Pharmaceutical grade water is critical material for equipment cleaning, as well as ingredient water in drug products, so it deserves our attention. Commonly expressed misconceptions held by pharmaceutical professionals including microbiologists, who should know better, about water monitoring include:
- Potable water does not contain coliform bacteria.
- Incoming potable water from a water authority must be monitored at least weekly.
- The USP chapters <61> and <62> contain suitable tests for monitoring water.
- Purified water should routinely be screened for the absence of the USP specified bacteria E. coli, P. aeruginosa, and S. aureus, and the objectionable bacterium B. cepacia.
- AWWA/APHA Standard Methods must be validated for use in the pharmaceutical industry.
- The microbial counts recommended in USP <1231> for purified water and water for injection are scientifically justified.
- Points of use must be sanitized with alcohol prior to sampling.
- Thermophilic bacteria can be found in pharmaceutical grade waters.
- Purified water must not contain any Gram-negative bacteria.
- Water for Injection must be produced by distillation.
- Exceeding the bacterial count level of less than 10 CFU per 100 mL for water for injection will result in bacterial endotoxin contamination.
- Rapidly circulating water in a purified water system will control the formation of bacterial biofilms.
This review article will explore some of these common misconceptions and offer other positions.
Description and Uses of Potable and Pharmaceutical Grade Water
In addition to the usual usage for drinking water, food preparation, and sanitary activities, potable water may be used for equipment and facility cleaning and is the starting material for pharmaceutical grade water production. The widely used microbial count of not more than 500 CFU/mL is not an EPA regulatory requirement but used to evaluate potable water distribution system in terms of circulation, dead legs, and residual chlorine. A U.S. survey of drinking water quality found in 969 public water supplies, an heterotrophic plate count equal to or less than 10 colony-forming units per mL, occurred with 60% of the distribution systems that contain a residual chlorine level (Haas et al, 1991). Details of the production of pharmaceutical grade water from potable water may be found in USP <1231>.
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special offers
from American Pharmaceutical Review – all delivered right to your inbox! Sign up now!
What are the U.S. Federal Current Good Manufacturing Practice (CGMP) requirements? 21 CFR 211.48 (a) states “Potable water shall be supplied under continuous positive pressure in a plumbing system free of defects that could contribute contamination to any drug product. Potable water shall meet the standards prescribed in the Environmental Protection Agency’s Primary Drinking Water Regulations set out in 40 CFR Part 141.” Potable water used in manufacturing facilities outside the U.S. would be expected to comply with local and national standards of that country or World Health Organization (WHO) compliance standards.
Microbiologists working in the pharmaceutical industry, tend to assume that the regulations require an absence of coliforms in potable water. What are the actual requirements? They are tested for absence or presence of total coliform bacteria in the samples collected: For systems, which collect 40 or more per month no more than 5.0% shall be positive; and for systems, which collect fewer than 40 samples per month no more than 1 sample shall be positive. However, there is a zero tolerance for fecal coliforms or E. coli. It is strongly recommended that your company routinely receive quarterly water monitoring reports from the water authority that supplies your manufacturing plant and boil alerts when fecal coliforms are detected and the water distribution system is treated with additional chlorination.
The most widely used pharmaceutical grade waters for equipment cleaning, laboratory testing, and ingredient water are Purified Water (PW) and Water For Injection (WFI). The USP monographs for these waters contain Total Organic Content (TOC) and Conductivity requirements with WFI having the additional bacterial endotoxin requirement of less than 0.25 USP Endotoxin Units per mL, but, surprisingly to some microbiologists, there is no microbial count requirement in the monographs. This is because the microbial count in a non-sterile packaged water is uncontrolled. The recommended total aerobic microbial counts are found in USP <1231> Water for Pharmaceutical Purposes states “Users should establish their own quantitative microbial specifications suited to their water uses. But these values should not be greater than 100 cfu/mL for Purified Water or 10 cfu/100 mL for Water for Injection unless specifically justified, because these values generally represent the highest microbial levels for pharmaceutical water that are still suitable for manufacturing use.”
Although these USP <1231> microbial count requirements have a long history of use in the pharmaceutical industry, the statement that they are the highest levels suitable for manufacturing use is difficult to support on scientific grounds. Experience has shown that PW meeting these requirements typically has proven suitable for the routine manufacture of non-sterile drug products, but there are many exceptions based on microbial infection outbreaks and product recalls due to the presence of objectionable microorganisms that originated from the water system that presumably met a NMT 100 cfu/mL requirement. The WFI microbial count requirement is more stringent than is needed to control bacterial endotoxin and sterility assurance of sterile filtered and certainly terminally sterilized drug products. The author believes that it is preferable to conduct a comprehensive risk assessment based on the microbial counts, bacterial species isolated, dosage form, formulation, manufacturing process and intended use of the drug product than relying on an arbitrary numerical cutoff as recommended in <1231>.
Pharmaceutical Grade Water Product Systems
Until 2017, the European Union GMPs disallowed the production of water for injections by non-distillation methods because of concerns with biofilm production and endotoxin contamination. This recently implemented change was discussed in Questions and answers on production of water for injections by non-distillation methods –Reverse osmosis and biofilms and control strategies EMA/INS/GMP/443117/2017.
The Ph. Eur. WFI monograph now states: “Water for injections in bulk is obtained from water that complies with the regulations on water intended for human consumption laid down by the competent authority or from purified water. “
“It is produced either by: Distillation in an apparatus of which the parts in contact with the water are of neutral glass, quartz or a suitable metal and which is fitted with an effective device to prevent the entrainment of droplets; a purification process that is equivalent to distillation. Reverse osmosis, which may be single-pass or double-pass, coupled with other appropriate techniques such as electro-deionization, ultrafiltration or nano-filtration, is suitable. Notice is given to the supervisory authority of the manufacturer before implementation.”
One perceived advantage in using distillation to produce WFI, over and above the exclusion of bacterial endotoxins, is the use of the heat generated to maintain a hot water distribution system. Facilities implementing a change from distillation to reverse osmosis will need to change their WFI management strategies.
Microbial Monitoring Methods – Total Viable Microbial Count, Coliform Screening, Objectionable Microorganisms Screening, Biofilm Detection and Bacterial Endotoxin Assay
USP <1231> states “Microbes in water systems can be detected as exampled in this section or by methods adapted from <61> Microbial Enumeration Tests and <62> Tests for Specified Microorganisms or the current edition of Standard Methods for the Examination of Water and Wastewater by the American Public Health Association.”
As the full name of the USP chapters indicates, i.e., Microbiological Examination of Non-sterile Products, these methods in terms of media, incubation conditions, and QC organism selections were never designed for water testing. The American Water Works Association/American Public Health Association (AWWA/APHA) Standard Method 9215 Heterotrophic Plate Count (HPC) that this author supports describes the pour plate, spread plate and membrane filtration methods, sample collection, storage and preparation, media selection, incubation, and counting and recording results. As originally reported by the U.S. Federal Environmental Protection Agency (EPA) microbiologists who developed the medium (Reasoner and Geldreich, 1985), the highest counts typically will be obtained using membrane filtration, R2A agar, incubated at 20 to 28°C for 5 to 7 days. Presumably as a compromise to obtain results as quickly as possible, the European Pharmacopeia selected R2A agar, incubated at 30-35°C for 48 to 72 hours to obtain microbial counts in pharmaceutical grade water. Plate Count Agar, that is less rich in nutrients than soybean-casein digest (SCD) agar but more that R2A agar, may be a good compromise as the subculture of bacteria from R2A to SCD agar can be difficult.
A perennial question asked in pharmaceutical discussion groups is whether membrane filters of 0.22 or 0.45 micron pore size should be used for water monitoring. A study conducted by the Millipore Corporation on the recovery of Brevundimonas diminuta, the challenge bacterium for the validation of sterilizing-grade membrane filters showed that the use of the 0.22 micron filters had no advantage over 0.45 micron filters (Carter, 1996) and there is evidence that colonies grow faster and larger on 0.45 micron filters, presumably due to superior wicking of nutrients.
As an official method, the AWWA/APHA standard method is considered validated by the 1985 Standard Methods Committee. In addition, as pharmaceutical grade water has no added substances that could inhibit or enhance the bacterial recovery, no method suitability test is required. Many pharmaceutical microbiologists waste their company’s resources in this unnecessary activity. However, if your quality control unit feels more comfortable, you can qualify the method by spiking growth-promotion bacteria into water samples and demonstrate the recovery of these challenge organisms.
Coliform monitoring is not recommended for pharmaceutical grade waters. If coliform monitoring is conducted for incoming potable water when the AWWA/APHA Standard Method 9233 Enzyme Substrate Coliform Test, which is widely used by U.S. water authorities, is strongly recommended.
Routine monitoring of PW for objectionable microorganisms is not recommended. An exception may be monitoring for the presence of Burkholderia cepacia in PW used as ingredient water for topical liquids, ointments and creams, and nasal sprays. The recently published in-process revision USP <60> Microbiological Examination of Non-sterile Products: Test for Burkholderia cepacia complex could be used for PW monitoring.
It should be expected that biofilms would form within water storage and distribution systems. Currently there is no satisfactory method for monitoring water systems for biofilms. The occasional high counts seen when monitoring may be due to biofilms sloughing off the internal surfaces of the water distribution system. Individual isolates may be monitored for their ability to form biofilms in a micro-titer plate. For example, bacterial cultures are incubated overnight and transferred into fresh TSB to an OD600 nm of 0.05. Aliquots (1 mL) were transferred into 24-well plates and plates were incubated overnight at 37 °C. Attachment/biofilm was evaluated by crystal violet staining after washing to quantify attached cells and measurements were taken spectrophotometrically at OD595 nm (O’Toole and Kolter, 1998). Contrary to widely held beliefs, increased flow rates and turbulence promotes biofilm production in water systems by accessing more nutrients and minerals but results in more dense biofilms (Tsagkari and Sloan, 2018).
The Limulus ambeobocyte lysate (LAL) - bacterial endotoxin assays as described in USP <85> are suitable to WFI monitoring.
Monitoring for Thermophiles in Hot Pharmaceutical Grade Water Systems
Another perennial question asked in pharmaceutical discussion groups is whether a WFI system maintained at 80°C needs to be monitored for thermophilic bacteria. Thermophiles (Kristjansson and Stetter, 1991) are a diverse group of bacteria and archaea that have specific and variable nutritional requirements, i.e., rich nutrients, vitamins, light, electron acceptors other than oxygen, anaerobic conditions and elevated temperatures, making it highly unlikely that thermophiles will exist in hot WFI. If they persisted in hot water they would not grow in the human body, which has a temperature around 37°C. Given this situation, monitoring pharmaceutical grade water for thermophiles is not recommended.
Regulatory Expectations for Water Monitoring
The 1993 FDA Guide to Inspections of High Purity Water Systems does contain examples of the FDA expectations. For example with respect to incoming potable water monitoring the guide states: “A water system should be designed to operate within these anticipated extremes. Obviously, the only way to know the extremes is to periodically monitor feed water. If the feed water is from a municipal water system, reports from the municipality testing can be used in lieu of in-house testing.”
The overall inspection strategy recommended to FDA investigators was:
“Manufacturers typically will have periodic printouts or tabulations of results for their purified water systems. These printouts or data summaries should be reviewed. Additionally, investigation reports, when values exceed limits, should be reviewed.”
Selection of Monitoring Methods, Growth Media and Incubation Conditions
The scientific consensus, first proposed by Reason and Geldreich (1985), is that the use of less nutrient-rich microbiological growth medium, the membrane filter technology, lower incubation temperatures, and longer incubation times will give the highest recovery rates (Cundell, 2004). Despite the use of the medium R2A agar being the industry practice, USP <1231> recommends Plate Count Agar and recommends growth promotion microorganisms used at a minimum Pseudomonas aeruginosa ATCC 9027 and Bacillus subtilis ATCC 6633. The chapter continues that additional organisms should be used to represent those that are considered objectionable and/or typically isolated from the water system (house isolates).
Although neither of these recommended bacteria is expected to be found in pharmaceutical grade water, it is customary to use well described laboratory cultures as QC microorganisms to demonstrate the consistent level of performance of microbiological growth media.
For example, the Difco/BD Manual recommends inoculation with 20-200 colonies of Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus incubated at 35°C for up to 48 hours for growth promotion testing. In contrast, the Japanese Pharmacopeia recommends the use of stressed waterborne bacteria Methylobacterium extorquens and Pseudomonas fluorescens. MilliporeSigma in their product literature for R2A Agar recommends the following growth-promotion testing (see Table 1).
Microbial Identification Methods
The predominant microorganisms in pharmaceutical grade water are Gram-negative, oxidase-positive rod-shaped bacteria (Cundell, 2004). These may include Sphingomonas paucimobilis, Comamonas acidovorans, Xanthomonas multophilia, Ralstonia picketii, Burkholderia cepacia and Pseudomonas vesicularis. General guidance on microbial identification can be found in USP <1117> Microbiological characterization, identification and strain typing.
Continuous Verses Grab Sample Testing
General experience with both chemical and microbiological testing of water shows that the processing of sampling, shipment and storage in a sterile container for laboratory testing changes the test results usually for the worst and delays obtaining the results until after the manufacturing step is long completed. Sanitization of points of use with 70% isopropyl alcohol prior to sample collection is discouraged as it does not reflect water usage during manufacturing and would contaminate samples for TOC determinations.
Emerging Water Testing Technologies
The most promising water testing technology is LASER-induced fluorescence viable particle monitoring. This technology may be used for at line continuous monitoring of the loops of both PW and WFI systems. The challenges associated with the implementation of this technology is lack of equivalency of colony-forming units to LASER induced fluorescence viable particles and the increased data level when transitioning from daily or weekly grab sample monitoring to continuous monitoring. Compliance-minded quality units will want to react to isolated out-of-limit counts that do not represent adverse trends in the water distribution system. Continuous monitoring should be viewed as process analytical technology. A paradigm shift must occur in terms of response to the collection and analysis of monitoring data.
Water for Equipment and Packaging Component Cleaning and Ingredient Water Potable water can be used for equipment cleaning with either PW for non-sterile manufacturing or WFI for sterile manufacturing used as the final rinse. This conserves more expensive pharmaceutical grade water. Although microbial contaminated PW used for cleaning may result in contaminated processing equipment, the largest source of contaminated non-sterile drug products is ingredient water from poorly designed and operated water systems.
Objectionable Microorganisms in Non-Sterile Drug Products
Based on a survey of U.S. product recalls, Burkholderia cepacia complex is the most frequently implicated microorganism with the recall of nonsterile drug products (Jimenez and Sutton, 2011). For a comprehensive discussion of objectionable microorganisms the reader is referred to the PDA Technical Report No. 67 for the industry practices related to the CGMP regulations.
Conclusions
Given the intensity of water monitoring in the pharmaceutical industry, it is surprising that there are so many misconceptions around pharmaceutical grade water. The author hopes that this review article will help clear up many of these common misconceptions.
References
Carter, J. Evaluation of recovery filters for use in bacterial retention testing of sterilizing-grade filters PDA J. Pharm. Sci. & Technol. 50(3): 147-153 1996
Cundell, A. M. Microbial Monitoring of Potable Water and Water for Pharmaceutical Purposes In Microbial Contamination Control in the Pharmaceutical Industry Luis Jimenez (Editor) Marcel Dekker pp45-75 2004
Haas C. D., M. A. Meyer and M. S. Paller 1982 Analytical note: evaluation of the m-SPC method as a substitute for standard plate count in water microbiology. J. AWWA 74: 322
Jimenez, L and S. Sutton, 2011 A Review of Reported Recalls Involving Microbiological Control 2004-2011 with Emphasis on FDA Considerations of “Objectionable Organisms” Amer. Pharm. Rev. 15(1):42-57.
Kristjansson, J.K. and K. O. Stetter Thermophilic Bacteria In Thermophilic Bacteria. Kristjansson, J.K. (editor) CRC Press, pp2-13 1991
O’Toole G.A. and R. Kolter. 1998 Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol. Microbiol. 28(3): 449–61
PDA Technical Report No. 67 Exclusion of Objectionable Microorganisms from Non-sterilePharmaceutical and OTC Drug Products, Medical Devices and Cosmetics 2012
Reason, D. J. and E. E. Geldreich, 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 39(10): 1-7
Tsagkari, E and W. T. Sloan 2018 Turbulence accelerates the growth of drinking water biofilms Biopro. Biosyst. Eng. 41:757–770
Author Biography
Dr. Tony Cundell consults with a number of pharmaceutical, consumer health and dietary supplement companies, microbiology instrument manufacturers, contract testing laboratories and sterile compounding pharmacies in the areas of microbial risk assessment, regulatory affairs, and microbiological testing. Prior to November 2013 he worked for Merck Research Laboratories in Summit, New Jersey, as the Senior Principal Scientist in early phase drug development. Earlier in his career, Tony Cundell worked at a director level in Quality Control and Product Development organizations at the New York Blood Center, Lederle Laboratories, Wyeth Pharmaceuticals and Schering-Plough.
He is a member of the 2015-2020 U.S.P. Microbiology Committee of Experts where he takes a leadership role in the area of modern microbiology methods, co-chairing the USP Expert Panel that published a stimuli article in the Sept-Oct. 2017 Pharmacopeial Forum entitled The Development of Compendial Rapid Sterility Tests.
Tony Cundell chaired the PDA task force responsible for the groundbreaking 2000 Technical Report No. 33 The Development, Validation, and Implementation of New (Rapid) Microbiological Methods. In June 2009, he co-edited with Anthony Fontana a book entitled Water Activity Applications in the Pharmaceutical Industry and contributed two chapters to the book. He was co-chair of the PDA task force responsible for 2014 Technical Report No. 67 Exclusion of Objectionable Microorganisms from Non-Sterile Drug Products. In 2015 he was appointed to the Advisory Committee of Sterile Compounding to the State of Massachusetts Board of Pharmacy. He received the 2016 PDA Martin Van Trieste Pharmaceutical Science Award for outstanding contributions to the advancement of pharmaceutical science. More recently Tony Cundell co-authored a review article entitled Data Integrity in the Microbial Testing in the September-October, 2017 issue of American Pharmaceutical Review.
Tony Cundell has a Ph.D. in Microbiology from the Lincoln University, New Zealand.