste recycle

- Periodic thermal and/or chemical sanitization

- Periodic (time based) membrane "swap" with externally cleaned membranes (every six months)

- Sanitary product tubing

- Lower feed water temperature (< 20-25°C)
15. Storage Tank Microbial Control

When storage tanks are not provided with continuous microbial control provisions (ozone or water temperature > 65 – 80°C), design, operating, and maintenance parameters must be considered. USP Water for Injection Storage Systems operating at ambient or cold recirculation temperatures are generally heated to ? 80°C daily for a time period demonstrated to provide TVB control. USP Purified Water Storage Systems may be either periodically thermally sanitized or chemical sanitized. Since the make-up water to USP Purified Water Storage Tanks is not "sterile", periodic chemical sanitization to remove biofilm should be performed periodically (every six months) to augment periodic hot water sanitization. Tanks should be of sanitary design with internal spay ball system. A non intrusive multipoint level monitoring and control system is preferred. Tank volume should consider the make-up flow rate, the maximum instantaneous demand, and the maximum daily demand from the distribution loop(s).
16. Storage Tank Hydrophobic Vent Filtration Systems

Storage tank hydrophobic vent filtration systems remove bacteria from displacement air during tank drawdown. Generally the 0.2 micron hydrophobic membrane area is inadequate to support drawdown or, more commonly, the condensation of water vapor during thermal cycling of a tank. This condition eventually results in loss of filter integrity and/or observation of Gram-positive bacteria at points-of-use.

Another matter of concern is the use of valves and tubing to "isolate" a hydrophobic vent filter for testing. While heating provisions are generally supplied on the filter housing, they are either not included or inappropriately sized for the large thermal mass of the valves and tubing. This can result in condensation of water, "blinding" of the hydrophobic vent filter and microbial introduction to the stored water.
17. Pump Seals, Sanitary Ferrule Gaskets, and Valve Diaphragms

Distribution pump seal type and materials should be selected for the application. Many USP Water for Injection Systems employ a double mechanical seal with Water for Injection flush. USP Purified Water Systems often employ a mechanical seal of silicon carbide and carbon material. Pump seal replacement should be included as part of a periodic maintenance program. Replacement of a pump seal during a non maintenance shutdown period is an intrusive operation requiring subsequent system sanitization/sterilization. Operation with a leaking seal may appear to be only an annoyance. However, it is possible that bacteria can be aspirated into the recirculating water stream as loop flow and pressure conditions change with distribution loop normal operation.

Sanitary ferrule gaskets should also be replaced as part of a periodic maintenance program. This is particularly important for ozonated or thermally cycled systems. EPDM, Viton, and Silicone may all become a bit "brittle" with time, resulting in a possible leak. Teflon, or Teflon envelope gaskets, generally requires periodic tightening of the sanitary clamps. The sealing area of the Teflon surface exhibits "engagement memory". A very slight "shift" in sealing position during clamp tightening can result in a leak.

Valve diaphragms should also be replaced as part of a periodic maintenance program. The suggested replacement frequency should not exceed every two years. Annual replacement should be considered for automatic and manual valves that are cycled frequently. Leaking diaphragms can result in system back contamination by aspiration or retention of water along the discharge/delivery side of the valve weir.
18. Passivation of Stainless Steel Storage and Distribution Systems

The procedure employed during execution of storage and distribution system passivation can produce microbial issues. On occasions, passivation of a distribution system will be performed using a "passivator supplied" tank and pump. This is most prevalent for new system with a new storage tank that has been passivated by the tank manufacturer. When the temporary tank and pump are disconnected, system integrity is compromised. Subsequently, system sanitization of the reassembled tank/loop is required prior to operation.

A second item of concern is the source of water used for the post passivation rinse operation, particularly final rinse. Passivation is performed during facility shutdown. Frequently, maintenance is simultaneously being performed on the water purification system. As a result, product from the system is not available for rinsing. Rechargeable mixed bed canisters, provided by the passivation contractor, are often used as the source of rinse water. As discussed in Item 13, these canisters are a source of microbial contamination. It is suggested that passivation operations performed when product water is not available from the compendial water system, be scheduled at the end of the shutdown when the system is available. This situation also applies to systems with low water purification make-up capability and large volume storage tanks.
19. Distribution Tubing Chemical Sanitization Provisions

Periodic chemical sanitization of all USP Purified Water storage and distribution systems for biofilm control should be considered. A storage tank in systems employing ozone for TVB is continuously exposed to ozone, eliminating this requirement. However, extended exposure of the distribution loop in these systems should also be considered.

As discussed in earlier section of the multi-part article, critical chemical sanitization parameters include elimination or the ability to flush sanitizing agent through dead legs, selection of sanitizing agent, sanitizing agent concentration, dynamic and static exposure, and sanitization time period.
20. Distribution Loops – Point-of-Use Concerns

Points-of-use may be "hard piped" to distribution system. The possibility for loop back contamination is significant. The use of a properly designed and installed conventional "double block and bleed" system or "satellite" storage tank with air break will eliminate back contamination. Hoses may also be used at points-of-use. A validated hose management program should be established to insure that the quality of the "delivered water" meets the chemical and microbial attributes of the recirculating water.